The Journal of Immunology recent issues

IN THIS ISSUE [IN THIS ISSUE]

Wed, 18 Nov 2009 13:05:59 PST

Comment on "TLR9-Dependent Activation of Dendritic Cells by DNA from Leishmania major Favors Th1 Cell Development and the Resolution of Lesions" [LETTERS TO THE EDITOR]

Bogdan, C., Schleicher, U. — Wed, 18 Nov 2009 13:06:00 PST

Response to Comment on "TLR9-Dependent Activation of Dendritic Cells by DNA from Leishmania major Favors Th1 Cell Development and the Resolution of Lesions" [LETTERS TO THE EDITOR]

Doyen, N. — Wed, 18 Nov 2009 13:06:00 PST

A Crucial Door to the Mast Cell Mystery Knocked In [PILLARS OF IMMUNOLOGY]

Kawakami, T. — Wed, 18 Nov 2009 13:06:00 PST

Pillars Article: Fate of Bone Marrow-Derived Cultured Mast Cells After Intracutaneous, Intraperitoneal, And Intravenous Transfer Into Genetically Mast Cell-Deficient W/Wv Mice. Evidence That Cultured Mast Cells Can Give Rise To Both Connective Tissue Type And Mucosal Mast Cells. The Journal of Experimental Medicine. 1985. 162: 1025-1043 [PILLARS OF IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:00 PST

What Role Does the Route of Immunization Play in the Generation of Protective Immunity against Mucosal Pathogens? [BRIEF REVIEWS]

Belyakov, I. M., Ahlers, J. D. — Wed, 18 Nov 2009 13:06:00 PST

The route of vaccination is important in influencing immune responses at the initial site of pathogen invasion where protection is most effective. Immune responses required for mucosal protection can differ vastly depending on the individual pathogen. For some mucosal pathogens, including acute self-limiting infections, high-titer neutralizing Abs that enter tissue parenchyma or transude into the mucosal lumen are sufficient for clearing cell-free virus. However, for pathogens causing chronic infections such as HIV, hepatitis C virus, herpes viruses, mycobacteria, and fungal and parasitic infections, a single arm of the immune response generated by systemic vaccination may be insufficient for protection. Induction of the mucosal innate and adaptive immune systems, including CD4⁺ T help, Th17, high avidity CD8⁺ CTL, and secretory IgA and IgG1 neutralizing Abs, at the site of pathogen entry may be required for effective protection against highly invasive pathogens that lead to chronic infection and may be generated predominantly by mucosal vaccination.

Cutting Edge: Requirement of MARCH-I-Mediated MHC II Ubiquitination for the Maintenance of Conventional Dendritic Cells [CUTTING EDGE]

Wed, 18 Nov 2009 13:06:00 PST

MARCH-I (membrane-associated RING-CH I) has been suggested as a physiological E3 ubiquitin ligase for both MHC class II (MHC II) and B7-2. In this study, we show that MARCH-I-mediated MHC II ubiquitination is necessary for the maintenance of conventional dendritic cell (cDC) functions in the steady state. MARCH-I-deficient cDCs accumulated MHC II and B7-2 and exhibited low Ag-presenting ability for exogenous Ags and low cytokine-producing ability upon stimulation in vivo. Importantly, MHC II, but not B7-2, was required for impaired cDC function induced by loss of MARCH-I in vivo. Moreover, MHC II knockin mice whose MHC II was not ubiquitinated showed dysfunction of cDC similar to that of MARCH-I knockout mice. These results suggest that the accumulation of MHC II resulting from loss of ubiquitination caused cDC abnormality; therefore, MARCH-I may function as a housekeeper of cDC in the steady state.

Cutting Edge: Memory CD8 T Cell Compartment Grows in Size with Immunological Experience but Nevertheless Can Lose Function [CUTTING EDGE]

Wed, 18 Nov 2009 13:06:00 PST

The size of the adaptive immune system is considered to be kept constant by the attrition of pre-existing memory. However, recently it was shown that the CD8 memory compartment can grow in size and the number of pre-existing memory is largely preserved, predicting that pre-existing immunity should be maintained (Vezys et al.; Nature 457: 196–199). Experimental proof for this assumption is still lacking. We address this question in the Listeria monocytogenes (L.m.) infection model and confirm the growth of size of the memory compartment by subsequent vaccination with modified vaccinia virus Ankara. We also find only modest attrition of pre-existing L.m.-specific memory CD8 T cells. However, pre-existing protective immunity toward L.m. is not preserved. Pre-existing L.m.-specific effector-memory cells, in contrast to central memory cells, become altered, and this results in a significant loss of pre-existing protective immunity. Our findings are clinically relevant for vaccines introducing new CD8 memory cells in high numbers, as this might influence pre-existing immunity.

Peroxisome Proliferator-Activated Receptor {gamma} Ligands Enhance Human B Cell Antibody Production and Differentiation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:00 PST

Protective humoral immune responses critically depend on the optimal differentiation of B cells into Ab-secreting cells. Because of the important role of Abs in fighting infections and in successful vaccination, it is imperative to identify mediators that control B cell differentiation. Activation of B cells through TLR9 by CpG-DNA induces plasma cell differentiation and Ab production. Herein, we examined the role of the peroxisome proliferator-activated receptor (PPAR)/RXR pathway on human B cell differentiation. We demonstrated that activated B cells up-regulate their expression of PPAR. We also show that nanomolar levels of natural (15-deoxy-^12,14-prostaglandin J₂) or synthetic (rosiglitazone) PPAR ligands enhanced B cell proliferation and significantly stimulated plasma cell differentiation and Ab production. Moreover, the addition of GW9662, a specific PPAR antagonist, abolished these effects. Retinoid X receptor (RXR) is the binding partner for PPAR and is required to produce an active transcriptional complex. The simultaneous addition of nanomolar concentrations of the RXR ligand (9-cis-retinoic acid) and PPAR ligands to CpG-activated B cells resulted in additive effects on B cell proliferation, plasma cell differentiation, and Ab production. Furthermore, PPAR ligands alone or combined with 9-cis-retinoic acid enhanced CpG-induced expression of Cox-2 and the plasma cell transcription factor BLIMP-1. Induction of these important regulators of B cell differentiation provides a possible mechanism for the B cell-enhancing effects of PPAR ligands. These new findings indicate that low doses of PPAR/RXR ligands could be used as a new type of adjuvant to stimulate Ab production.

Impaired NK Cytolytic Activity and Enhanced Tumor Growth in NK Lytic-Associated Molecule-Deficient Mice [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Hoover, R. G., Gullickson, G., Kornbluth, J. — Wed, 18 Nov 2009 13:06:00 PST

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells. It is weakly expressed in resting NK cells but upon target cell stimulation or after incubation with cytokines that enhance NK killing, NKLAM mRNA levels increase and protein is synthesized and is targeted to cytoplasmic granule membranes. We have previously shown that NKLAM plays a role in perforin/granzyme-mediated cytolysis in vitro. To further investigate the function of NKLAM in NK cell-mediated cytotoxicity, we generated, by gene targeting, NKLAM-deficient mice. These mice have normal numbers of NK cells and other lymphoid populations in the spleen. They also have no alterations in NK maturation or NK receptor repertoire. NK cells from NKLAM-deficient and WT mice have comparable amounts of perforin, granzyme B, and lysosomal membrane-associated protein 1 (CD107a) in their cytotoxic granules and comparable levels of granule exocytosis are induced by PMA and calcium ionophore A23187. However, NKLAM-deficient NK cells display significantly less NK cytotoxic activity in vitro than WT NK cells. They also secrete less IFN- upon target cell stimulation, In addition, NKLAM-deficient mice exhibit greater numbers of pulmonary metastases after i.v. injection with B16 melanoma cells. These studies indicate that NKLAM-deficient mice have diminished capacity to control tumor metastases and support the role for NKLAM in NK function both in vitro and in vivo.

NOD2 Ligation Subverts IFN-{alpha} Production by Liver Plasmacytoid Dendritic Cells and Inhibits Their T Cell Allostimulatory Activity via B7-H1 Up-Regulation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Castellaneta, A., Sumpter, T. L., Chen, L., Tokita, D., Thomson, A. W. — Wed, 18 Nov 2009 13:06:00 PST

The nucleotide-binding oligomerization domain (NOD)2/CARD15 protein, which senses muramyl dipeptide (MDP), a product of bacterial peptidoglycan, appears to play an important role in regulating intestinal immunity. Although the liver is exposed to gut-derived MDP, the influence of NOD2 ligation on hepatic APC, in particular dendritic cells (DC), is unknown. Freshly isolated mouse liver and spleen plasmacytoid (p)DC expressed higher levels of NOD2 message than conventional myeloid (m)DC. Following MDP stimulation in vivo, liver pDC, but not mDC, up-regulated expression of IFN regulatory factor 4 (IRF-4), a negative regulator of TLR signaling, and induced less allogeneic T cell proliferation and IFN- production. The adoptive transfer of liver pDC from MDP-treated mice failed to prime allogeneic T cells in vivo. By contrast, splenic DC IRF-4 levels and T cell stimulatory activity remained unchanged. Liver pDC from MDP-stimulated mice also displayed greater IB, cell surface B7-H1, and B7-H1 relative to CD86 than control liver pDC. No similar effects were observed for liver mDC or spleen DC. Absence of B7-H1 on liver pDC reversed the inhibitory effect of MDP. After ex vivo stimulation with LPS or CpG, liver pDC but not mDC from MDP-treated animals secreted less IL-12p70, IL-6, and TNF- and induced weaker allogeneic T cell proliferation than those from controls. Moreover, CpG-stimulated liver pDC from MDP-treated mice secreted less IFN- than their splenic counterparts, and systemic levels of IFN- were reduced in MDP-treated animals after CpG administration. These findings suggest that differential effects of NOD2 ligation on liver pDC may play a role in regulating hepatic innate and adaptive immunity.

Intranasal Immunization Promotes Th17 Immune Responses [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zygmunt, B. M., Rharbaoui, F., Groebe, L., Guzman, C. A. — Wed, 18 Nov 2009 13:06:00 PST

Th17 cells are a lineage of CD4⁺ T cells characterized by IL-17 secretion, which plays a crucial role in immune responses against important respiratory pathogens, such as Mycobacterium tuberculosis. In this study, we demonstrated that intranasal (i.n.) immunization leads per se to Th17-biased immune responses, regardless of the adjuvant used. The activated CD4⁺ T cells also showed an up-regulated expression of the chemokine receptor CCR6, which is a marker for murine Th17 cells. These results have important implications in the context of optimizing rational vaccine design, since i.n. immunization appears to be the strategy of choice for situations where the induction of a Th17 phenotype would be beneficial.

Chimeric NKG2D Expressing T Cells Eliminate Immunosuppression and Activate Immunity within the Ovarian Tumor Microenvironment [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Barber, A., Rynda, A., Sentman, C. L. — Wed, 18 Nov 2009 13:06:00 PST

Adoptive transfer of T cells expressing chimeric NKG2D (chNKG2D) receptors, a fusion of NKG2D and CD3, can lead to long-term, tumor-free survival in a murine model of ovarian cancer. To determine the mechanisms of chNKG2D T cell antitumor efficacy, we analyzed how chNKG2D T cells altered the tumor microenvironment, including the tumor-infiltrating leukocyte populations. chNKG2D T cell treatment of mice bearing ID8 tumor cells increased the number and activation of NK cells and increased the activation of host CD8⁺ T cells within the tumor. Foxp3⁺ regulatory T cells at the tumor site decreased more than 300-fold after chNKG2D T cell treatment. Tumor-associated regulatory T cells expressed cell surface NKG2D ligands and were killed by chNKG2D T cells in a perforin-dependent manner. chNKG2D T cells also altered the function of myeloid cells at the tumor site, changing these cells from being immunosuppressive to enhancing T cell responses. Cells isolated from the tumor produced elevated amounts of IFN-, NO, and other proinflammatory cytokines after chNKG2D T cell treatment. ChNKG2D T cells required perforin, IFN-, and GM-CSF to induce a full response at the tumor site. In addition, transfer of chNKG2D T cells into mice bearing tumors that were established for 5 weeks led to long-term survival of the mice. Thus, chNKG2D T cells altered the ovarian tumor microenvironment to eliminate immunosuppressive cells and induce infiltration and activation of antitumor immune cells and production of inflammatory cytokines. This induction of an immune response likely contributes to chNKG2D T cells’ ability to eliminate established tumors.

RREB-1 Is a Transcriptional Repressor of HLA-G [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Flajollet, S., Poras, I., Carosella, E. D., Moreau, P. — Wed, 18 Nov 2009 13:06:00 PST

The nonclassical HLA-G is a molecule specifically involved in immune tolerance with highly restricted tissue distribution in healthy conditions. Yet it is overexpressed in numerous tumors and in allografts with better acceptance. Major mechanisms involved in regulation of HLA-G transcription are still poorly described. Thus, to characterize these mechanisms we have developed a specific proteomic approach to identify proteins that bind differentially to the HLA-G gene promoter by promoter pull-down assay followed by spectrometry mass analysis. Among specific binding factors, we focused on RREB-1, a ras-responsive element binding protein 1. We demonstrated that RREB-1 represses HLA-G transcriptional activity and binds three ras response elements within the HLA-G promoter. RREB-1 protein, specifically in HLA-G-negative cells, interacts with subunits of CtBP complex implicated in chromatin remodeling. This demonstration is the first of a repressor factor of HLA-G transcriptional activity taking part in HLA-G repression by epigenetic mechanisms.

TLR2 Promotes Th2/Th17 Responses via TLR4 and TLR7/8 by Abrogating the Type I IFN Amplification Loop [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:00 PST

TLR2 plays an important role in the removal of Gram-positive bacteria; contrastingly, it also appears to have important protective effects against unrestrained inflammation and subsequent organ injury during infection and autoimmunity. We hypothesized that TLR2 tunes the phenotype of dendritic cells (DCs) activated through other TLRs, thereby fulfilling a crucial role in the modulation of the immune response. TLR2 potently inhibited TLR4- and TLR7/8-induced cytokine production by human DCs. The inhibitory effect of TLR2 on the release of TNF- but not of IL-12p70 was mediated by PI3K. TLR2 inhibits the production of IL-12p70 by dampening the type 1 IFN amplification loop. When DCs were triggered with the potent synergistic combination of LPS (TLR4) and R848 (TLR7/8) in conjunction with a TLR2 ligand, a clear shift to more Th2- and Th17-prone responses in the naive and memory T cell subpopulations was observed. This shift in T cell responses was inherent to the inability of TLR2-stimulated DCs to produce IL-12p70 and was dependent on the production of IL-1 and IL-6.

Innate Immune CD11b+Gr-1+ Cells, Suppressor Cells, Affect the Immune Response during Theiler's Virus-Induced Demyelinating Disease [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Bowen, J. L., Olson, J. K. — Wed, 18 Nov 2009 13:06:00 PST

Multiple sclerosis is a demyelinating disease associated with an inflammatory immune response in the CNS. Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a relevant mouse model for the study of multiple sclerosis. TMEV infection of susceptible mice leads to a persistent virus infection of the CNS which contributes to development of demyelinating disease. We have previously shown that the innate immune response can affect the development and progression of demyelinating disease. In the current studies, we determined that the predominant infiltrating cells during the innate immune response are CD11b⁺Ly6C⁺ cells. CD11b⁺Ly6C⁺ cells are immature myeloid cells that have exited the bone marrow without maturing and have been shown to suppress CD4⁺ and CD8⁺ T cell responses. Therefore, we wanted to determine what role these cells play in development and progression of demyelinating disease. TMEV-infected mice depleted of CD11b⁺Ly6C⁺ cells during the innate immune response developed a reduced demyelinating disease which was associated with a decreased myelin-specific CD4⁺ T cell response and a decreased inflammatory immune response in the CNS. TMEV-infected mice depleted of CD11b⁺Ly6C⁺ cells had increased virus-specific CD4⁺ and CD8⁺ T cell responses during early virus infection associated with increased expression of IFN- and IL-17 and decreased expression of IL-10 in the CNS. These results suggest that CD11b⁺Ly6C⁺ cells which infiltrate into the CNS during the innate immune response are myeloid-derived suppressor cells that suppress virus-specific T cell responses and contribute to the development of demyelinating disease.

Prostacyclin Inhibits IFN-{gamma}-Stimulated Cytokine Expression by Reduced Recruitment of CBP/p300 to STAT1 in a SOCS-1-Independent Manner [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Strassheim, D., Riddle, S. R., Burke, D. L., Geraci, M. W., Stenmark, K. R. — Wed, 18 Nov 2009 13:06:00 PST

Increasing evidence indicates that pulmonary arterial hypertension is a vascular inflammatory disease. Prostacyclin (PGI₂) is widely used to treat pulmonary arterial hypertension and is believed to benefit patients largely through vasodilatory effects. PGI₂ is also increasingly believed to have anti-inflammatory effects, including decreasing leukocyte cytokine production, yet few mechanistic details exist to explain how these effects are mediated at the transcriptional level. Because activated monocytes are critical sources of MCP-1 and other cytokines in cardiovascular inflammation, we examined the effects of iloprost on IFN-- and IL-6-stimulated cytokine production in human monocytes. We found that iloprost inhibited IFN-- and IL-6-induced MCP-1, IL-8, RANTES, and TNF- production in monocytes, indicating wide-ranging anti-inflammatory action. We found that activation of STAT1 was critical for IFN--induced MCP-1 production and demonstrated that iloprost inhibited STAT1 activation by several actions as follows: 1) iloprost inhibited the phosphorylation of STAT1-S727 in the transactivation domain, thereby reducing recruitment of the histone acetylase and coactivator CBP/p300 to STAT1; 2) iloprost selectively inhibited activation of JAK2 but not JAK1, both responsible for activation of STAT1 via phosphorylation of STAT1-Y701, resulting in reduced nuclear recruitment and activation of STAT1; and 3) SOCS-1, which normally terminates IFN--signaling, was not involved in iloprost-mediated inhibition of STAT1, indicating divergence from the classical pathway for terminating IFN--signaling. We conclude that PGI₂ exerts anti-inflammatory action by inhibiting STAT1-induced cytokine production, in part by targeting the transactivation domain-induced recruitment of the histone acetylase CBP/p300.

Role of Double-Stranded RNA Pattern Recognition Receptors in Rhinovirus-Induced Airway Epithelial Cell Responses [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:00 PST

Rhinovirus (RV), a ssRNA virus of the picornavirus family, is a major cause of the common cold as well as asthma and chronic obstructive pulmonary disease exacerbations. Viral dsRNA produced during replication may be recognized by the host pattern recognition receptors TLR-3, retinoic acid-inducible gene (RIG)-I, and melanoma differentiation-associated gene (MDA)-5. No study has yet identified the receptor required for sensing RV dsRNA. To examine this, BEAS-2B human bronchial epithelial cells were infected with intact RV-1B or replication-deficient UV-irradiated virus, and IFN and IFN-stimulated gene expression was determined by quantitative PCR. The separate requirements of RIG-I, MDA5, and IFN response factor (IRF)-3 were determined using their respective small interfering RNAs (siRNA). The requirement of TLR3 was determined using siRNA against the TLR3 adaptor molecule Toll/IL-1R homologous region-domain-containing adapter-inducing IFN-β (TRIF). Intact RV-1B, but not UV-irradiated RV, induced IRF3 phosphorylation and dimerization, as well as mRNA expression of IFN-β, IFN-1, IFN-2/3, IRF7, RIG-I, MDA5, 10-kDa IFN--inducible protein/CXCL10, IL-8/CXCL8, and GM-CSF. siRNA against IRF3, MDA5, and TRIF, but not RIG-I, decreased RV-1B-induced expression of IFN-β, IFN-1, IFN-2/3, IRF7, RIG-I, MDA5, and inflammatory protein-10/CXCL10 but had no effect on IL-8/CXCL8 and GM-CSF. siRNAs against MDA5 and TRIF also reduced IRF3 dimerization. Finally, in primary cells, transfection with MDA5 siRNA significantly reduced IFN expression, as it did in BEAS-2B cells. These results suggest that TLR3 and MDA5, but not RIG-I, are required for maximal sensing of RV dsRNA and that TLR3 and MDA5 signal through a common downstream signaling intermediate, IRF3.

Estrogen Regulates Transcription Factors STAT-1 and NF-{kappa}B to Promote Inducible Nitric Oxide Synthase and Inflammatory Responses [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Dai, R., Phillips, R. A., Karpuzoglu, E., Khan, D., Ahmed, S. A. — Wed, 18 Nov 2009 13:06:01 PST

Estrogen regulation of inflammatory responses has broad physiological and pathological consequences. However, the molecular mechanism of estrogen regulation of inflammation is still poorly understood. In this study, we report that activation of both STAT-1 and NF-B signaling is essential for Con A-induced inducible NO synthase (iNOS) and NO in murine splenocytes. Estrogen enhances STAT-1 DNA-binding activity without increasing the expression of phosphorylated and total STAT-1 protein. We have recently reported that estrogen blocks the nuclear expression of NF-B p65 and modifies nuclear NF-Bp50. Here, we demonstrated that both nuclear STAT-1 and NF-B are modified by serine protease-mediated proteolysis, which resulted in altered STAT-1 and NF-B activity/signaling in splenocytes from estrogen-treated mice. Inhibition of serine protease activity with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) restores the nuclear expression of full-length STAT-1 and NF-B proteins, and resulted in decreased STAT-1 DNA-binding activity and formation of NF-B p65/p50 binding complexes in nuclei of splenocytes from estrogen-treated mice. Consequently, there is significantly decreased iNOS and IFN- production in AEBSF-treated splenocytes from estrogen-treated mice, which suggests a positive regulatory role of truncated STAT-1 and/or NF-B. Interestingly, there is increased production of MCP-1 in STAT-1 or NF-B small interfering RNA-transfected cells, as well as in AEBSF-treated splenocytes from estrogen-treated mice. These data suggest a differential role of truncated STAT-1 and NF-B in regulation of various inflammatory molecules in splenocytes from estrogen-treated mice. Together, our data reveal a novel molecular mechanism of estrogen-mediated promotion of inflammatory responses, which involves posttranslational modification of STAT-1 and NF-B proteins.

IL-9 Regulates Pathology during Primary and Memory Responses to Respiratory Syncytial Virus Infection [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Dodd, J. S., Lum, E., Goulding, J., Muir, R., Van Snick, J., Openshaw, P. J. M. — Wed, 18 Nov 2009 13:06:01 PST

IL-9 is a cytokine of great current interest associated with allergic/Th2 responses. High levels of IL-9 are present in bronchial secretions from infants with respiratory syncytial virus (RSV) bronchiolitis. To test its effects in RSV disease with a Th2 profile, BALB/c mice were vaccinated with recombinant vaccinia virus expressing the RSV G protein. On RSV challenge, immunized mice developed augmented disease characterized by enhanced pulmonary Th2 and local IL-9 production peaking on days 7–10 of RSV infection. Depletion with anti-IL-9 Ab at vaccination or RSV challenge enhanced viral clearance. Depletion only at challenge had no effect on disease severity, whereas depletion at immunization and challenge enhanced Th1 responses, inhibited virus-specific IgG1 production, and enhanced disease severity. By contrast, depletion of IL-9 at immunization boosted IgG2a and inhibited the Th2 response and disease during subsequent infection without a concomitant increase in type 1 cytokines. Adoptive transfer of secondary memory CD4 T cells from the spleens of IL-9-depleted mice into naive recipients replicated many of the effects of depletion, indicating that IL-9 acts via CD4 T cells. Therefore, IL-9 is a previously unknown but key modulator of antiviral immunity, regulating T and B cell responses and having potent and specific effects on viral lung disease.

Limited Role of CD4+Foxp3+ Regulatory T Cells in the Control of Experimental Cerebral Malaria [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Steeg, C., Adler, G., Sparwasser, T., Fleischer, B., Jacobs, T. — Wed, 18 Nov 2009 13:06:01 PST

Cerebral malaria (CM) associated with Plasmodium berghei ANKA (PbA) infection is an accepted model of human CM. CM during PbA infection critically depends on sequestration of T cells into the brain. Several studies aimed to address the role of regulatory T cells (T_reg) in modulating this pathogenic T cell response. However, these studies are principally hampered due to the fact that until recently no reagents were available to deplete Foxp3⁺ T_reg specifically. To study the function of T_reg in the genesis of CM, we used depletion of T_reg mice that are transgenic for a bacterial artificial chromosome expressing a diphtheria toxin receptor-enhanced GFP fusion protein under the control of the foxp3 gene locus. These mice allow for a selective depletion of Foxp3⁺ T_reg by diphtheria toxin injection, and also their specific detection and purification during an ongoing infection. Using depletion of T_reg mice, we found only a small increase in the absolute numbers of Foxp3⁺ T_reg during PbA infection and, consequently, the ratio of T_reg to T effector cells (T_eff) decreased due to the rapid expansion of T_eff. Although the latter sequester in the brains of infected mice, almost no T_reg were found in the brains of infected mice. Furthermore, we demonstrate that depletion of T_reg has no influence on sequestration of T_eff and on the clinical outcome, and only minor influence on T cell activation. Using ex vivo analysis of purified T_reg from either naive mice or PbA-infected mice, we found that both exhibit similar inhibitory capacity on T_eff.

Systemic Increase in the Ratio between Foxp3+ and IL-17-Producing CD4+ T Cells in Healthy Pregnancy but Not in Preeclampsia [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

Preeclampsia is the leading cause of morbidity and mortality in pregnancy. Although the etiology of preeclampsia is still unclear, it is believed to involve rejection of the fetus, possibly due to an imbalance between regulatory (Treg) and effector T cells. To test this, we compared the frequencies of circulating CD4⁺ T cells expressing Foxp3, IFN-, IL-10, or IL-17 at the end of the third trimester of healthy and preeclamptic pregnancies. The size of the Treg cell compartment, defined by the frequency of CD4⁺CD25^high, CD4⁺CD127^lowCD25⁺, and CD4⁺Foxp3⁺ cells was significantly higher in normal compared with preeclamptic pregnancies. CD4⁺CD25^high and CD4⁺CD127^lowCD25⁺ populations in preeclampsia were not significantly different from those in nonpregnant controls, whereas CD4⁺Foxp3⁺ cells numbersre slightly lower in preeclampsia. The suppressive activity of ex vivo-sorted CD4⁺CD127^lowCD25⁺ Treg cells was not significantly different between the three study groups. The percentage of CD4⁺IL-17-producing T cells decreased significantly in healthy compared with preeclamptic pregnancies and nonpregnant controls, whereas CD4⁺IL-10- and CD4⁺IFN--producing cells remained unchanged. Consequently, the ratio of Foxp3⁺ Treg to IL-17-expressing CD4⁺ T cells was significantly increased in healthy but not in preeclamptic pregnancies. Thus, preeclampsia is associated with the absence of normal systemic skewing away from IL-17 production toward Foxp3⁺ expression. Finally, preeclamptic women had significantly higher levels of soluble endoglin, an inhibitor of TGF-β receptor signaling, which may bias toward IL-17 production. These results suggest that homeostasis between regulatory and proinflammatory CD4⁺ T cells might be pivotal for the semiallogeneic fetus to be tolerated within the maternal environment.

Female and Male Sex Hormones Differentially Regulate Expression of Ifi202, an Interferon-Inducible Lupus Susceptibility Gene within the Nba2 Interval [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Panchanathan, R., Shen, H., Bupp, M. G., Gould, K. A., Choubey, D. — Wed, 18 Nov 2009 13:06:01 PST

Increased expression of IFN-inducible Ifi202 gene in certain strains of female mice is associated with susceptibility to systemic lupus erythematosus (SLE). Although, the development of SLE is known to have a strong sex bias, the molecular mechanisms remain unknown. Here we report that in vivo treatment of orchiectomized (NZB x NZW)F₁ male mice with the female sex hormone 17β-estradiol significantly increased steady-state levels of Ifi202 mRNA in splenic cells, whereas treatment with the male hormone dihydrotestosterone decreased the levels. Moreover, increased expression of Ifi202 in B6.Nba2 B cells and reduced expression in T cells were associated with increased levels of estrogen receptor- (ER) and androgen receptor, respectively. Furthermore, the steady-state levels of Ifi202 mRNA were higher in splenic cells from C57BL/6, B6.Nba2, NZB, and (NZB x NZW)F₁ female mice as compared with males. 17β-estradiol treatment of B cells and WT276 cells increased Ifi202 mRNA levels, whereas treatment with dihydrotestosterone decreased the levels. Interestingly, overexpression of ER in WT276 cells increased the expression of Ifi202 and stimulated the activity of the 202-luc-reporter through the c-Jun/AP-1 DNA-binding site. Accordingly, ER preferentially associated with the regulatory region of the Ifi202 gene in female B6.Nba2 B cells than in males. Furthermore, Ifi202 mRNA levels were detectable in splenic cells of wild-type (Esr1^+/+), but not null (Esr1^–/–), (NZB x NZW)F₁ female mice. Collectively, our observations demonstrate that the female and male sex hormones differentially regulate the expression of Ifi202, thus providing support for the role of Ifi202 in sex bias in SLE.

Absence of the Transcriptional Repressor Blimp-1 in Hematopoietic Lineages Reveals Its Role in Dendritic Cell Homeostatic Development and Function [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

Dendritic cells (DCs) are important for the initiation and regulation of immune responses. In this study, we demonstrate that DC homeostatic development in peripheral lymphoid organs is negatively regulated by the transcriptional repressor, Blimp-1, which is critical for regulation of plasma cell differentiation and T cell homeostasis and function. Deletion of Prdm1, the gene encoding Blimp-1, in mouse hematopoietic lineages resulted in an increase in the steady-state number of conventional DCs (cDCs). Specifically, Prdm1 deletion increased immediate CD8^– cDC precursors in peripheral lymphoid organs, causing selective expansion of the CD8^– cDC population. Upon stimulus-induced maturation, Blimp-1 was up-regulated in bone marrow-derived DCs via the p38 MAPK and NF-B pathways. Notably, Blimp-1-deficient DCs matured poorly upon stimulation in vitro and in vivo. Blimp-1 binds to the proinflammatory cytokine/chemokine genes, Il-6 and Ccl2, and negatively regulates their expression. Collectively, our findings reveal two new roles for Blimp-1: negative regulation of a select subset of cDCs during homeostatic development, and enhancement of DC maturation.

SOCS3 in T and NKT Cells Negatively Regulates Cytokine Production and Ameliorates ConA-Induced Hepatitis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

Suppressor of cytokine signaling 3 (SOCS3), a negative-feedback molecule for cytokine signaling, has been implicated in protection against liver injury. Previous studies have shown that overexpression of SOCS3 in the liver by adenovirus or membrane permeable recombinant protein protected the liver from various injuries. However it remained uncertain in which type of cells SOCS3 suppresses liver injury. In this study, we demonstrated that forced expression of SOCS3 in T and NKT cells suppressed ConA-induced hepatitis using T and NKT cell-specific SOCS3 transgenic (Lck-SOCS3 Tg) mice. IFN- and IL-4 production was reduced in Lck-SOCS3 Tg mice as well as splenocytes treated with ConA. IFN- and IL-4 levels were also reduced in Lck-SOCS3 Tg mice administrated with -galactosylceramide, suggesting that SOCS3 in NKT cells has suppressive function. Sustained expression of SOCS3 in an NKT cell line also resulted in reduced expression of various cytokines and transcription factors. In contrast, T and NKT cell-specific SOCS3 conditional knockout (Lck-SOCS3 cKO) mice were hypersensitive to ConA-mediated hepatitis. Isolated SOCS3-deficient NKT cells produced higher levels of IFN- and IL-4. These data indicate that SOCS3 plays a negative regulatory role in NKT cell activation and that forced expression of SOCS3 in NKT cells is effective in preventing hepatitis.

Dendritic Cells Secrete the Immunosuppressive HLA-G Molecule upon CTLA4-Ig Treatment: Implication in Human Renal Transplant Acceptance [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

CTLA4-Ig (Belatacept) is a new recombinant molecule that interferes with the signal of T lymphocyte activation and prevents acute rejection after renal transplantation. HLA-G acts as a naturally tolerogenic molecule in humans. In this study, we analyzed whether HLA-G contributes to CTLA4-Ig-mediated graft acceptance. Our results demonstrate that patients treated with CTLA4-Ig displayed significantly higher soluble HLA-G (sHLA-G) plasma concentrations (72 ± 14 ng/ml) than patients treated with calcineurin inhibitors (5 ± 1 ng/ml) or healthy donors (5 ± 5 ng/ml). Notably, sHLA-G purified from plasma of CTLA4-Ig-treated patients was biologically active as it inhibited allogeneic T cell proliferation in vitro. Dendritic cells (DC) were identified as one of the cellular sources of sHLA-G in CTLA4-Ig-treated patients. Supporting this observation, we showed that DC generated in vitro in presence of CTLA4-Ig released sHLA-G in response to allostimulation. These CTLA4-Ig-treated DC acted as tolerogenic APC through sHLA-G secretion as they suppressed T cell alloproliferation, which could be restored by using a neutralizing anti-HLA-G Ab. These data define a novel pathway by which CTLA4-Ig immunomodulates allogenic response through posttranscriptional regulation of HLA-G expression in DC. CTLA4-Ig-mediated HLA-G release appears as a critical factor in T cell alloresponse inhibition, thereby contributing to the immunosuppressive effect and graft acceptance.

Interplay between Chromatin Remodeling and Epigenetic Changes during Lineage-Specific Commitment to Granzyme B Expression [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

The role of chromatin remodeling and histone posttranslational modifications and how they are integrated to control gene expression during the acquisition of cell-specific functions is poorly understood. We show here that following in vitro activation of CD4⁺ and CD8⁺ T lymphocytes, both cell types show rapid histone H3 loss at the granzyme B (gzmB) proximal promoter region. However, despite the gzmB proximal promoter being remodeled in both T cell subsets, only CD8⁺ T cells express high levels of gzmB and display a distinct pattern of key epigenetic marks, notably differential H3 acetylation and methylation. These data suggest that for high levels of transcription to occur a distinct set of histone modifications needs to be established in addition to histone loss at the proximal promoter of gzmB.

Mast Cell Differentiation and Activation Is Closely Linked to Expression of Genes Coding for the Serglycin Proteoglycan Core Protein and a Distinct Set of Chondroitin Sulfate and Heparin Sulfotransferases [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Duelli, A., Ronnberg, E., Waern, I., Ringvall, M., Kolset, S. O., Pejler, G. — Wed, 18 Nov 2009 13:06:01 PST

Serglycin (SG) proteoglycan consists of a small core protein to which glycosaminoglycans of chondroitin sulfate or heparin type are attached. SG is crucial for maintaining mast cell (MC) granule homeostasis through promoting the storage of various basic granule constituents, where the degree of chondroitin sulfate/heparin sulfation is essential for optimal SG functionality. However, the regulation of the SG core protein expression and of the various chondroitin sulfate/heparin sulfotransferases during MC differentiation and activation are poorly understood. Here we addressed these issues and show that expression of the SG core protein, chondroitin 4-sulfotransferase (C4ST)-1, and GalNAc(4S)-6-O-sulfotransferase (GalNAc4S6ST) are closely linked to MC maturation. In contrast, the expression of chondroitin 6-sulfotransferase correlated negatively with MC maturation. The expression of N-deacetylase/N-sulfotransferase (NDST)-2, a key enzyme in heparin synthesis, also correlated strongly with MC maturation, whereas the expression of the NDST-1 isoform was approximately equal at all stages of maturation. MC activation by either calcium ionophore or IgE ligation caused an up-regulated expression of the SG core protein, C4ST-1, and GalNAc4S6ST, accompanied by increased secretion of chondroitin sulfate as shown by biosynthetic labeling experiments. In contrast, NDST-2 was down-regulated after MC activation, suggesting that MC activation modulates the nature of the glycosaminoglycan chains attached to the SG core protein. Taken together, these data show that MC maturation is associated with the expression of a distinct signature of genes involved in SG proteoglycan synthesis, and that MC activation modulates their expression.

Sex Steroid Ablation Enhances Hematopoietic Recovery following Cytotoxic Antineoplastic Therapy in Aged Mice [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

Cytotoxic antineoplastic therapy is widely used in the clinic as a treatment for malignant diseases. The treatment itself, however, leads to long-term depletion of the adaptive immune system, which is more pronounced in older patients, predominantly due to thymic atrophy. We and others have previously shown that withdrawal of sex steroids is able to regenerate the aged thymus and enhance recovery from autologous and allogeneic hematopoietic stem cell transplant. In this study we have examined the effects of sex steroid ablation (SSA) on the recovery of lymphopoiesis in the bone marrow (BM) and thymus following treatment with the chemotherapeutic agent cyclophosphamide (Cy) in middle-aged and old mice. Furthermore, we have also examined the impact of this regeneration on peripheral immunity. SSA enhanced the recovery of BM resident hematopoietic stem cells and lymphoid progenitors and promoted lymphopoiesis. Interestingly, Cy alone caused a profound increase in the recently described common lymphoid progenitor 2 (CLP-2) population in the BM. In the thymus, SSA caused a profound increase in cellularity as well as all intrathymic T-lineage progenitors including early T-lineage progenitors (ETPs) and non-canonical T cell progenitors such as the CLP-2. We also found that these transferred into numerical increases in the periphery with enhanced B and T cell numbers. Furthermore, these lymphocytes were found to have an enhanced functional capacity with no perturbation of the TCR repertoire. Taken together, these results provide the basis for the use of SSA in the clinic to enhance treatment outcomes from cytotoxic antineoplastic therapy.

Dendritic Cells Matured by Inflammation Induce CD86-Dependent Priming of Naive CD8+ T Cells in the Absence of Their Cognate Peptide Antigen [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Maroof, A., Beattie, L., Kirby, A., Coles, M., Kaye, P. M. — Wed, 18 Nov 2009 13:06:01 PST

Dendritic cells (DC) licensed by the interaction between pathogen products and pattern recognition receptors can activate naive T cells to undergo Ag-dependent proliferation and cytokine production. In contrast, DC induced to mature by trans-acting inflammatory stimuli are believed to only be capable of supporting Ag-dependent proliferative responses. In this study, we show that uninfected DC matured as a consequence of Leishmania-induced inflammation induce CD8⁺ T cells to proliferate in the absence of their cognate Ag. We separated splenic DC from Leishmania donovani-infected mice into those that contained parasites and had been activated to induce IL-12p40, from those that had undergone only partial maturation, measured by increased CD86 expression in the absence of IL-12p40 induction. We then showed that these partially matured DC could induce exogenous peptide-independent proliferation of OT-I and F5 CD8⁺ TCR transgenic T cells, as well as polyclonal CD8⁺ T cells. Proliferation of OT-I cells was significantly inhibited in vitro and in vivo by anti-CD86 mAb but not by anti-CD80 mAb and could also be inhibited by cyclosporine A. Proliferating OT-I cells did not produce IFN-, even when re-exposed to mature DC. However, these primed OT-I cells subsequently produced effector cytokines, not just on exposure to their cognate peptide but, more importantly, to weak exogenous TCR agonists that otherwise failed to induce IFN-. We further showed that OT-I cells undergoing locally driven proliferation to another pathogen, Streptococcus pneumoniae, rapidly seeded other lymphoid tissues, suggesting that CD8⁺ T cells primed in this way may play a role in rapidly countering pathogen dissemination.

Apoptotic Dendritic Cells Induce Tolerance in Mice through Suppression of Dendritic Cell Maturation and Induction of Antigen-Specific Regulatory T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kushwah, R., Oliver, J. R., Zhang, J., Siminovitch, K. A., Hu, J. — Wed, 18 Nov 2009 13:06:01 PST

Dendritic cell (DC) apoptosis has been shown to play a role in maintaining a balance between tolerance and immunity. However, the mechanisms of how DC apoptosis affects the immune response are unclear. We have shown that in vitro culture of apoptotic DCs with immature DCs, results in their uptake by immature DCs, which subsequently turn into tolerogenic DCs, which then secrete TGF-β1 and induce Foxp3⁺ regulatory T cells (T_regs). In this study we looked at the effects of apoptotic DCs in vivo. Here we show that apoptotic DCs are taken up by viable DCs in vivo, which suppresses the ability of viable DCs to undergo maturation and subsequent migration to the lymph nodes in response to LPS. Additionally, delivery of apoptotic DCs to LPS inflamed lungs results in resolution of inflammation, which is mediated by the ability of apoptotic DCs to suppress response of viable DCs to LPS. Additionally, apoptotic DCs also induce TGF-β1 secretion in the mediastinal lymph nodes, which results in expansion of Foxp3⁺ T_regs. Most importantly, we show that delivery of apoptotic DCs followed by OVA in CFA to mice suppresses T cell response to OVA and instead induces de novo generation of OVA-specific T_regs. Furthermore, delivery of apoptotic DCs followed by OVA in CFA results in expansion of T_regs in TCR transgenic (OT-II) mice. These findings demonstrate that apoptotic DCs are taken up by viable DCs in vivo, which promotes tolerance through suppression of DC maturation and induction of T_regs.

Deciphering the Immune Function and Regulation by a TLR of the Cytokine EMAPII in the Lesioned Central Nervous System Using a Leech Model [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

A highly conserved ortholog of the human complex p43/endothelial monocyte-activating polypeptide II (EMAPII) was characterized in the CNS of the leech Hirudo medicinalis. As observed in mammals, the leech complex is processed to release the cytokine HmEMAPII. Taking advantages of these similarities, we have attempted to elucidate the role of EMAPII in the CNS using the leech model. Although EMAPII is considered a modulator of inflammatory reactions within the peripheral innate immune response in humans, its function in CNS immunity has yet to be described. Chemotaxis assays were conducted, revealing the ability of EMAPII to exert a chemoattractant effect on both leech and human microglial cells, indicating a novel function of this cytokine in the human brain. Quantitative RT-PCR analysis together with in situ hybridization and immunohistochemistry approaches showed that bacterial challenge induced the expression of HmEMAPII at the lesion site where microglial cells accumulated. Moreover, gene silencing experiments have demonstrated that the gene expression of HmEMAPII is under the control of a signaling pathway associated with the TLR HmTLR1, newly characterized in the CNS of our model. To the best of our knowledge, this is the first report showing evidence for (1) the chemoattractant properties of EMAPII on leech and human microglial cells, (2) the regulation by a TLR of the expression of a gene encoding a cytokine in the CNS of an invertebrate, and (3) an immune function of a TLR in a lophotrochozoan model.

Dominant Expression of the Inhibitory Fc{gamma}RIIB Prevents Antigen Presentation by Murine Plasmacytoid Dendritic Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:01 PST

Plasmacytoid dendritic cells (pDCs) are key regulators of the innate immune response, yet their direct role as APCs in the adaptive immune response is unclear. We found that unlike conventional DCs, immune complex (IC) exposed murine pDCs neither up-regulated costimulatory molecules nor activated Ag-specific CD4⁺ and CD8⁺ T cells. The inability of murine pDCs to promote T cell activation was due to inefficient proteolytic processing of internalized ICs. This defect in the IC processing capacity of pDCs results from a lack of activating FcR expression (FcRI, III, IV) and the dominant expression of the inhibitory receptor FcRIIB. Consistent with this idea, transgenic expression of the activating human FcRIIA gene, not present in the mouse genome, recapitulated the human situation and rescued IC antigenic presentation capacity by murine pDCs. The selective expression of FcRIIB by murine pDCs was not strain dependent and was maintained even following stimulation with TLR ligands and inflammatory cytokines. The unexpected difference between the mouse and human in the expression of activating/inhibitory FcRs has implications for the role of pDCs in Ab-modulated autoimmunity and anti-viral immunity.

Plasmacytoid Dendritic Cells Regulate Autoreactive B Cell Activation via Soluble Factors and in a Cell-to-Cell Contact Manner [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Ding, C., Cai, Y., Marroquin, J., Ildstad, S. T., Yan, J. — Wed, 18 Nov 2009 13:06:01 PST

Plasmacytoid dendritic cells (pDCs) are specialized type I IFN producers, which play an important role in pathogenesis of autoimmune disorders. Dysregulated autoreactive B cell activation is a hallmark in most autoimmune diseases. This study was undertaken to investigate interactions between pDCs and autoreactive B cells. After coculture of autoreactive B cells that recognize self-Ag small nuclear ribonucleoprotein particles with activated pDCs, we found that pDCs significantly enhance autoreactive B cell proliferation, autoantibody production, and survival in response to TLR and BCR stimulation. Neutralization of IFN-/β and IL-6 abrogated partially pDC-mediated enhancement of autoreactive B cell activation. Transwell studies demonstrated that pDCs could provide activation signals to autoreactive B cells via a cell-to-cell contact manner. The involvement of the ICAM-1-LFA-1 pathway was revealed as contributing to this effect. This in vitro enhancement effect was further demonstrated by an in vivo B cell adoptive transfer experiment, which showed that autoreactive B cell proliferation and activation were significantly decreased in MyD88-deficient mice compared with wild-type mice. These data suggest the dynamic interplay between pDCs and B cells is required for full activation of autoreactive B cells upon TLR or BCR stimulation.

Neonatal Innate TLR-Mediated Responses Are Distinct from Those of Adults [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

The human neonate and infant are unduly susceptible to infection with a wide variety of microbes. This susceptibility is thought to reflect differences from adults in innate and adaptive immunity, but the nature of these differences is incompletely characterized. The innate immune response directs the subsequent adaptive immune response after integrating information from TLRs and other environmental sensors. We set out to provide a comprehensive analysis defining differences in response to TLR ligation between human neonates and adults. In response to most TLR ligands, neonatal innate immune cells, including monocytes and conventional and plasmacytoid dendritic cells produced less IL-12p70 and IFN- (and consequently induced less IFN-), moderately less TNF-, but as much or even more IL-1β, IL-6, IL-23, and IL-10 than adult cells. At the single-cell level, neonatal innate cells generally were less capable of producing multiple cytokines simultaneously, i.e., were less polyfunctional. Overall, our data suggest a robust if not enhanced capacity of the neonate vs the adult white-blood cell TLR-mediated response to support Th17- and Th2-type immunity, which promotes defense against extracellular pathogens, but a reduced capacity to support Th1-type responses, which promote defense against intracellular pathogens.

Tc17 CD8 T Cells: Functional Plasticity and Subset Diversity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

IL-17-secreting CD8 T cells (Tc17) have been described in several settings, but little is known regarding their functional characteristics. While Tc1 cells produced IFN- and efficiently killed targets, Tc17 cells lacked lytic function in vitro. Interestingly, the small numbers of IFN--positive or IL-17/IFN--double-positive cells generated under Tc17 conditions also lacked lytic activity and expressed a similar pattern of cell surface proteins to IL-17-producing cells. As is the case for Th17 (CD4) cells, STAT3 is important for Tc17 polarization, both in vitro and in vivo. Adoptive transfer of highly purified, Ag-specific IL-17-secreting Tc17 cells into Ag-bearing hosts resulted in near complete conversion to an IFN--secreting phenotype and substantial pulmonary pathology, demonstrating functional plasticity. Tc17 also accumulated to a greater extent than did Tc1 cells, suggesting that adoptive transfer of CD8 T cells cultured in Tc17 conditions may have therapeutic potential for diseases in which IFN--producing cells are desired.

Th1, Th17, and Th9 Effector Cells Induce Experimental Autoimmune Encephalomyelitis with Different Pathological Phenotypes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Jager, A., Dardalhon, V., Sobel, R. A., Bettelli, E., Kuchroo, V. K. — Wed, 18 Nov 2009 13:06:02 PST

Experimental autoimmune encephalomyelitis (EAE) is a model of human multiple sclerosis induced by autoreactive Th cells that mediate tissue inflammation and demyelination in the CNS. Initially, IFN--producing Th1 cells and, more recently, IL-17-producing Th17 cells with specificity for myelin Ags have been implicated in EAE induction, but whether Th17 cells are encephalitogenic has been controversial. Moreover, a new effector T cell subset, Th9 cells, has been identified; however, the ability of this T cell subset to induce EAE has not been investigated. Here, we have developed protocols to generate myelin oligodendrocyte glycoprotein-specific Th17, Th1, Th2, and Th9 cells in vitro, so that we could directly compare and characterize the encephalitogenic activity of each of these subsets upon adoptive transfer. We show that myelin oligodendrocyte glycoprotein-specific Th1, Th17, and Th9 cells but not Th2 cells induce EAE upon adoptive transfer. Importantly, each T cell subset induced disease with a different pathological phenotype. These data demonstrate that different effector T cell subsets with specificity for myelin Ags can induce CNS autoimmunity and that the pathological heterogeneity in multiple sclerosis lesions might in part be due to multiple distinct myelin-reactive effector T cells.

Recruitment of Sprouty1 to Immune Synapse Regulates T Cell Receptor Signaling [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

TCR stimulation not only initiates positive signals for T cell activation, but also induces negative signals that down-regulate T cells. We previously reported that Sprouty1, a negative regulator of Ras-MAPK pathway of receptor tyrosine kinases, was induced by TCR signal and inhibited TCR signaling in CD4⁺ T cell clones. In this study, we addressed the mechanism underlying Sprouty1 inhibition of T cells. When overexpressed in Jurkat T cells, Sprouty1 inhibited TCR signal-induced IL-2 transcription, and also AP-1, NFAT, and NF-B activation, which suggests that Sprouty1 acts at proximal TCR signalosome. Accordingly, we found that Sprouty1 translocated to immune synapse upon TCR engagement in both Jurkat cells and activated primary T cells and interacted with various signaling molecules in the TCR signalosome, such as linker for activation of T cells (LAT), phospholipase C-1 (PLC-1), c-Cbl/Cbl-b, and HPK1. Sprouty1 inhibited LAT phosphorylation, leading to decreased MAPK activation and IL-2 production. Deletion of C-terminal 54 amino acids in Sprouty1 abolished its inhibitory effect and this deletion mutant was unable to translocate to immune synapse and interact with LAT. Overall, our data suggest that Sprouty1 induced by TCR signal negatively regulates further TCR signaling by interacting with proximal signaling molecules in immune synapse, providing a novel regulatory mechanism of T cells.

Processing in the Endoplasmic Reticulum Generates an Epitope on the Insulin A Chain that Stimulates Diabetogenic CD8 T Cell Responses [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

RIP-B7.1 mice express the costimulator molecule B7.1 (CD80) on pancreatic β cells and are a well-established model for studying de novo induction of diabetogenic CD8 T cells. Immunization of RIP-B7.1 mice with preproinsulin (ppins)-encoding plasmid DNA efficiently induces experimental autoimmune diabetes (EAD). EAD is associated with an influx of CD8 T cells specific for the K^b/A_12–21 epitope into the pancreatic islets and the subsequent destruction of β cells. In this study, we used this model to investigate how ppins-derived Ags are expressed and processed to prime diabetogenic, K^b/A_12–21-specific CD8 T cells. Targeting the K^b/A_12–21 epitope, the insulin A chain, or the ppins to the endoplasmic reticulum (ER) (but not to the cytosol and/or nucleus) efficiently elicited K^b/A_12–21-specific CD8 T cell responses. The K^b/A_12–21 epitope represents the COOH terminus of the ppins molecule and, hence, did not require COOH-terminal processing before binding its restriction element in the ER. However, K^b/A_12–21-specific CD8 T cells were also induced by COOH-terminally extended ppins-specific polypeptides expressed in the ER, indicating that the epitope position at the COOH terminus is less important for its diabetogenicity than is targeting the Ag to the ER. The K^b/A_12–21 epitope had a low avidity for K^b molecules. When epitopes of unrelated Ags were coprimed at the same site of Ag delivery, "strong" K^b-restricted (but not D^b-restricted) CD8 T cell responses led to the suppression of K^b/A_12–21-specific CD8 T cell priming and reduced EAD. Thus, direct expression and processing of the "weak" K^b/A_12–21 epitope in the ER favor priming of autoreactive CD8 T cells.

Ineffective Vaccination against Solid Tumors Can Be Enhanced by Hematopoietic Cell Transplantation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

Vaccination with tumor Ags has not been an effective treatment for solid tumors. The goal of the current study was to determine whether a combination of vaccination and hematopoietic cell transplantation (HCT) can effectively treat primary, disseminated, or metastatic CT26 and MC38 murine colon tumors. Vaccination of tumor-bearing mice with irradiated tumor cells and CpG adjuvant failed to alter progressive tumor growth. However, mice bearing primary, disseminated lung, or metastatic liver tumors were uniformly cured after administration of total body irradiation, followed by the transplantation of hematopoietic progenitor cells and T cells from syngeneic, but not allogeneic vaccinated donors. Requirements for effective treatment of tumors included irradiation of hosts, vaccination of donors with both tumor cells and CpG, transfer of both CD4⁺ and CD8⁺ T cells along with progenitor cells, and ability of donor cells to produce IFN-. Irradiation markedly increased the infiltration of donor T cells into the tumors, and the combined irradiation and HCT altered the balance of tumor-infiltrating cells to favor CD8⁺ effector memory T cells as compared with CD4⁺CD25⁺FoxP3⁺ T regulatory cells. The combination of vaccination and autologous hematopoietic cell transplantation was also effective in treating tumors. In conclusion, these findings show that otherwise ineffective vaccination to solid nonhematologic tumors can be dramatically enhanced by HCT.

P-Selectin Glycoprotein Ligand-1 Negatively Regulates T-Cell Immune Responses [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Matsumoto, M., Miyasaka, M., Hirata, T. — Wed, 18 Nov 2009 13:06:02 PST

Cell surface sialomucins often act as antiadhesive molecules by virtue of their extended structure and negative charge. CD43 is one such sialomucin, expressed on most leukocytes. P-selectin glycoprotein ligand-1 (PSGL-1) is another sialomucin expressed by leukocytes. It serves as a major selectin ligand, but no antiadhesive role for it has been described. In this study, we showed that PSGL-1-deficient T cells, like CD43-deficient T cells, exhibited increased adhesion and proliferation compared with wild-type cells. The loss of both PSGL-1 and CD43 led to a further increase in T cell adhesion and proliferation. The reexpression of full-length PSGL-1 or CD43 in double-deficient CD4⁺ T cells reversed their increased adhesion and proliferation phenotype. Using chimeric constructs of human CD8 and either PSGL-1 or CD43, we demonstrated that the intracellular domain of PSGL-1 or CD43 is required for suppressing proliferation but not adhesion. Furthermore, in a mouse model of inflammatory bowel disease induced by the adoptive transfer of naive T cells into RAG-deficient hosts, a PSGL-1 deficiency exacerbated the development of inflammation. These results reveal a novel regulatory role for PSGL-1 in T cell adhesion and proliferation and suggest that PSGL-1 negatively regulates T cell immune responses in vivo.

Dynamic Regulation of Notch 1 and Notch 2 Surface Expression during T Cell Development and Activation Revealed by Novel Monoclonal Antibodies [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4^–CD8^– thymocytes. Notch1 is progressively down-regulated at the CD4⁺CD8⁺ and mature CD4⁺ or CD8⁺ thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1⁺CD25^– cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117⁺ early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

Inhibition of RANK Expression and Osteoclastogenesis by TLRs and IFN-{gamma} in Human Osteoclast Precursors [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

TLRs have been implicated in promoting osteoclast-mediated bone resorption associated with inflammatory conditions. TLRs also activate homeostatic mechanisms that suppress osteoclastogenesis and can limit the extent of pathologic bone erosion associated with infection and inflammation. We investigated mechanisms by which TLRs suppress osteoclastogenesis. In human cell culture models, TLR ligands suppressed osteoclastogenesis by inhibiting expression of receptor activator of NF-B (RANK), thereby making precursor cells refractory to the effects of RANKL. Similar but less robust inhibition of RANK expression was observed in murine cells. LPS suppressed generation of osteoclast precursors in mice in vivo, and adsorption of LPS onto bone surfaces resulted in diminished bone resorption. Mechanisms that inhibited RANK expression were down-regulation of RANK transcription, and inhibition of M-CSF signaling that is required for RANK expression. TLRs inhibited M-CSF signaling by rapidly down-regulating cell surface expression of the M-CSF receptor c-Fms by a matrix metalloprotease- and MAPK-dependent mechanism. Additionally, TLRs cooperated with IFN- to inhibit expression of RANK and of the CSF1R gene that encodes c-Fms, and to synergistically inhibit osteoclastogenesis. Our findings identify a new mechanism of homeostatic regulation of osteoclastogenesis that targets RANK expression and limits bone resorption during infection and inflammation.

SHP-2 Expression Negatively Regulates NK Cell Function [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Purdy, A. K., Campbell, K. S. — Wed, 18 Nov 2009 13:06:02 PST

Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) is required for full activation of Ras/ERK in many cytokine and growth factor receptor signaling pathways. In contrast, SHP-2 inhibits activation of human NK cells upon recruitment to killer cell Ig-like receptors (KIR). To determine how SHP-2 impacts NK cell activation in KIR-dependent or KIR-independent signaling pathways, we employed knockdown and overexpression strategies in NK-like cell lines and analyzed the consequences on functional responses. In response to stimulation with susceptible target cells, SHP-2-silenced NK cells had elevated cytolytic activity and IFN- production, whereas cells overexpressing wild-type or gain-of-function mutants of SHP-2 exhibited dampened activities. Increased levels of SHP-2 expression over this range significantly suppressed microtubule organizing center polarization and granzyme B release in response to target cells. Interestingly, NK-target cell conjugation was only reduced by overexpressing SHP-2, but not potentiated in SHP-2-silenced cells, indicating that conjugation is not influenced by physiological levels of SHP-2 expression. KIR-dependent inhibition of cytotoxicity was unaffected by significant reductions in SHP-2 levels, presumably because KIR were still capable of recruiting the phosphatase under these limiting conditions. In contrast, the general suppressive effect of SHP-2 on cytotoxicity and cytokine release was much more sensitive to changes in cellular SHP-2 levels. In summary, our studies have identified a new, KIR-independent role for SHP-2 in dampening NK cell activation in response to tumor target cells in a concentration-dependent manner. This suppression of activation impacts microtubule organizing center-based cytoskeletal rearrangement and granule release.

MHC Drives TCR Repertoire Shaping, but not Maturation, in Recent Thymic Emigrants [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Houston, E. G., Fink, P. J. — Wed, 18 Nov 2009 13:06:02 PST

After developing in the thymus, recent thymic emigrants (RTEs) enter the lymphoid periphery and undergo a maturation process as they transition into the mature naive (MN) T cell compartment. This maturation presumably shapes RTEs into a pool of T cells best fit to function robustly in the periphery without causing autoimmunity; however, the mechanism and consequences of this maturation process remain unknown. Using a transgenic mouse system that specifically labels RTEs, we tested the influence of MHC molecules, key drivers of intrathymic T cell selection and naive peripheral T cell homeostasis, in shaping the RTE pool in the lymphoid periphery. We found that the TCRs expressed by RTEs are skewed to longer CDR3 regions compared with those of MN T cells, suggesting that MHC does streamline the TCR repertoire of T cells as they transition from the RTE to the MN T cell stage. This conclusion is borne out in studies in which the representation of individual TCRs was followed as a function of time since thymic egress. Surprisingly, we found that MHC is dispensable for the phenotypic and functional maturation of RTEs.

Ectopic T-bet Expression Licenses Dendritic Cells for IL-12-Independent Priming of Type 1 T Cells In Vitro [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

T-bet (TBX21) is a transcription factor required for the optimal development of type 1 immune responses. Although initially characterized for its intrinsic role in T cell functional polarization, endogenous T-bet may also be critical to the licensing of type 1-biasing APCs. Here, we investigated whether human dendritic cells (DC) genetically engineered to express high levels of T-bet (i.e., DC.Tbet) promote superior type 1 T cell responses in vitro. We observed that DC.Tbet were selective activators of type 1 effector T cells developed from the naive pool of responder cells, whereas DC.Tbet and control DC promoted type 1 responses equitably from the memory pool of responder cells. Naive T cells primed by (staphylococcal enterotoxin B or tumor-associated protein-loaded) DC.Tbet exhibited an enhancement in type 1- and a concomitant reduction in Th2- and regulatory T cell-associated phenotype/function. Surprisingly, DC.Tbets were impaired in their production of IL-12 family member cytokines (IL-12p70, IL-23, and IL-27) when compared with control DC, and the capacity of DC.Tbet to preferentially prime type 1 T cell responses was only minimally inhibited by cytokine (IL-12p70, IL-23, IFN-) neutralization or receptor (IL-12Rβ2, IL-27R) blockade during T cell priming. The results of transwell assays suggested the DC.Tbet-mediated effects are predominantly the result of direct DC-T cell contact or their close proximity, thereby implicating a novel, IL-12-independent mechanism by which DC.Tbets promote improved type 1 functional polarization from naive T cell responders. Given their superior type 1 polarizing capacity, DC.Tbet may be suitable for use in vaccines designed to prevent/treat cancer or infectious disease.

SLAT/Def6 Plays a Critical Role in the Development of Th17 Cell-Mediated Experimental Autoimmune Encephalomyelitis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Canonigo-Balancio, A. J., Fos, C., Prod'homme, T., Becart, S., Altman, A. — Wed, 18 Nov 2009 13:06:02 PST

SWAP-70-like adapter of T cells (SLAT; also known as Def6) is a novel guanine nucleotide exchange factor for Rho GTPases that has been previously shown to play a role in CD4⁺ T cell activation and Th1/Th2 differentiation. However, the role of SLAT/Def6 in autoimmunity and its associated Th1- and Th17-specific responses has not yet been clearly elucidated. We used a prototypical and pathologically relevant Th1/Th17-mediated autoimmune model, that is, experimental autoimmune encephalomyelitis, to assess the role of SLAT/Def6 in autoantigen-specific T cell response. We found that T cell-expressed SLAT/Def6 was critical for experimental autoimmune encephalomyelitis development and pathogenesis, as evidenced by the resistance of Def6-deficient (Def6^–/–) mice to clinical signs of the disease associated with a lack of CNS inflammation and demyelination in myelin oligodendrocyte glycoprotein-immunized Def6^–/– mice. Moreover, Def6 deficiency resulted in a severely diminished myelin oligodendrocyte glycoprotein-specific CD4⁺ T cell proliferation as well as a defect in IFN- and IL-17 production in secondary lymphoid organs and the CNS. Lastly, Def6^–/– CD4⁺ T cells were grossly deficient in their ability to differentiate into Th17 cells both in vitro and in vivo in a T cell-intrinsic manner. Therefore, our study establishes T cell-expressed SLAT/Def6 as a pivotal positive regulator of Th17 inflammatory responses and, thus, essential in controlling autoimmune and inflammatory diseases.

A Nonadjuvanted Polypeptide Nanoparticle Vaccine Confers Long-Lasting Protection against Rodent Malaria [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)₂D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2^b, H-2^d, and H-2^k alleles. Immunized mice produced a CD4⁺ T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.

Autophagic Compartments Gain Access to the MHC Class II Compartments in Thymic Epithelium [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kasai, M., Tanida, I., Ueno, T., Kominami, E., Seki, S., Ikeda, T., Mizuochi, T. — Wed, 18 Nov 2009 13:06:02 PST

The presentation of self-peptides in the context of MHC molecules by thymic epithelial cells (TECs) is essential for T cell repertoire selection in the thymus. However, the underlying mechanisms of this process have not been fully elucidated. To address whether autophagy, a catabolic process involving the degradation of a cell’s components through the lysosomal machinery, intersects the MHC class II-restricted Ag presentation pathway in TECs, we investigated the colocalization of LC3, a peculiar autophagy marker molecule, with MHC class II compartments in in vitro-established TEC lines by immunofluorescence microscopy and Western blotting analyses. We found that in both cortical and medullary TEC lines, LC3 was colocalized with the H2-DM-positive lysosomal compartments, in which MHC class II plus class II-associated invariant chain peptides complexes are formed. Furthermore, our analysis of thymic cryosections from 1-day-old mice revealed that LC3 colocalizes with the H2-DM-positive compartments in TECs. These results strongly suggest that the cytoplasmic self-Ags gain access to the H2-DM-positive compartments via the autophagic process in the thymus.

T Cell Intrinsic Heterodimeric Complexes between HVEM and BTLA Determine Receptivity to the Surrounding Microenvironment [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

The inhibitory cosignaling pathway formed between the TNF receptor herpesvirus entry mediator (HVEM, TNFRSF14) and the Ig superfamily members, B and T lymphocyte attenuator (BTLA) and CD160, limits the activation of T cells. However, BTLA and CD160 can also serve as activating ligands for HVEM when presented in trans by adjacent cells, thus forming a bidirectional signaling pathway. BTLA and CD160 can directly activate the HVEM-dependent NF-B RelA transcriptional complex raising the question of how NF-B activation is repressed in naive T cells. In this study, we show BTLA interacts with HVEM in cis, forming a heterodimeric complex in naive T cells that inhibits HVEM-dependent NF-B activation. The cis-interaction between HVEM and BTLA is the predominant form expressed on the surface of naive human and mouse T cells. The BTLA ectodomain acts as a competitive inhibitor blocking BTLA and CD160 from binding in trans to HVEM and initiating NF-B activation. The TNF-related ligand, LIGHT (homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14) binds HVEM in the cis-complex, but NF-B activation was attenuated, suggesting BTLA prevents oligomerization of HVEM in the cis-complex. Genetic deletion of BTLA or pharmacologic disruption of the HVEM-BTLA cis-complex in T cells promoted HVEM activation in trans. Interestingly, herpes simplex virus envelope glycoprotein D formed a cis-complex with HVEM, yet surprisingly, promoted the activation NF-B RelA. We suggest that the HVEM-BTLA cis-complex competitively inhibits HVEM activation by ligands expressed in the surrounding microenvironment, thus helping maintain T cells in the naive state.

Role of T Cell TGF{beta} Signaling and IL-17 in Allograft Acceptance and Fibrosis Associated with Chronic Rejection [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:02 PST

Chronic allograft rejection (CR) is the main barrier to long-term transplant survival. CR is a progressive disease defined by interstitial fibrosis, vascular neointimal development, and graft dysfunction. The underlying mechanisms responsible for CR remain poorly defined. TGFβ has been implicated in promoting fibrotic diseases including CR, but is beneficial in the transplant setting due to its immunosuppressive activity. To assess the requirement for T cell TGFβ signaling in allograft acceptance and the progression of CR, we used mice with abrogated T cell TGFβ signaling as allograft recipients. We compared responses from recipients that were transiently depleted of CD4⁺ cells (that develop CR and express intragraft TGFβ) with responses from mice that received anti-CD40L mAb therapy (that do not develop CR and do not express intragraft TGFβ). Allograft acceptance and suppression of graft-reactive T and B cells were independent of T cell TGFβ signaling in mice treated with anti-CD40L mAb. In recipients transiently depleted of CD4⁺ T cells, T cell TGFβ signaling was required for the development of fibrosis associated with CR, long-term graft acceptance, and suppression of graft-reactive T and B cell responses. Furthermore, IL-17 was identified as a critical element in TGFβ-driven allograft fibrosis. Thus, IL-17 may provide a therapeutic target for preventing graft fibrosis, a measure of CR, while sparing the immunosuppressive activity of TGFβ.

TGF{beta} Neutralization within Cardiac Allografts by Decorin Gene Transfer Attenuates Chronic Rejection [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Faust, S. M., Lu, G., Wood, S. C., Bishop, D. K. — Wed, 18 Nov 2009 13:06:03 PST

Chronic allograft rejection (CR) is the leading cause of late graft failure following organ transplantation. CR is a progressive disease, characterized by deteriorating graft function, interstitial fibrosis, cardiac hypertrophy, and occlusive neointima development. TGFβ, known for its immunosuppressive qualities, plays a beneficial role in the transplant setting by maintaining alloreactive T cells in a hyporesponsive state, but has also been implicated in promoting graft fibrosis and CR. In the mouse vascularized cardiac allograft model, transient depletion of CD4⁺ cells promotes graft survival but leads to CR, which is associated with intragraft TGFβ expression. Decorin, an extracellular matrix protein, inhibits both TGFβ bioactivity and gene expression. In this study, gene transfer of decorin into cardiac allografts was used to assess the impact of intragraft TGFβ neutralization on CR, systemic donor-reactive T cell responses, and allograft acceptance. Decorin gene transfer and neutralization of TGFβ in cardiac allografts significantly attenuated interstitial fibrosis, cardiac hypertrophy, and improved graft function, but did not result in systemic donor-reactive T cell responses. Thus, donor-reactive T and B cells remained in a hyporesponsive state. These findings indicate that neutralizing intragraft TGFβ inhibits the cytokine’s fibrotic activities, but does not reverse its beneficial systemic immunosuppressive qualities.

Taking Advantage: High-Affinity B Cells in the Germinal Center Have Lower Death Rates, but Similar Rates of Division, Compared to Low-Affinity Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:03 PST

B lymphocytes producing high-affinity Abs are critical for protection from extracellular pathogens, such as bacteria and parasites. The process by which high-affinity B cells are selected during the immune response has never been elucidated. Although it has been shown that high-affinity cells directly outcompete low-affinity cells in the germinal center (GC), whether there are also intrinsic differences between these cells has not been addressed. It could be that higher affinity cells proliferate more rapidly or are more likely to enter cell cycle, thereby outgrowing lower affinity cells. Alternatively, higher affinity cells could be relatively more resistant to cell death in the GC. By comparing high- and low-affinity B cells for the same Ag, we show here that low-affinity cells have an intrinsically higher death rate than do cells of higher affinity, even in the absence of competition. This suggests that selection in the GC reaction is due at least in part to the control of survival of higher affinity B cells and not by a proliferative advantage conferred upon these cells compared with lower affinity B cells. Control over survival rather than proliferation of low- and high-affinity B cells in the GC allows greater diversity not only in the primary response but also in the memory response.

Endogenous IL-21 Restricts CD8+ T Cell Expansion and Is not Required for Tumor Immunity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 13:06:03 PST

IL-21 has antitumor activity through actions on NK cells and CD8⁺ T cells, and is currently in clinical development for the treatment of cancer. However, no studies have addressed the role of endogenous IL-21 in tumor immunity. In this study, we have studied both primary and secondary immune responses in IL-21^–/– and IL-21R^–/– mice against several experimental tumors. We found intact immune surveillance toward methylcholanthrene-induced sarcomas in IL-21^–/– and IL-21R^–/– mice compared with wild-type mice and B16 melanomas showed equal growth kinetics and development of lung metastases. IL-21R^–/– mice showed competent NK cell-mediated rejection of NKG2D ligand (Rae1β) expressing H-2b^– RMAS lymphomas and sustained transition to CD8⁺ T cell-dependent memory against H-2b⁺ RMA lymphomas. -Galactosylceramide stimulation showed equal expansion and activation of NKT and NK cells and mounted a powerful antitumor response in the absence of IL-21 signaling, despite reduced expression of granzyme B in NKT, NK, and CD8⁺ T cells. Surprisingly, host IL-21 significantly restricted the expansion of Ag-specific CD8⁺ T cells and inhibited primary CD8⁺ T cell immunity against OVA-expressing EG7 lymphomas, as well as the secondary expansion of memory CD8⁺ T cells. However, host IL-21 did not alter the growth of less immunogenic MC38 colon carcinomas with dim OVA expression. Overall, our results show that endogenous IL-21/IL-21R is not required for NK, NKT, and CD8⁺ T cell-mediated tumor immunity, but restricts Ag-specific CD8⁺ T cell expansion and rejection of immunogenic tumors, indicating novel immunosuppressive actions of this cytokine.

Dynamic Regulation of CXCR1 and CXCR2 Homo- and Heterodimers [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:03 PST

Although homo- and heterodimerization are reported for some chemokine receptors, it remains unclear whether these functional states are in dynamic equilibrium and how receptor/ligand levels influence oligomerization. In human neutrophils and in cell lines that coexpress the chemokine receptors CXCR1 and CXCR2, we used fluorescence resonance energy transfer techniques to show that these two receptors form homo- and heterodimers. Receptor expression and ligand activation were found to regulate the balance between these complexes, adapting the response to changes in the milieu. CXCL8, a ligand for both receptors, alters heterodimeric complexes, whereas it stabilizes homodimers and promotes receptor internalization. Oligomerization of receptors, together with the regulation of their expression and desensitization, could thus contribute to the fine control of chemokine functions.

Coexistence of Closed and Open Conformations of Complement Factor B in the Alternative Pathway C3bB(Mg2+) Proconvertase [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Torreira, E., Tortajada, A., Montes, T., Rodriguez de Cordoba, S., Llorca, O. — Wed, 18 Nov 2009 13:06:03 PST

Complement factor B (fB) circulates in plasma as a proenzyme that, upon binding to C3b in the presence of Mg²⁺, is cleaved by factor D to produce Ba and Bb fragments. Activated Bb remains bound to C3b organizing the alternative pathway C3 convertase (C3bBb). Recently, we have visualized the stable C3bB(Ni²⁺) proconvertase using electron microscopy, revealing a large conformational change of the C3b-bound fB likely exposing the fD-cleavage site. In contrast, the crystal structure of the proconvertase formed by human fB and the cobra venom factor reveals fB in the closed conformation of the proenzyme. In this study, we have used single-particle electron microscopy and image processing to examine the C3bB(Mg²⁺) proconvertase. We describe two C3bB(Mg²⁺) conformations, one resembling cobra venom factor, likely representing the loading state of fB to C3b, and another identical with C3bB(Ni²⁺). These data illustrate the coexistence of C3b-bound fB in closed and open conformations that either exist in equilibrium or represent structural transitions during the assembly of the C3bB proconvertase.

Hematopoietic Lineage Cell-Specific Protein 1 Is Recruited to the Immunological Synapse by IL-2-Inducible T Cell Kinase and Regulates Phospholipase C{gamma}1 Microcluster Dynamics during T Cell Spreading [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:03 PST

Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk’s Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1^–/– T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLC1 phosphorylation was intact, indicating that HS1’s role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLC1 and altered formation of PLC1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLC1 signaling complexes.

Serine 649 Phosphorylation within the Protein Kinase C-Regulated Domain Down-Regulates CARMA1 Activity in Lymphocytes [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:03 PST

Phosphorylation of CARMA1 is a crucial event initiating the assembly of IB kinase and JNK signaling complexes downstream of activated Ag receptors. We previously mapped three protein kinase C (PKC) target sites in murine CARMA1 in vitro, and demonstrated that mutation of two of these serines (S564 and S657) resulted in reduced NF-B activation, whereas mutation of the third serine (S649) had no clear effect. In this study, we report that when low concentrations of Ag receptor activators are used, loss of S649 (by mutation to alanine) promotes enhanced IB kinase and JNK activation in both B and T cell lines. Reconstitution of CARMA1^–/– DT40 B cells with CARMA1 S649A leads to increased cell death and reduced cell growth in comparison to wild-type CARMA1, likely a result of enhanced JNK activation. To directly determine whether S649 is modified in vivo, we generated phospho-specific Abs recognizing phospho-S649, and phospho-S657 as a positive control. Although phospho-S657 peaked and declined rapidly after Ag receptor stimulation, phospho-S649 occurred later and was maintained for a significantly longer period poststimulation in both B and T cells. Interestingly, phospho-S657 was completely abolished in PKCβ-deficient B cells, whereas delayed phosphorylation at S649 was partially intact and depended, in part, upon novel PKC activity. Thus, distinct PKC-mediated CARMA1 phosphorylation events exert opposing effects on the activation status of CARMA1. We propose that early phosphorylation events at S657 and S564 promote the initial assembly of the CARMA1 signalosome, whereas later phosphorylation at S649 triggers CARMA1 down-regulation.

MAp44, a Human Protein Associated with Pattern Recognition Molecules of the Complement System and Regulating the Lectin Pathway of Complement Activation [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:03 PST

Essential effector functions of innate immunity are mediated by complement activation initiated by soluble pattern recognition molecules: mannan-binding lectin (MBL) and the ficolins. We present a novel, phylogenetically conserved protein, MAp44, which is found in human serum at 1.4 µg/ml in Ca²⁺-dependent complexes with the soluble pattern recognition molecules. The affinity for MBL is in the nanomolar range (K_D = 0.6 nM) as determined by surface plasmon resonance. The first eight exons of the gene for MAp44 encode four domains shared with MBL-associated serine protease (MASP)-1 and MASP-3 (CUB1-EGF-CUB2-CCP1), and a ninth exon encodes C-terminal 17 aa unique to MAp44. mRNA profiling in human tissues shows high expression in the heart. MAp44 competes with MASP-2 for binding to MBL and ficolins, resulting in inhibition of complement activation. Our results add a novel mechanism to those known to control the innate immune system.

Cytosolic Aminopeptidases Influence MHC Class I-Mediated Antigen Presentation in an Allele-Dependent Manner [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Kim, E., Kwak, H., Ahn, K. — Wed, 18 Nov 2009 13:06:03 PST

Antigenic peptides presented by MHC class I molecules are generated mainly by the proteasome in the cytosol. Several cytosolic aminopeptidases further trim proteasomal products to form mature epitopes or individual amino acids. However, the distinct function of cytosolic aminopeptidases in MHC class I Ag processing remains to be elucidated. In this study, we show that cytosolic aminopeptidases differentially affect the cell surface expression of MHC class I molecules in an allele-dependent manner in human cells. In HeLa cells, knockdown of puromycin-sensitive aminopeptidase (PSA) by RNA interference inhibited optimal peptide loading of MHC class I molecules, and their cell surface expression was correspondingly reduced. In contrast, depletion of bleomycin hydrolase (BH) enhanced optimal peptide loading and cell surface expression of MHC class I molecules. We did not find evidence on the effect of leucine aminopeptidase knockdown on the MHC class I Ag presentation. Moreover, we demonstrated that PSA and BH influence the peptide loading and surface expression of MHC class I in an allele-specific manner. In the absence of either PSA or BH, the surface expression and peptide-dependent stability of HLA-A68 were reduced, whereas those of HLA-B15 were enhanced. The surface expression and peptide-dependent stability of HLA-A3 were enhanced by BH knockdown, although those of HLA-B8 were increased in PSA-depleted conditions.

MAPK, Phosphatidylinositol 3-Kinase, and Mammalian Target of Rapamycin Pathways Converge at the Level of Ribosomal Protein S6 Phosphorylation to Control Metabolic Signaling in CD8 T Cells [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Salmond, R. J., Emery, J., Okkenhaug, K., Zamoyska, R. — Wed, 18 Nov 2009 13:06:03 PST

Ribosomal protein S6 (rpS6) is a key component of the translational machinery in eukaryotic cells and is essential for ribosome biogenesis. rpS6 is phosphorylated on evolutionarily conserved serine residues, and data indicate that rpS6 phosphorylation might regulate cell growth and protein synthesis. Studies in cell lines have shown an important role for the serine kinase mammalian target of rapamycin (mTOR) in rpS6 phosphorylation, further linking rpS6 to control of cellular metabolism. rpS6 is essential in T cells because its deletion in mouse double-positive thymocyte cells results in a complete block in T cell development; however, the signaling pathway leading to rpS6 phosphorylation downstream of TCR stimulation has yet to be fully characterized. We show that maximal TCR-induced rpS6 phosphorylation in CD8 T cells requires both Lck and Fyn activity and downstream activation of PI3K, mTOR, and MEK/ERK MAPK pathways. We demonstrate that there is cross-talk between the PI3K and MAPK pathways as well as PI3K-independent mTOR activity, which result in differential phosphorylation of specific rpS6 serine residues. These results place rpS6 phosphorylation as a point of convergence for multiple crucial signaling pathways downstream of TCR triggering.

The DC-SIGN of Zebrafish: Insights into the Existence of a CD209 Homologue in a Lower Vertebrate and Its Involvement in Adaptive Immunity [IMMUNOGENETICS]

Lin, A.-F., Xiang, L.-X., Wang, Q.-L., Dong, W.-R., Gong, Y.-F., Shao, J.-Z. — Wed, 18 Nov 2009 13:06:03 PST

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN/CD209) has become hot topic in recent studies because of its important roles in immune responses and immune escape. CD209 has been well characterized in humans and several other mammals, but little documentation exists about it in lower vertebrates. This is the first report on the identification and functional characterization of a fish DC-SIGN/CD209 molecule. The zebrafish DC-SIGN/CD209 cDNA translates into 343 aa organized into three domains structurally conserved among vertebrates. An EPN motif essential for interacting with Ca²⁺ and for recognizing mannose-containing motifs has been identified. Several conserved motifs crucial for internalization and signal transduction are also present within the cytoplasmic tail. Phylogenetic analysis supports the hypothesis that CD209 family members diverged from a common ancestor. The expression of DC-SIGN/CD209 in immune-related tissues can be significantly up-regulated by exogenous Ags and IL-4. This molecule associates with various APCs, including macrophages, B lymphocytes, and a possible dendritic cell-like (CD83⁺/CD80⁺CD209⁺) population. Functionally, T cell activation, Ab (IgM) production, and bacterial vaccination-elicited immunoprotection can be dramatically inhibited by a CD209 blockade after stimulation with keyhole limpet hemocyanin (KLH) in vivo or challenged with Aeromonas hydrophila, suggesting that DC-SIGN/CD209 in zebrafish is crucial for the initiation and development of adaptive immunity. Phagocytosis analysis showed that DC-SIGN/CD209 does not participate in the uptake of KLH Ag, suggesting that other mechanisms might exist that underlie DC-SIGN/CD209 involvement. We hope that the present study will contribute to a better cross-species understanding of the evolutionary history of the DC-SIGN/CD209 family.

Increased Inflammation, Impaired Bacterial Clearance, and Metabolic Disruption after Gram-Negative Sepsis in Mkp-1-Deficient Mice [HOST DEFENSE]

Wed, 18 Nov 2009 13:06:03 PST

MAPKs are crucial for TNF- and IL-6 production by innate immune cells in response to TLR ligands. MAPK phosphatase 1 (Mkp-1) deactivates p38 and JNK, abrogating the inflammatory response. We have previously demonstrated that Mkp-1^–/– mice exhibit exacerbated inflammatory cytokine production and increased mortality in response to challenge with LPS and heat-killed Staphylococcus aureus. However, the function of Mkp-1 in host defense during live Gram-negative bacterial infection remains unclear. We challenged Mkp-1^+/+ and Mkp-1^–/– mice with live Escherichia coli i.v. to examine the effects of Mkp-1 deficiency on animal survival, bacterial clearance, metabolic activity, and cytokine production. We found that Mkp-1 deficiency predisposed animals to accelerated mortality and was associated with more robust production of TNF-, IL-6 and IL-10, greater bacterial burden, altered cyclooxygenase-2 and iNOS expression, and substantial changes in the mobilization of energy stores. Likewise, knockout of Mkp-1 also sensitized mice to sepsis caused by cecal ligation and puncture. IL-10 inhibition by neutralizing Ab or genetic deletion alleviated increased bacterial burden. Treatment with the bactericidal antibiotic gentamicin, given 3 h after Escherichia coli infection, protected Mkp-1^+/+ mice from septic shock but had no effect on Mkp-1^–/– mice. Thus, during Gram-negative bacterial sepsis Mkp-1 not only plays a critical role in the regulation of cytokine production but also orchestrates the bactericidal activities of the innate immune system and controls the metabolic response to stress.

Early Response of Mucosal Epithelial Cells during Toxoplasma gondii Infection [HOST DEFENSE]

Ju, C.-H., Chockalingam, A., Leifer, C. A. — Wed, 18 Nov 2009 13:06:03 PST

The innate immune response of mucosal epithelial cells during pathogen invasion plays a central role in immune regulation in the gut. Toxoplasma gondii is a protozoan intracellular parasite that is usually transmitted through oral infection. Although much of the information on immunity to T. gondii has come from i.p. infection models, more recent studies have revealed the importance of studying immunity following infection through the natural peroral route. Oral infection studies have identified many of the key players in the intestinal response; however, they have relied on responses detected days to weeks following infection. Much less is known about how the gut epithelial layer senses and reacts during initial contact with the pathogen. Given the importance of epithelial cells during pathogen invasion, this study uses an in vitro approach to isolate the key players and examine the early response of intestinal epithelial cells during infection by T. gondii. We show that human intestinal epithelial cells infected with T. gondii elicit rapid MAPK phosphorylation, NF-B nuclear translocation, and secretion of IL-8. Both ERK1/2 activation and IL-8 secretion responses were shown to be MyD88 dependent and TLR2 was identified to be involved in the recognition of the parasite regardless of the parasite genotype. Furthermore, we were able to identify additional T. gondii-regulated genes in the infected cells using a pathway-focused array. Together, our findings suggest that intestinal epithelial cells were able to recognize T. gondii during infection, and the outcome is important for modulating intestinal immune responses.

Immune Gene and Cell Enrichment Is Associated with a Good Prognosis in Ependymoma [HOST DEFENSE]

Wed, 18 Nov 2009 13:06:03 PST

Approximately 50% of children with ependymoma will suffer from tumor recurrences that will ultimately lead to death. Development of more effective therapies and patient stratification in ependymoma mandates better prognostication. In this study, tumor gene expression microarray profiles from pediatric ependymoma clinical samples were subject to ontological analyses to identify outcome-associated biological factors. Histology was subsequently used to evaluate the results of ontological analyses. Ontology analyses revealed that genes associated with nonrecurrent ependymoma were predominantly immune function-related. Additionally, increased expression of immune-related genes was correlated with longer time to progression in recurrent ependymoma. Of those genes associated with both the nonrecurrent phenotype and that positively correlated with time to progression, 95% were associated with immune function. Histological analysis of a subset of these immune function genes revealed that their expression was restricted to a subpopulation of tumor-infiltrating cells. Analysis of tumor-infiltrating immune cells showed increased infiltration of CD4⁺ T cells in the nonrecurrent ependymomas. No genomic sequences for SV40, BK, JC, or Merkel polyomaviruses were found in nonrecurrent ependymoma. This study reveals that up-regulation of immune function genes is the predominant ontology associated with a good prognosis in ependymoma and it provides preliminary evidence of a beneficial host proinflammatory and/or Ag-specific immune response.

Neutrophils Ameliorate Lung Injury and the Development of Severe Disease during Influenza Infection [HOST DEFENSE]

Wed, 18 Nov 2009 13:06:03 PST

The clinical response to influenza infection ranges from mild disease to severe pneumonia and it remains unclear whether the inflammatory response to infection is protective or pathogenic. We have defined a novel role for neutrophils in ameliorating lung injury during influenza infection, thereby limiting development of severe disease. Infection of neutrophil-depleted mice with influenza virus HKx31 (H3N2) led to rapid weight loss, pneumonia, and death. Neutropenia was associated with enhanced virus replication in the respiratory tract; however, viral titers were declining at the time of death, leading us to investigate other factors contributing to mortality. In addition to thymic atrophy, lymphopenia, and viremic spread, depletion of neutrophils led to exacerbated pulmonary inflammation, edema, and respiratory dysfunction. Thus, while it is well established that neutrophils contribute to lung injury in a range of pathological conditions, reduced numbers or impaired neutrophil function can facilitate progression of mild influenza to severe clinical disease.

Identification of Lipoteichoic Acid as a Ligand for Draper in the Phagocytosis of Staphylococcus aureus by Drosophila Hemocytes [HOST DEFENSE]

Wed, 18 Nov 2009 13:06:03 PST

Phagocytosis is central to cellular immunity against bacterial infections. As in mammals, both opsonin-dependent and -independent mechanisms of phagocytosis seemingly exist in Drosophila. Although candidate Drosophila receptors for phagocytosis have been reported, how they recognize bacteria, either directly or indirectly, remains to be elucidated. We searched for the Staphylococcus aureus genes required for phagocytosis by Drosophila hemocytes in a screening of mutant strains with defects in the structure of the cell wall. The genes identified included ltaS, which encodes an enzyme responsible for the synthesis of lipoteichoic acid. ltaS-dependent phagocytosis of S. aureus required the receptor Draper but not Eater or Nimrod C1, and Draper-lacking flies showed reduced resistance to a septic infection of S. aureus without a change in a humoral immune response. Finally, lipoteichoic acid bound to the extracellular region of Draper. We propose that lipoteichoic acid serves as a ligand for Draper in the phagocytosis of S. aureus by Drosophila hemocytes and that the phagocytic elimination of invading bacteria is required for flies to survive the infection.

TLR Cross-Talk Specifically Regulates Cytokine Production by B Cells from Chronic Inflammatory Disease Patients [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Chronic systemic inflammation links periodontal disease and diabetes to increased incidence of serious comorbidities. Activation of TLRs, particularly TLR2 and TLR4, promotes chronic systemic inflammation. Human B cells have been generally thought to lack these TLRs. However, recent work showed that an increased percentage of circulating B cells from inflammatory disease patients express TLR2 and TLR4, and that TLR engagement on B cells resulted in unexpected changes in gene expression. New data show that B cells from inflammatory disease patients secrete multiple cytokines in response to different classes of TLR ligands. Furthermore, the B cell response to combinations of TLR ligands is cytokine- and ligand-specific. Some cytokines (IL-1β and IL-10) are predominantly regulated by TLR4, but others (IL-8 and TNF-) are predominantly regulated by TLR2, due in part to TLR-dictated changes in transcription factor/promoter association. TLR2 and TLR9 also regulate B cell TLR4 expression, demonstrating that TLR cross-talk controls B cell responses at multiple levels. Parallel examination of B cells from periodontal disease and diabetes patients suggested that outcomes of TLR cross-talk are influenced by disease pathology. We conclude that disease-associated alteration of B cell TLR responses specifically regulates cytokine production and may influence chronic inflammation.

Lipopolysaccharide Sensitizes Neonatal Hypoxic-Ischemic Brain Injury in a MyD88-Dependent Manner [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Neurological deficits in children, including cerebral palsy, are associated with prior infection during the perinatal period. Experimentally, we have shown that pre-exposure to the Gram-negative component LPS potentiates hypoxic-ischemic (HI) brain injury in newborn animals. LPS effects are mediated by binding to TLR4, which requires recruitment of the MyD88 adaptor protein or Toll/IL-1R domain-containing adapter inducing IFN-β for signal transduction. In this study, we investigated the role of MyD88 in neonatal brain injury. MyD88 knockout (MyD88 KO) and wild-type mice were subjected to left carotid artery ligation and 10% O₂ for 50 min on postnatal day 9. LPS or saline were administered i.p. at 14 h before HI. At 5 days after HI in wild-type mice, LPS in combination with HI caused a significant increase in gray and white matter tissue loss compared with the saline-HI group. By contrast, in the MyD88 KO mice there was no potentiation of brain injury with LPS-HI. MyD88 KO mice exhibited reduced NFB activation and proinflammatory cytokine-chemokine expression in response to LPS. The number of microglia and caspase-3 activation was increased in the brain of MyD88 KO mice after LPS exposure. Collectively, these findings indicate that MyD88 plays an essential role in LPS-sensitized HI neonatal brain injury, which involves both inflammatory and caspase-dependent pathways.

CCR5 Ligands Modulate CXCL12-Induced Chemotaxis, Adhesion, and Akt Phosphorylation of Human Cord Blood CD34+ Cells [INFLAMMATION]

Basu, S., Broxmeyer, H. E. — Wed, 18 Nov 2009 13:06:04 PST

CXCL12 and its receptor CXCR4 play an important role in hematopoietic stem/progenitor cell (HSPC) migration from and retention within the bone marrow. HSPCs are very selective in their chemotactic response and undergo chemotaxis only in response to CXCL12. In addition to CXCR4, HSPCs express receptors for various other chemokines; however, the role of these receptors is not well understood. Freshly isolated CD34⁺ cells (highly enriched for HSPCs) from cord blood (CB) express low levels of CCR5; however, if the cells were washed with acidic buffer before Ab staining to remove any ligand bound to CCR5, then nearly 80% of CD34⁺ CB cells were found to express CCR5 on the cell surface. Although none of the CCR5 ligands investigated in this study (CCL3, CCL4, and CCL5) induced chemotaxis, at relatively high concentrations they transiently enhanced CXCL12-mediated chemotaxis of CD34⁺ CB cells. In contrast, CXCL12-mediated adhesion of cells to VCAM-1-coated surfaces was reduced if CD34⁺ CB cells were pretreated with these CCR5 ligands for 15 min. The effect of these chemokines on CXCL12-mediated responses was not at the level of CXCR4 expression, but on downstream signaling pathways elicited by CXCL12. Pretreatment with CCR5 chemokines enhanced CXCL12-mediated Akt phosphorylation, but down-modulated calcium flux in CD34⁺ CB cells. Modulation of CXCL12-mediated responses of CD34⁺ cells by CCR5 chemokines provides a possible mechanism that underlies movement of HSPCs during inflammation.

The Oxazolidinone Derivative Locostatin Induces Cytokine Appeasement [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF- production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

Calcium-Independent Phospholipase A2{beta}-Akt Signaling Is Involved in Lipopolysaccharide-Induced NADPH Oxidase 1 Expression and Foam Cell Formation [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Foam cell formation is the most important process in atherosclerosis, and low density lipoprotein oxidation by reactive oxygen species (ROS) is the key step in the conversion of macrophages to foam cells. This study reveals the control mechanism of the gene for NADPH oxidase 1 (Nox1), which produces ROS in the formation of foam cells by stimulating TLR4. Treatment of macrophages by the TLR4 agonist LPS stimulated ROS production and ROS-mediated macrophage to foam cell conversion. This LPS-induced ROS production and foam cell formation could be abrogated by pretreatment of macrophages with N-acetyl cysteine or apocynin. LPS increased Nox1 promoter activity, and resultant expression of mRNA and protein. Small interfering RNA mediated inhibition of Nox1 expression decreased LPS-induced ROS production and foam cell formation. LPS-mediated Nox1 expression and the responses occurred in a calcium-independent phospholipase A₂ (iPLA₂)-dependent manner. The iPLA₂β-specific inhibitor S-BEL or iPLA₂β small interfering RNA attenuated LPS-induced Nox1 expression, ROS production, and foam cell formation. In addition, activation of iPLA₂β by LPS caused Akt phosphorylation and was followed by increased Nox1 expression. These results suggest that the binding of LPS and TLR4 increases Nox1 expression through the iPLA₂β-Akt signaling pathway, and control ROS production and foam cell formation.

CD80 Blockade Enhance Glucocorticoid-Induced Leucine Zipper Expression and Suppress Experimental Autoimmune Encephalomyelitis [INFLAMMATION]

Dudhgaonkar, S. P., Janardhanam, S. B., Kodumudi, K. N., Srinivasan, M. — Wed, 18 Nov 2009 13:06:04 PST

Designing mimetic of the interface functional groups of known receptor-ligand complexes is an attractive strategy for developing potential therapeutic agents that interfere with target protein-protein interactions. The CD80/CD86-CD28/CD152 costimulatory interactions transmit signals for CD4⁺ T cell activation and suppression and are critically involved in the initiation, progression, and reactivation of the immunopathology in multiple sclerosis. Differences in the pattern, levels, and kinetics of expression of CD80/CD86 molecules in conjunction with differences in the strength of the signals delivered upon binding CD28 or CD152 determine the outcome of the immune response. A temporal up-regulation of surface expression of CD80 relative to CD86 on APCs and CNS-infiltrating cells has been shown to correlate with disease progression in experimental autoimmune encephalomyelitis an animal model for multiple sclerosis. Hence blockade of the CD80 costimulatory axis has therapeutic potential in multiple sclerosis. In this study, we report the efficacy of a novel CD80-blocking agent CD80-competitive antagonist peptide (CD80-CAP) in suppressing clinical disease and relapse in experimental autoimmune encephalomyelitis. The CD80-CAP mediates protection by inhibiting proinflammatory cytokines and skewing toward anti-inflammatory response presumably by enhancing the expression of glucocorticoid-induced leucine zipper in activated CD4⁺ T cells.

G Protein-Coupled Receptor 43 Is Essential for Neutrophil Recruitment during Intestinal Inflammation [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Molecular danger signals attract neutrophilic granulocytes (polymorphonuclear leukocytes (PMNs)) to sites of infection. The G protein-coupled receptor (GPR) 43 recognizes propionate and butyrate and is abundantly expressed on PMNs. The functional role of GPR43 activation for in vivo orchestration of immune response is unclear. We examined dextrane sodium sulfate (DSS)-induced acute and chronic intestinal inflammatory response in wild-type and Gpr43-deficient mice. The severity of colonic inflammation was assessed by clinical signs, histological scoring, and cytokine production. Chemotaxis of wild-type and Gpr43-deficient PMNs was assessed through transwell cell chemotactic assay. A reduced invasion of PMNs and increased mortality due to septic complications were observed in acute DSS colitis. In chronic DSS colitis, Gpr43^–/– animals showed diminished PMN intestinal migration, but protection against inflammatory tissue destruction. No significant difference in PMN migration and cytokine secretion was detected in a sterile inflammatory model. Ex vivo experiments show that GPR43-induced migration is dependent on activation of the protein kinase p38, and that this signal acts in cooperation with the chemotactic cytokine keratinocyte chemoattractant. Interestingly, shedding of L-selectin in response to propionate and butyrate was compromised in Gpr43^–/– mice. These results indicate a critical role for GPR43-mediated recruitment of PMNs in containing intestinal bacterial translocation, yet also emphasize the bipotential role of PMNs in mediating tissue destruction in chronic intestinal inflammation.

Natural Occurring IL-17 Producing T Cells Regulate the Initial Phase of Neutrophil Mediated Airway Responses [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Effector Th17 cells are a major source of IL-17, a critical inflammatory cytokine in autoimmune diseases and in host defenses during bacterial infections. Recently, splenic lymphoid tissue inducer-like cells have been reported to be a source of T cell independent IL-17. In this study, we report that the immune system contains a unique set of natural occurring IL-17 producing cell, "natural" Th17 (nTh17), which are a memory-like T cell subset. The nTh17 cells can develop in the absence of the IL-6/STAT3 signaling axis required by inducible Th17 cells. The nTh17 cell population is distinct from conventional inducible Th17 cells, since nTh17 cells express substantial amounts of IL-17A (IL-17), but not IL-17F, under the control of the master regulator, RORt. The nTh17 cells simultaneously produce IFN-. DO11.10 transgenic mice with a Rag^–/– background (DO11.10 Rag^–/–) lack nTh17 cells, and, following intranasal administration of OVA, IL-17-dependent neutrophil infiltration occurs in DO11.10 transgenic mice, but not in DO11.10 Rag^–/– mice. The impaired neutrophil-dependent airway response is restored by adaptive transfer of nTh17 cells into DO11.10 Rag^–/– mice. These results demonstrate that a novel T cell subset, nTh17, facilitates the early phase of Ag-induced airway responses and host defenses against pathogen invasion before the establishment of acquired immunity.

Fms-Like Tyrosine Kinase 3 Ligand Regulates Migratory Pattern and Antigen Uptake of Lung Dendritic Cell Subsets in a Murine Model of Allergic Airway Inflammation [INFLAMMATION]

Shao, Z., Makinde, T. O., McGee, H. S., Wang, X., Agrawal, D. K. — Wed, 18 Nov 2009 13:06:04 PST

Fms-like tyrosine kinase 3 ligand (Flt3L) reverses the features of allergic airway inflammation and increases a Th2-suppressive regulatory lung CD11c^highCD11b^low dendritic cell (DC) subset in a mouse model. We examined the migratory pattern and Ag uptake efficiency of lung DC subsets in the therapeutic effect of Flt3L. Lung CD11c^highCD11b^low and CD11c^lowCD11b^high DCs from PBS-treated, OVA-sensitized, and Flt3L-treated/OVA-sensitized BALB/c mice were sorted using MACS and FACS for phenotype analysis. Lymphatic chemokine expression in thoracic lymph nodes was determined by immunohistochemistry. Migration of two lung DC subsets to lymphatic chemokines was examined in vitro using a Transwell chemotaxis assay. Labeled Ag was intranasally delivered into mouse lung to track the migration and Ag uptake of lung DCs. The in vitro cytokine secretion of mediastinal lymph node cells was determined using ELISA. CD11c^lowCD11b^high DCs have higher expression of CCR5, CCR6, and CCR7, but lower expression of CCR2 than CD11c^highCD11b^low DCs. CD11c^lowCD11b^high DCs in Flt3L-treated/OVA-sensitized mice demonstrated a less mature phenotype, inefficiency in Ag uptake, and impaired migration in vitro to lymphatic chemokine than those in OVA-sensitized mice. Administration of Flt3L decreased the expression of CCR5 and CCR7 in CD11c^lowCD11b^high DCs in OVA-sensitized mice. Fewer Ag-carrying cells were detected in the lungs and lymph nodes in Flt3L-treated/OVA-sensitized mice than OVA-sensitized mice with a greater decrease in CD11c^lowCD11b^high DCs. Mediastinal lymph node cells from Flt3L-treated mice secreted higher levels of Th1 cytokines and IL-10 than OVA-sensitized mice in vitro. In conclusion, Flt3L-generated lung immunogenic CD11c^lowCD11b^high DCs have a less mature phenotype, impaired Ag uptake, and impaired migration to draining lymph nodes.

Tyrosine Kinase 2 Plays Critical Roles in the Pathogenic CD4 T Cell Responses for the Development of Experimental Autoimmune Encephalomyelitis [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Tyrosine kinase 2 (Tyk2), a member of the JAK family, is involved in IL-12- and IL-23-mediated signaling. In the present study, we examined the roles of Tyk2 in the development of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by using Tyk2 knockout (KO) mice. In vitro differentiation of Th1 but not Th17 cells was severely impaired in Tyk2 KO CD4 T cells, although Tyk2 KO Th17 cells did not respond to IL-23. Tyk2 KO mice showed complete resistance against EAE with no infiltration of CD4 T cells in the spinal cord. Surprisingly, the number of MOG-specific Th17 cells in the periphery was comparable between KO and wild-type (WT) mice, whereas Th1 cells were greatly reduced in Tyk2 KO mice. Adoptive transfer of MOG-primed WT T cells induced EAE in Tyk2 KO recipients, indicating that Tyk2 in the environment was dispensable for the infiltration of effector T cells into the spinal cord. A reduced but significant number of Tyk2 KO T cells were detected in the spinal cord of mice with EAE, which had been reconstituted with bone marrow cells of WT and KO mice. Furthermore, MOG-immunized Tyk2 KO mice developed EAE after adoptive transfer of MOG-primed WT Th1 cells, which might trigger local inflammation that recruits Th17 cells. Taken together, these results indicate that Tyk2 is critically involved in the pathogenic CD4 T cell responses and thus could be a target molecule for the treatment of autoimmune diseases.

Unlike Th1, Th17 Cells Mediate Sustained Autoimmune Inflammation and Are Highly Resistant to Restimulation-Induced Cell Death [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Both Th1 and Th17 T cell subsets can mediate inflammation, but the kinetics of the pathogenic processes mediated by these two subsets have not been investigated. Using an experimental system in which TCR-transgenic Th1 or Th17 cells specific for hen egg lysozyme induce ocular inflammation in recipient mice expressing eye-restricted hen egg lysozyme, we found important differences in the in vivo behavior of these two subsets. Th1 cells initially proliferated considerably faster and invaded the eye more quickly than their Th17 counterparts, but then disappeared rapidly. By contrast, Th17 cells accumulated and remained the majority of the infiltrating CD4⁺ cells in the eye for as long as 25 days after transfer, mediating more long-lasting pathological changes. Unlike Th1, Th17 cells were highly resistant to restimulation-induced apoptosis, a major pathway by which autoimmune and chronically restimulated Th1 cells are eliminated. Th17 cells had reduced Fas ligand production and resistance to Fas-induced apoptosis, relative to Th1 cells, despite similar surface expression of Fas. Th17-induced ocular inflammation also differed from Th1-induced inflammation by consisting of more neutrophils, whereas Th1-induced disease had higher proportions of CD8 cells. Taken together, our data show that pathogenic processes triggered by Th17 lag behind those induced by Th1, but then persist remarkably longer, apparently due to the relative resistance of Th17 cells to restimulation-induced cell death. The long-lasting inflammation induced by Th17 cells is in accord with these cells being involved in chronic conditions in humans.

Selective Down-Regulation of Neutrophil Mac-1 in Endotoxemic Hepatic Microcirculation via IL-10 [INFLAMMATION]

Wed, 18 Nov 2009 13:06:04 PST

Hepatic neutrophil adhesion during endotoxemia is an integrin-independent, CD44-dependent process. Because integrins function in other endotoxemic vasculatures, we used spinning disk confocal intravital microscopy to assess whether LPS down-modulated integrin functions in sinusoids. First, we applied fMLP onto the liver surface, and compared it with systemic LPS administration. Local fMLP caused neutrophil adhesion, crawling, and emigration for at least 2 h. Surprisingly, the number of adherent and crawling neutrophils was markedly reduced in Mac-1^–/– and ICAM-1^–/– mice, but not in mice treated with anti-CD44 mAb. By contrast, systemic LPS injection induced a robust accumulation of neutrophils in sinusoids, which was dependent on CD44, but not on integrins. Strikingly, local fMLP could not induce any integrin-dependent adhesion in endotoxemic mice treated with anti-CD44 mAb, indicating that Mac-1-dependent neutrophil adhesion was inhibited by LPS. This response was localized to the hepatic microvasculature because neutrophils still adhered via integrins in brain microvasculature. ICAM-1/ICAM-2 levels were not decreased, but following LPS treatment, Mac-1 was down-regulated in neutrophils localized to liver, but not in the circulation. Mac-1 down-regulation in neutrophils was not observed in IL-10^–/– mice. In vitro neutrophil incubation with IL-10 induced direct decrease of Mac-1 expression and adhesivity in LPS-stimulated neutrophils. Therefore, our data suggest that Mac-1 is necessary for neutrophil adhesion and crawling during local inflammatory stimuli in sinusoids, but during systemic inflammation, neutrophils are exposed to high concentrations of IL-10, leading to a CD44-dependent, integrin-independent adhesion. This may be a mechanism to keep neutrophils in sinusoids for intravascular trapping.

Systematic Classification of Primary Immunodeficiencies Based on Clinical, Pathological, and Laboratory Parameters [CLINICAL IMMUNOLOGY]

Samarghitean, C., Ortutay, C., Vihinen, M. — Wed, 18 Nov 2009 13:06:04 PST

The classification of diseases has several important applications ranging from diagnosis and choice of treatment to demographics. To date, classifications have been successfully created manually, often within international consortia. Some groups of diseases, such as primary immunodeficiencies (PIDs), are especially hard to nosologically cluster due, on one hand, to the presence of a wide variety of disorders and, in contrast, because of overlapping characteristics. More than 200 PIDs affecting components of the innate and adaptive immune systems have been described. Clinical, pathological, and laboratory characteristics were collected and used to group PIDs. A consensus of at least five independent methods provided a novel classification of 11 groups, which revealed previously unknown features and relationships of PIDs. Comparison of the classification to independent features, including the severity and therapy of the diseases, functional classification of proteins, and network vulnerability, indicated a strong statistical support. The method can be applied to any group of diseases.

The Cutaneous Biochemical Redox Barrier: A Component of the Innate Immune Defenses against Sensitization by Highly Reactive Environmental Xenobiotics [CLINICAL IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:04 PST

Contact allergy to environmental xenobiotics is a common and important problem, but it is unclear why some chemicals are potent sensitizers and others weak/nonsensitizers. We explored this by investigating why similar chemicals, 2,4-dinitrochlorobenzene (DNCB) and 2,4-dinitrothiocyanobenzene (DNTB), differ in their ability to induce contact hypersensitivity (CHS). DNCB induced CHS in humans, whereas at similar doses DNTB did not. However, following DNCB sensitization, DNTB elicited CHS in vivo and stimulated DNCB-responsive T cells in vitro, suggesting that differences in response to these compounds lie in the sensitization phase. In contrast to DNCB, DNTB failed to induce emigration of epidermal Langerhans cells in naive individuals. Examination for protein dinitrophenylation in skin revealed that DNCB penetrated into the epidermis, whereas DNTB remained bound to a thiol-rich band within the stratum corneum. DNTB reacted rapidly with reduced glutathione in vitro and was associated with a decrease in the free thiol layer in the stratum corneum, but not in the nucleated epidermis. By contrast, DNCB required GST facilitation to react with gluthathione and, following penetration through the stratum corneum, depleted thiols in the viable epidermis. Chemical depletion of the thiol-rich band or removing it by tape stripping allowed increased penetration of DNTB into the epidermis. Our results suggest that the dissimilar sensitizing potencies of DNCB and DNTB in humans are determined by a previously undescribed outer epidermal biochemical redox barrier, a chemical component of the innate immune defense mechanisms that defend against sensitization by highly reactive environmental chemicals.

{beta} Cell-Specific CD4+ T Cell Clonotypes in Peripheral Blood and the Pancreatic Islets Are Distinct [CLINICAL IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:04 PST

Type 1 diabetes is an autoimmune disease mediated by β cell-specific CD4⁺ and CD8⁺ T cells. Tracking β cell-specific T cells is one approach to monitor the diabetogenic response in at risk or diabetic individuals. Such analyses, however, are limited to PBL because T cells infiltrating the pancreatic islets are normally inaccessible. A key issue is whether peripheral β cell-specific T cells accurately reflect those cells infiltrating the target tissue. We investigated the properties of CD4⁺ T cells specific for a mimetic epitope recognized by the BDC2.5 clonotypic TCR in NOD mice. Soluble IA^g7-Ig (sIA^g7-Ig) multimer complexes covalently linked to a mimetic BDC peptide (sIA^g7-mBDC) were used to identify or isolate CD4⁺ T cells from PBL and the islets of NOD mice. A temporal increase in sIA^g7-mBDC binding (g7-mBDC⁺) T cells corresponding with the progression of β cell autoimmunity was detected in both PBL and islets in NOD female mice. In contrast to T cells in PBL, however, the majority of islet g7-mBDC⁺ T cells exhibited a type 1 phenotype, and mediated diabetes upon transfer into NOD.scid recipients. TCR-β and CDR-β gene usage of single islet-infiltrating g7-mBDC⁺ CD4⁺ T cells from individual NOD mice showed a restricted repertoire dominated by one or two clones typically expressing TCR β-chain variable TRBV-15. In contrast, a distinct and diverse TCR repertoire was detected for PBL-derived g7-mBDC⁺ T cells. These results demonstrate that PBL and islet CD4⁺ T cells specific for a given β cell epitope can differ regarding pathogenicity and TCR repertoire.

Immunomodulation of Nasal Epithelial Cells by Staphylococcus aureus-Derived Serine Proteases [CLINICAL IMMUNOLOGY]

Rudack, C., Sachse, F., Albert, N., Becker, K., von Eiff, C. — Wed, 18 Nov 2009 13:06:05 PST

The impact of Staphylococcus aureus in the pathogenesis of chronic rhinosinusitis is not well understood. Therefore, we investigated primary human nasal epithelial cell cultures for their ability to produce IL-8, growth-related oncogene-, and IL-6 via stimulation with trypsin and culture supernatants of different S. aureus strains and phenotypes. Inhibition of cytokine synthesis was performed using a glucocorticoid, a serine protease inhibitor, and a cysteine protease inhibitor. Finally, signal transduction pathways were analyzed by quantifying phosphorylated forms of MAPKs (PI3K, ERK, and p38) and DNA-binding assays that quantified NF-B and its inhibition using BAY11-7085. In vitro studies showed that the induction of IL-8, growth-related oncogene-, and IL-6 by S. aureus culture supernatants was significantly inhibited by the serine protease inhibitor. In contrast, steroids and the cysteine protease inhibitor had little effect. Activation of NF-B was observed after cell treatment with trypsin and bacterial supernatants, and was inhibited by BAY11-7085 and the serine protease inhibitor. S. aureus serine proteases were identified to modulate chemokine synthesis and activate NF-B in nasal epithelial cells, and may therefore be relevant for the pathophysiology of chronic rhinosinusitis.

CD39+Foxp3+ Regulatory T Cells Suppress Pathogenic Th17 Cells and Are Impaired in Multiple Sclerosis [CLINICAL IMMUNOLOGY]

Wed, 18 Nov 2009 13:06:05 PST

Despite the fact that CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg cells) play a central role in maintaining self-tolerance and that IL-17-producing CD4⁺ T cells (Th17 cells) are pathogenic in many autoimmune diseases, evidence to date has indicated that Th17 cells are resistant to suppression by human Foxp3⁺ Treg cells. It was recently demonstrated that CD39, an ectonucleotidase which hydrolyzes ATP, is expressed on a subset of human natural Treg cells. We found that although both CD4⁺CD25^highCD39⁺ and CD4⁺CD25^highCD39^– T cells suppressed proliferation and IFN- production by responder T cells, only the CD4⁺CD25^highCD39⁺, which were predominantly FoxP3⁺, suppressed IL-17 production, whereas CD4⁺CD25^highCD39^– T cells produced IL-17. An examination of T cells from multiple sclerosis patients revealed a normal frequency of CD4⁺CD25⁺CD127^lowFoxP3⁺, but interestingly a deficit in the relative frequency and the suppressive function of CD4⁺CD25⁺CD127^lowFoxP3⁺CD39⁺ Treg cells. The mechanism of suppression by CD39⁺ Treg cells appears to require cell contact and can be duplicated by adenosine, which is produced from ATP by the ectonucleotidases CD39 and CD73. Our findings suggest that CD4⁺CD25⁺Foxp3⁺CD39⁺ Treg cells play an important role in constraining pathogenic Th17 cells and their reduction in multiple sclerosis patients might lead to an inability to control IL-17 mediated autoimmune inflammation.

HIV-1 Nef promotes endocytosis of cell surface MHC class II molecules via a constitutive pathway [CORRECTIONS]

Wed, 18 Nov 2009 13:06:05 PST

Adenovirus vector-induced immune responses in nonhuman primates: responses to prime boost regimens [CORRECTIONS]

Wed, 18 Nov 2009 13:06:05 PST

IN THIS ISSUE [IN THIS ISSUE]

Wed, 04 Nov 2009 13:05:37 PST

Comment on "Ym1/2 Promotes Th2 Cytokine Expression by Inhibiting 12/15(S)-Lipoxygenase: Identification of a Novel Pathway for Regulating Allergic Inflammation" [LETTERS TO THE EDITOR]

Mabalirajan, U., Agrawal, A., Ghosh, B. — Wed, 04 Nov 2009 13:05:38 PST

Response to Comment on "Ym1/2 Promotes Th2 Cytokine Expression by Inhibiting 12/15(S)-Lipoxygenaes: Identification of a Novel Pathway for Regulating Allergic Inflammation" [LETTERS TO THE EDITOR]

Webb, D. C. — Wed, 04 Nov 2009 13:05:38 PST

Comment on "Reduced Cytotoxic Function of Effector CD8+ T Cells Is Responsible for Indoleamine 2,3-Dioxygenase-Dependent Immune Suppression" [LETTERS TO THE EDITOR]

Sorensen, R. B., Straten, P. t., Andersen, M. H. — Wed, 04 Nov 2009 13:05:38 PST

Role of Gut Commensal Microflora in the Development of Experimental Autoimmune Encephalomyelitis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Mucosal tolerance has been considered a potentially important pathway for the treatment of autoimmune disease, including human multiple sclerosis and experimental conditions such as experimental autoimmune encephalomyelitis (EAE). There is limited information on the capacity of commensal gut bacteria to induce and maintain peripheral immune tolerance. Inbred SJL and C57BL/6 mice were treated orally with a broad spectrum of antibiotics to reduce gut microflora. Reduction of gut commensal bacteria impaired the development of EAE. Intraperitoneal antibiotic-treated mice showed no significant decline in the gut microflora and developed EAE similar to untreated mice, suggesting that reduction in disease activity was related to alterations in the gut bacterial population. Protection was associated with a reduction of proinflammatory cytokines and increases in IL-10 and IL-13. Adoptive transfer of low numbers of IL-10-producing CD25⁺CD4⁺ T cells (>75% FoxP3⁺) purified from cervical lymph nodes of commensal bacteria reduced mice and in vivo neutralization of CD25⁺ cells suggested the role of regulatory T cells maintaining peripheral immune homeostasis. Our data demonstrate that antibiotic modification of gut commensal bacteria can modulate peripheral immune tolerance that can protect against EAE. This approach may offer a new therapeutic paradigm in the treatment of multiple sclerosis and perhaps other autoimmune conditions.

TNFR2-Deficient Memory CD8 T Cells Provide Superior Protection against Tumor Cell Growth [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kim, E. Y., Teh, S.-J., Yang, J., Chow, M. T., Teh, H.-S. — Wed, 04 Nov 2009 13:05:38 PST

TNF receptor-2 (TNFR2) plays a critical role in promoting the activation and survival of naive T cells during the primary response. Interestingly, anti-CD3 plus IL-2 activated TNFR2^–/– CD8 T cells are highly resistant to activation-induced cell death (AICD), which correlates with high expression levels of prosurvival molecules such as Bcl-2, survivin, and CD127 (IL-7R). We determined whether the resistance of activated TNFR2^–/– CD8 T cells to AICD contributes to more effective protection against tumor cell growth. We found that during a primary tumor challenge, despite initial inferiority in controlling tumor cell growth, TNFR2^–/– mice were able to more effectively control tumor burden over time compared with wild-type (WT) mice. Furthermore, vaccination of TNFR2^–/– mice with recombinant Listeria monocytogenes that express OVA confers better protection against the growth of OVA-expressing E.G7 tumor cells relative to similarly vaccinated WT mice. The enhanced protection against tumor cell growth was not due to more effective activation of OVA-specific memory CD8 T cells in vaccinated TNFR2^–/– mice. In vitro studies indicate that optimally activated OVA-specific TNFR2^–/– CD8 T cells proliferated to the same extent and possess similar cytotoxicity against E.G7 tumor cells as WT CD8 T cells. However, relative to WT cells, activated OVA-specific TNFR2^–/– CD8 T cells were highly resistant to AICD. Thus, the enhanced protection against E.G7 in TNFR2^–/– mice is likely due to the recruitment and activation of OVA-specific memory TNFR2^–/– CD8 T cells and their prolonged survival at the tumor site.

Dendritic Cell Function in Allostimulation Is Modulated by C5aR Signaling [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Regulation of T cell immunity by C5a has been suggested from recent studies. However, the underlying mechanisms, particularly the involved cells and biochemical basis, are not well defined. In this study, the direct modulation of dendritic cell (DC) activation and its function in T cell stimulation by C5a-C5aR interaction and the involved signaling pathways were investigated. We show that DCs from C5aR^–/– mice and normal DCs treated with C5aR antagonist have less-activated phenotype characterized with increased IL-10 and decreased IL-12p70 production in response to LPS stimulation, lowered surface expression of MHC class II, B7.2, and consequently have reduced capacity to stimulate allospecific T cells. Conversely, C5a stimulation up-regulates DC activation and its function in allostimulation. Furthermore, stimulation of C5aR mediates the inhibition of cAMP production and protein kinase A activity and is involved in activation of PI3K/AKT and NF-B signaling in DCs. These results demonstrate that C5a acts directly on C5aR expressed on DCs resulting in the cell activation and subsequently enhances its capacity for allospecific T cell stimulation. It also suggests that NF-B signaling induced by down-regulation of cAMP/ protein kinase A pathway and up-regulation of PI3K/AKT pathway following C5a stimulation may contribute to up-regulation of DC function.

Analysis of the Role of Tripeptidyl Peptidase II in MHC Class I Antigen Presentation In Vivo [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kawahara, M., York, I. A., Hearn, A., Farfan, D., Rock, K. L. — Wed, 04 Nov 2009 13:05:38 PST

Previous experiments using enzyme inhibitors and RNA interference in cell lysates and cultured cells have suggested that tripeptidyl peptidase II (TPPII) plays a role in creating and destroying MHC class I-presented peptides. However, its precise contribution to these processes has been controversial. To elucidate the importance of TPPII in MHC class I Ag presentation, we analyzed TPPII-deficient gene-trapped mice and cell lines from these animals. In these mice, the expression level of TPPII was reduced by >90% compared with wild-type mice. Thymocytes from TPPII gene-trapped mice displayed more MHC class I on the cell surface, suggesting that TPPII normally limits Ag presentation by destroying peptides overall. TPPII gene-trapped mice responded as well as did wild-type mice to four epitopes from lymphocytic choriomeningitis virus. The processing and presentation of peptide precursors with long N-terminal extensions in TPPII gene-trapped embryonic fibroblasts was modestly reduced, but in vivo immunization with recombinant lentiviral or vaccinia virus vectors revealed that such peptide precursors induced an equivalent CD8 T cell response in wild-type and TPPII-deficient mice. These data indicate that while TPPII contributes to the trimming of peptides with very long N-terminal extensions, TPPII is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags in vivo.

Immunostimulatory RNA Oligonucleotides Induce an Effective Antitumoral NK Cell Response through the TLR7 [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

RNA oligonucleotides containing immune-activating sequences promote the development of cytotoxic T cell and B cell responses to Ag. In this study, we show for the first time that immunostimulatory RNA oligonucleotides induce a NK cell response that prevents growth of NK-sensitive tumors. Treatment of mice with immunostimulatory RNA oligonucleotides activates NK cells in a sequence-dependent manner, leading to enhanced IFN- production and increased cytotoxicity. Use of gene-deficient mice showed that NK activation is entirely TLR7-dependent. We further demonstrate that NK activation is indirectly induced through IL-12 and type I IFN production by dendritic cells. Reconstitution of TLR7-deficient mice with wild-type dendritic cells restores NK activation upon treatment with immunostimulatory RNA oligonucleotides. Thus, by activating both NK cells and CTLs, RNA oligonucleotides stimulate two major cellular effectors of antitumor immunity. This dual activation may enhance the efficacy of immunotherapeutic strategies against cancer by preventing the development of tumor immune escape variants.

Cupressaceae Pollen Grains Modulate Dendritic Cell Response and Exhibit IgE-Inducing Adjuvant Activity In Vivo [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Pollen is considered a source of not only allergens but also immunomodulatory substances, which could play crucial roles in sensitization and/or the exacerbation of allergies. We investigated how allergenic pollens from different plant species (Japanese cedar and Japanese cypress, which belong to the Cupressaceae family, and birch, ragweed, and grass) modulate murine bone marrow-derived dendritic cell (DC) responses and examined the effect of Cupressaceae pollen in vivo using mice. DCs were stimulated with pollen extracts or grains in the presence or absence of LPS. Cell maturation and cytokine production in DCs were analyzed by flow cytometry, ELISA, and/or quantitative PCR. Pollen extracts suppressed LPS-induced IL-12 production and the effect was greatest for birch and grass. Without LPS, pollen grains induced DC maturation and cytokine production without IL-12 secretion and the response, for which TLR 4 was dispensable, was greatest for the Cupressaceae family. Intranasal administration of Cupressaceae pollen in mice induced an elevation of serum IgE levels and airway eosinophil infiltration. Coadministration of ovalbumin with Cupressaceae pollen grains induced ovalbumin-specific IgE responses associated with eosinophil infiltration. The results suggest that modulation of DC responses by pollen differs among the plant families via (1) the promotion of DC maturation and cytokine production by direct contact and/or (2) the inhibition of IL-12 production by soluble factors. The strong DC stimulatory activity in vitro and IgE-inducing activity in mice support the clinical relevance of Cupressaceae pollen to allergies in humans.

Anergic T Cells Are Metabolically Anergic [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zheng, Y., Delgoffe, G. M., Meyer, C. F., Chan, W., Powell, J. D. — Wed, 04 Nov 2009 13:05:38 PST

Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function. In this report we demonstrate that anergic T cells are metabolically anergic, in that upon full stimulation (signals 1 plus 2) they fail to up-regulate the machinery necessary to support increased metabolism. These findings suggest that one mechanism responsible for the maintenance of T cell anergy is failure to up-regulate the metabolic machinery. Furthermore, we demonstrate that by blocking leucine, glucose, and energy metabolism, T cell activation is mitigated. Additionally, inhibition of these metabolic pathways during T cell activation leads to anergy in Th1-differentiated cells. Overall, our findings extend the role of T cell metabolism in regulating T cell function.

Ligand Binding but Undetected Functional Response of FcR after Their Capture by T Cells via Trogocytosis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Intercellular transfer of cell surface proteins by trogocytosis is common and could affect T cell responses. Yet, the role of trogocytosis in T cell function is still elusive, and it is unknown whether a molecule, once captured by T cells, harbors the same biological properties as in donor APC. In this study, we showed that FcR as well as the associated FcR subunit could be detected at high levels on murine and human T cells after their intercellular transfer from FcR-expressing APC. Capture of FcR occurred during coculture of T cells with FcR-expressing APC upon Ab- or Ag-mediated T cell stimulation. Once captured by T cells, FcR were expressed in a conformation compatible with physiological function and conferred upon T cells the ability to bind immune complexes and to provision B cells with this source of Ag. However, we were unable to detect downstream signal or signaling-dependent function following the stimulation of FcR captured by T cells, and biochemical studies suggested the improper integration of FcR in the recipient T cell membrane. Thus, our study demonstrates that T cells capture FcR that can efficiently exert ligand-binding activity, which, per se, could have functional consequences in T cell-B cell cooperation.

Activation-Induced CD154 Expression Abrogates Tolerance Induced by Apoptotic Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Gurung, P., Kucaba, T. A., Ferguson, T. A., Griffith, T. S. — Wed, 04 Nov 2009 13:05:38 PST

The decision to generate a productive immune response or tolerance often depends on the context in which T cells first see Ag. Using a classical system of tolerance induction, we examined the immunological consequence of Ag encountered in the presence of naive or activated apoptotic cells. Naive apoptotic cells induced tolerance when injected i.v.; however, previously activated apoptotic cells induced immunity. Further analysis revealed a key role for CD154, as tolerance resulted after i.v. injection of either naive or activated apoptotic CD154^–/– T cells, while coinjection of an agonistic anti-CD40 mAb with naive apoptotic T cells induced robust immunity. Dendritic cells fed activated apoptotic T cells in vitro produced IL-12p40 in a CD154-dependent manner, and the use of IL-12p40^–/– mice or mAb-mediated neutralization of IL-12 revealed a link between CD154, IL-12, and the ability of activated apoptotic T cells to induce immunity rather than tolerance. Collectively, these results show that CD154 expression on apoptotic T cells can determine the outcome of an immune response to Ag recognized within the context of the apoptotic cells and suggest that the balance between naive and activated apoptotic T cells may dictate whether a productive immune response is encouraged.

Protein Kinase B/Akt Signals Impair Th17 Differentiation and Support Natural Regulatory T Cell Function and Induced Regulatory T Cell Formation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:38 PST

Protein kinase B (PKB)/Akt signals control T cell proliferation and differentiation but their effect on the generation and function of regulatory T cells (Treg) and Th17 cells is not well understood. In this study, we show that elevated PKB signals antagonize the immunosuppressive effect of TGF-β1 on cell size, CD25 and CD98 expression, and proliferation of CD3-stimulated naive CD4⁺ T cells from wild-type and CD28-deficient mice. Conventional CD4⁺ T cells expressing active PKB are less susceptible to suppression by natural regulatory T cells. Although PKB signals do not affect the development of natural regulatory T cells, they enhance their suppressor capacity. Upon TCR triggering and TGF-β1 costimulation, wild-type and CD28-deficient CD4⁺ T cells transgenic for PKB readily express Foxp3, thereby acquiring suppressor capacity. These effects of elevated PKB signals on T cell function involve a marked and sustained activation of STAT5 and Foxp3 and reduction in nuclear NFATc1 levels. In contrast, PKB signals impair TGF-β1/IL-6-mediated differentiation of naive CD4⁺ T cells into the Th17 lineage. This correlates with an increased signaling of ERK, STAT5, and STAT6. Finally, elevated PKB signals reduced the severity of experimental autoimmune encephalomyelitis in wild-type mice but induced experimental autoimmune encephalomyelitis in mice deficient for CD28. Altogether, these data indicate an important role of PKB signals on control of TGF-β1-mediated T cell responses and, thereby, on tolerizing and inflammatory immune processes.

Thymic Regulation of Autoimmune Disease by Accelerated Differentiation of Foxp3+ Regulatory T Cells through IL-7 Signaling Pathway [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Chen, X., Fang, L., Song, S., Guo, T. B., Liu, A., Zhang, J. Z. — Wed, 04 Nov 2009 13:05:38 PST

The exact role of adult thymus in autoimmune disease state is poorly understood. We show here that thymus regulated experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, as evidenced by loss of spontaneous recovery in thymectomized EAE mice. There was progressive enrichment for CD4 single-positive Foxp3⁺ regulatory T cells in thymocytes during the course of EAE and they suppressed the disease when adoptively transferred. Thymus was shown to undergo an active process characterized by accelerated differentiation and proliferation of regulatory T (Treg) cells through a mechanism involving increased expression of IL-7 in stromal cells and dynamic expression of IL-7 receptor in thymic Treg cells. This process preceded EAE recovery and selectively affected Treg over non-Treg cells in the thymus, leading to increased output of thymic Treg cells and self-regulation of EAE. The study reveals a novel role of thymus in self-regulation of autoimmune condition.

The Proteasome Inhibitor Bortezomib Enhances the Susceptibility to Viral Infection [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Basler, M., Lauer, C., Beck, U., Groettrup, M. — Wed, 04 Nov 2009 13:05:38 PST

The proteasome, a multicatalytic protease, is responsible for the generation of most MHC class I ligands. Bortezomib, a proteasome inhibitor, is clinically approved for treatment of multiple myeloma and mantle cell myeloma. In the present study, we investigated the effect of bortezomib on viral infection. Infection of bortezomib-treated mice with the lymphocytic choriomeningitis virus (LCMV) led to a decreased cytotoxic T cell response to several LCMV-derived CD8⁺ T cell epitopes. Bortezomib treatment caused a reduced expansion of CD8⁺ T lymphocytes and increased viral titers in LCMV-infected mice. Administration of bortezomib during expansion of CD8⁺ T cells had no influence on the cytotoxic T cell response, suggesting that bortezomib interferes with priming of naive T cells. Indeed, determination of Ag load in spleen 4 days post infection, revealed a reduced presentation of LCMV-derived cytotoxic T cell epitopes on MHC class I molecules. In summary, we show that proteasome inhibition with bortezomib led to an increased susceptibility to viral infection, and demonstrate for the first time, that proteasome inhibitors can alter Ag processing in vivo.

A Major Role for the Minor Capsid Protein of Human Papillomavirus Type 16 in Immune Escape [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Fahey, L. M., Raff, A. B., Da Silva, D. M., Kast, W. M. — Wed, 04 Nov 2009 13:05:39 PST

High-risk human papillomavirus (HPV) infection of the cervical epithelium is causally linked with the generation of cervical cancer. HPV does not activate Langerhans cells (LC), the APC at the site of infection, leading to immune evasion. The HPV protein responsible for inducing this immune escape has not been determined. We demonstrate that LC exposed to the minor capsid protein L2 in HPV16L1L2 virus-like particles do not phenotypically or functionally mature. However, HPV16L1 virus-like particles significantly induce activation of LC. Our data suggest that the L2 protein plays a specific role in the induction of this immune escape of HPV16 through the manipulation of LC. This novel function is the first immune modulating action attributed to the L2 protein and adds significantly to our understanding of the mechanism of HPV immune escape.

Human Follicular Lymphoma CD39+-Infiltrating T Cells Contribute to Adenosine-Mediated T Cell Hyporesponsiveness [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Our previous work has demonstrated that human follicular lymphoma (FL) infiltrating T cells are anergic, in part due to suppression by regulatory T cells. In this study, we identify pericellular adenosine, interacting with T cell-associated G protein-coupled A_2A/B adenosine receptors (AR), as contributing to FL T cell hyporesponsiveness. In a subset of FL patient samples, treatment of lymph node mononuclear cells (LNMC) with specific A_2A/B AR antagonists results in an increase in IFN- or IL-2 secretion upon anti-CD3/CD28 Ab stimulation, as compared with that seen without inhibitors. In contrast, treatment with an A₁ AR antagonist had no effect on cytokine secretion. As the rate limiting step for adenosine generation from pericellular ATP is the ecto-ATPase CD39, we next show that inhibition of CD39 activity using the inhibitor ARL 67156 partially overcomes T cell hyporesponsiveness in a subset of patient samples. Phenotypic characterization of LNMC demonstrates populations of CD39-expressing CD4⁺ and CD8⁺ T cells, which are overrepresented in FL as compared with that seen in normal or reactive nodes, or normal peripheral blood. Thirty percent of the FL CD4⁺CD39⁺ T cells coexpress CD25^high and FOXP3 (consistent with regulatory T cells). Finally, FL or normal LNMC hydrolyze ATP in vitro, in a dose- and time-dependent fashion, with the rate of ATP consumption being associated with the degree of CD39⁺ T cell infiltration. Together, these results support the finding that the ATP-ectonucleotidase-adenosine system mediates T cell anergy in a human tumor. In addition, these studies suggest that the A_2A/B AR as well as CD39 are novel pharmacological targets for augmenting cancer immunotherapy.

Human Late Memory CD8+ T Cells Have a Distinct Cytokine Signature Characterized by CC Chemokine Production without IL-2 Production [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Late memory T cell skewing is observed in the setting of immune recovery after cord blood transplantation, and may be associated with inferior control of viral reactivation and cancers. Therefore, we sought to understand how late memory cells differ functionally from earlier stage memory T cells, and whether surface phenotypes associated with differentiation stages were predictably associated with functional signatures. Higher order cytokine flow cytometry allows characterization of human T cells based on complex phenotypic markers and their differential capacity to simultaneously secrete effector proteins, including cytokines and chemokines. We used 8-color, 10-parameter cytokine flow cytometry to characterize the functional activation of human late memory CD8⁺ T cells defined by CD45RA and CD27 expression (CD27^–CD45RA⁺). We assessed the 15 possible functional signatures of cells defined by production of IL-2, IFN-, TNF-, and MIP-1β alone or in combination, following activation with Ags stimulating bypassing surface proteins (PMA:ionomycin) or through the TCR (e.g., viral Ags). Late memory CD8⁺ T cells produced abundant amounts of CC chemokines (MIP-1β, MIP-1, and RANTES) but not IL-2. IL-2/IFN- coproduction, characteristic of protective immune responses to viral infections, was absent in late memory CD8⁺ T cells. These data demonstrate that functional cytokine signatures are predictably associated with CD8⁺ maturation stages, and that the polarization of late memory CD8⁺ T cells toward CC chemokine production and away from IL-2 production suggests a unique functional role for this subset.

C1q Differentially Modulates Phagocytosis and Cytokine Responses during Ingestion of Apoptotic Cells by Human Monocytes, Macrophages, and Dendritic Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Fraser, D. A., Laust, A. K., Nelson, E. L., Tenner, A. J. — Wed, 04 Nov 2009 13:05:39 PST

C1q, the first component of the classical complement pathway, is also a pattern recognition receptor involved in the recognition and clearance of apoptotic cells. C1q deficiency in humans leads to development of lupus-like autoimmune disease, and it has been speculated that impaired clearance of apoptotic cells may contribute to disease development. Since phagocytes initiate specific and appropriate immune responses as a result of initial ligand-receptor interactions, regulation of gene expression by C1q may also contribute to the sculpting of an immune response to the ingested "self-Ags." In this study, the role of C1q in apoptotic cell clearance and subsequent modulation of cytokine release by phagocytes was assessed including donor matched human monocytes, monocyte-derived macrophages (HMDMs), and dendritic cells (DCs). First, C1q binding is much greater to late compared with early apoptotic cells. Second, C1q binding to apoptotic cells significantly enhanced the levels of ingestion by monocytes but had no effect on HMDM and DC uptake. Third, in the presence of serum, C1q bound to apoptotic cells, activated the complement pathway, leading to C3b deposition, and enhancement of uptake of apoptotic cells by monocytes, HMDMs, and DCs. Finally, although C1q, either immobilized on a plate or bound to apoptotic cells, modulates the LPS-induced cytokine levels released by human monocytes, HMDMs, and DCs toward a more limited immune response, both the degree and direction of modulation differed significantly depending on the differentiation state of the phagocyte, providing further evidence of the integration of these cell- and environment-specific signals in determining appropriate immune responses.

AS04, an Aluminum Salt- and TLR4 Agonist-Based Adjuvant System, Induces a Transient Localized Innate Immune Response Leading to Enhanced Adaptive Immunity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4'-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-B activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4⁺ T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

Mcl-1-Mediated Impairment of the Intrinsic Apoptosis Pathway in Circulating Neutrophils from Critically Ill Patients Can Be Overcome by Fas Stimulation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

Critical Role of TLR2 and TLR4 in Autoantibody Production and Glomerulonephritis in lpr Mutation-Induced Mouse Lupus [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathogenic autoantibodies directed against nuclear Ags and immune complex deposits in damaged organs. Environmental factors have been thought to play a role in the onset of the disease. The recognition of these factors is mediated by TLRs, in particular TLR2 and TLR4 which bind pathogen-associated molecular patterns of Gram⁺ and Gram^– bacteria, respectively. We attempted to determine the role of these TLRs in SLE by creating TLR2- or TLR4-deficient C57BL/6^lpr/lpr mice. These mice developed a less severe disease and fewer immunological alterations. Indeed, in C57BL/6^lpr/lpr-TLR2 or -TLR4-deficient mice, glomerular IgG deposits and mesangial cell proliferation were dramatically decreased and antinuclear, anti-dsDNA, and anti-cardiolipin autoantibody titers were significantly reduced. However, the response against nucleosome remained unaffected, indicating a role of TLR2 and TLR4 in the production of Abs directed against only certain categories of SLE-related autoantigens. Analysis of B cell phenotype showed a significant reduction of marginal zone B cells, particularly in C57BL/6^lpr/lpr-TLR4-deficient mice, suggesting an important role of TLR4 in the sustained activation of these cells likely involved in autoantibody production. Interestingly, the lack of TLR4 also affected the production of cytokines involved in the development of lupus disease.

IL-27 Directly Restrains Lung Tumorigenicity by Suppressing Cyclooxygenase-2-Mediated Activities [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Ho, M.-Y., Leu, S.-J. J., Sun, G.-H., Tao, M.-H., Tang, S.-J., Sun, K.-H. — Wed, 04 Nov 2009 13:05:39 PST

Gene transfer of IL-27 to tumor cells has been proven to inhibit tumor growth in vivo by antiproliferation, antiangiogenesis, and stimulation of immunoprotection. To investigate the nonimmune mechanism of IL-27 that suppresses lung cancer growth, we have established a single-chain IL-27-transduced murine Lewis lung carcinoma (LLC-1) cell line (LLC-1/scIL-27) to evaluate its tumorigenic potential in vivo. Mice inoculated with LLC/scIL-27 displayed retardation of tumor growth. Production of IL-12, IFN-, and cytotoxic T cell activity against LLC-1 was manifest in LLC/scIL-27-injected mice. Of note, LLC-1/scIL-27 exhibited decreased expression of cyclooxygenase-2 (COX-2) and PGE₂. On the cellular level, the LLC/scIL-27 transfectants had reduced malignancy, including down-regulation of vimentin expression and reduction of cellular migration and invasion. The suppression of tumorigenesis by IL-27 on lung cancer cells was further confirmed by the treatment with rIL-27 on the murine LLC-1 and human non-small cell lung carcinoma (NSCLC) cell lines. PGE₂-induced vimentin expression, movement, and invasiveness were also suppressed by the treatment with rIL-27. Our data show that IL-27 not only suppresses expression of COX-2 and PGE₂ but also decreases the levels of vimentin and the abilities of cellular migration and invasion. Furthermore, inoculation of LLC/scIL-27 into immunodeficient NOD/SCID mice also exhibited reduced tumor growth. Our data indicate that IL-27-induced nonimmune responses can contribute to significant antitumor effects. Taken together, the results suggest that IL-27 may serve as an effective agent for lung cancer therapy in the future.

A NUP98-HOXD13 Fusion Gene Impairs Differentiation of B and T Lymphocytes and Leads to Expansion of Thymocytes with Partial TCRB Gene Rearrangement [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Choi, C. W., Chung, Y. J., Slape, C., Aplan, P. D. — Wed, 04 Nov 2009 13:05:39 PST

Expression of a NUP98-HOXD13 (NHD13) fusion gene leads to myelodysplastic syndrome in mice. In addition to ineffective hematopoiesis, we observed that NHD13 mice were lymphopenic; the lymphopenia was due to a decrease in both T and B lymphocytes. Although the pro-B cell (B220⁺/CD43⁺) populations from the NHD13 and wild-type mice were similar, the NHD13 mice showed decreased pre-B cells (B220⁺/CD43^–), indicating impaired differentiation at the pro-B to pre-B stage. Thymi from NHD13 mice were smaller and overexpressed Hoxa cluster genes, including Hoxa7, Hoxa9, and Hoxa10. In addition, the NHD13 thymi contained fewer thymocytes, with an increased percentage of CD4^–/CD8^– (double-negative (DN)) cells and a decreased percentage of CD4⁺/CD8⁺ (double-positive) cells; the DN1/DN2 population was increased and the DN3/DN4 population was decreased, suggesting a partial block at the DN2 to DN3 transition. To determine clonality of the thymocytes, we used degenerate RT-PCR to identify clonal Tcrb gene rearrangements. Five of six NHD13 thymi showed an unusual Tcrb gene rearrangement pattern with common, clonal DJ rearrangements, but distinct V-D junctions, suggesting a marked clonal expansion of thymocytes that had undergone a DJ rearrangement, but not completed a VDJ rearrangement. Taken together, these findings demonstrate that expression of the NHD13 transgene inhibits lymphoid as well as myeloid and erythroid differentiation, results in overexpression of Hoxa cluster genes, and leads to a precursor T cell lymphoblastic leukemia/lymphoma.

Regulation of Airway MUC5AC Expression by IL-1{beta} and IL-17A; the NF-{kappa}B Paradigm [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Fujisawa, T., Velichko, S., Thai, P., Hung, L.-Y., Huang, F., Wu, R. — Wed, 04 Nov 2009 13:05:39 PST

Mucin over-production is one of the hallmarks of chronic airway diseases such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis. NF-B activation in airway epithelial cells has been shown to play a positive inflammatory role in chronic airway diseases; however, the role of NF-B in mucin gene expression is unresolved. In this study, we have shown that the proinflammatory cytokines, IL-1β and IL-17A, both of which utilize the NF-B pathway, are potent inducers of mucin (MUC)5AC mRNA and protein synthesis by both well-differentiated primary normal human bronchial epithelial cells and the human bronchial epithelial cell line, HBE1. MUC5AC induction by these cytokines was both time- and dose-dependent and occurred at the level of promoter activation, as measured by a reporter gene assay. These effects were attenuated by the small molecule inhibitor NF-B inhibitor III, as well as p65 small-interfering RNA, suggesting that the regulation of MUC5AC expression by these cytokines is via an NF-B-based transcriptional mechanism. Further investigation of the promoter region identified a putative NF-B binding site at position-3594/-3582 in the promoter of MUC5AC as critical for the regulation of MUC5AC expression by both IL-1β and IL-17A. Chromatin immunoprecipitation analysis confirmed enhanced binding of the NF-B subunit p50 to this region following cytokine stimulation. We conclude that an NF-B-based transcriptional mechanism is involved in MUC5AC regulation by IL-1β and IL-17A in the airway epithelium. This is the first demonstration of the participation of NF-B and its specific binding site in cytokine-mediated airway MUC5AC expression.

HMGB1 Enhances the Proinflammatory Activity of Lipopolysaccharide by Promoting the Phosphorylation of MAPK p38 through Receptor for Advanced Glycation End Products [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Qin, Y.-H., Dai, S.-M., Tang, G.-S., Zhang, J., Ren, D., Wang, Z.-W., Shen, Q. — Wed, 04 Nov 2009 13:05:39 PST

High mobility group box-1 (HMGB1) protein was originally characterized as a nuclear DNA-binding protein, and was described to have an extracellular role when involved in cellular activation and proinflammatory responses. In the present study, we have found that the proinflammatory activity of recombinant HMGB1 proteins is determined by the containing endotoxin level, and HMGB1 that contains few endotoxins fails to stimulate macrophages to secrete proinflammatory cytokines. HMGB1 acts as a ligand of receptor for advanced glycation end products (RAGE) and works in synergy with LPS in activating the macrophages in vitro. In vivo, intra-articular injections of HMGB1 act in synergy with LPS to induce experimental arthritis in mice. HMGB1 promotes the phosphorylation of MAPK p38 and the activation of NF-B through RAGE, and then enhances the expression of proinflammatory cytokines. These results demonstrate that HMGB1 enhances the proinflammatory activity of LPS by promoting the phosphorylation of MAPK p38 and by the activation of NF-B through RAGE.

The Bile Acid Receptor FXR Is a Modulator of Intestinal Innate Immunity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Vavassori, P., Mencarelli, A., Renga, B., Distrutti, E., Fiorucci, S. — Wed, 04 Nov 2009 13:05:39 PST

The farnesoid X receptor (FXR) is a bile acid-regulated nuclear receptor expressed in enterohepatic tissues. In this study we investigated whether FXR is expressed by cells of innate immunity and regulates inflammation in animal models of colitis. Acute (7 days) and chronic (8 wk) colitis were induced in wild-type and FXR^–/– mice by intrarectal administration of trinitrobenzensulfonic acid or by 7-day administration of 5% dextran sulfate in drinking water. The results of this experiment demonstrate that FXR is expressed by and exerts counterregulatory effects on cells of innate immunity. Exposure of LPS-activated macrophages to 6-ethyl chenodeoxycholic acid (6E-CDCA; INT-747) a synthetic FXR ligand, results in a reciprocal regulation of NF-B dependent-genes (TNF-, IL-1β, IL-6, COX-1, COX-2, and iNOS) and induction of SHP, a FXR-regulated gene. FXR activation stabilizes the nuclear corepressor NCoR on the NF-B responsive element on the IL-1β promoter. Colon inflammation in Crohn’s disease patients and in rodent models of colitis is associated with a reduced expression of FXR mRNA. Using two rodent models of colon inflammation, we show that progression of these immune-mediated disorders is exacerbated in FXR^–/– mice (p < 0.01). In vivo treatment with INT-747 attenuates organ injury and immune cell activation. FXR activation increased the colon expression of I-BABP, FXR, and SHP while reducing IL-1β, IL-2, IL-6, TNF-, and IFN- mRNA expression and attenuating disease severity. In aggregate, these findings provide evidence that FXR is an essential component of a network of nuclear receptors that regulate intestinal innate immunity and homeostasis.

Differential Cytokine Production and Bystander Activation of Autoreactive B Cells in Response to CpG-A and CpG-B Oligonucleotides [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Synthetic oligonucleotides containing CpG motifs have been shown to induce proliferation, differentiation, and cytokine production in B cells, macrophages, and dendritic cells through a TLR9-dependent mechanism. A class (CpG-A) and B class (CpG-B) oligonucleotides display distinct physical properties. CpG-A, but not CpG-B, can multimerize to form exceedingly large lattices. CpG-A cannot effectively activate B cells but does induce plasmacytoid dendritic cells to produce high levels of IFN, while CpG-B is a potent B cell mitogen. In this study, we report that CpG-A is internalized by B cells, and CpG-A and CpG-B accumulate in distinct intracellular compartments. When present in the form of an immune complex (CpG-A IC), CpG-A is taken up more efficiently by AM14 IgG2a-specific B cells, and elicits a robust TLR9-dependent B cell proliferative response. B cells proliferating comparably and in a TLR9-dependent fashion in response to CpG-A IC and CpG-B exhibited distinct cytokine profiles. CpG-A IC induced enhanced production of RANTES and markedly reduced levels of IL-6 when compared with CpG-B. We also found that engagement of the AM14 BCR by a protein IC, which cannot by itself induce proliferation, promoted TLR9-dependent but BCR-independent proliferation by bystander CpG-A or fragments of mammalian dsDNA. These data identify direct and indirect mechanisms by which BCR engagement facilitates access of exogenous ligands to TLR9-associated compartments and subsequent B cell activation.

The PPE18 of Mycobacterium tuberculosis Interacts with TLR2 and Activates IL-10 Induction in Macrophage [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

The pathophysiological functions of proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family of proteins of Mycobacterium tuberculosis are not well understood. In this study, we demonstrate that one of the PPE proteins, PPE18 can stimulate macrophages to secrete IL-10, known to favor a Th2 type response. The recombinant PPE18 was found to specifically interact with the TLR2 leading to an early and sustained activation of p38 MAPK, which is critical for IL-10 induction. In silico docking analyses and mutation experiments indicate that PPE18 specifically interacts with the leucine rich repeat 11~15 domain of TLR2 and the site of interaction is different from that of a synthetic lipopeptide Pam₃CSK₄ known to activate predominantly ERK 1/2. When PMA-differentiated THP-1 macrophages were infected with a mutant Mycobacterium tuberculosis strain lacking the PPE18, produced poorer levels of IL-10 as compared with those infected with the wild-type strain. In contrast, an M. smegmatis strain overexpressing the PPE18 induced higher levels of IL-10 in infected macrophages. Our data indicate that the PPE18 protein may trigger an anti-inflammatory response by inducing IL-10 production.

CCR7-Dependent Stimulation of Survival in Dendritic Cells Involves Inhibition of GSK3{beta} [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Escribano, C., Delgado-Martin, C., Rodriguez-Fernandez, J. L. — Wed, 04 Nov 2009 13:05:39 PST

Chemokine receptor CCR7 regulates chemotaxis and survival in mature dendritic cells (DCs). We studied the role of glycogen synthase kinase-3β (GSK3β) in the regulation of CCR7-dependent survival. We show that GSK3β behaves as a proapoptotic regulator in cultured monocyte-derived human DCs and murine splenic DCs in vitro, and in lymph node DCs in vivo. In keeping with its prosurvival role, stimulation of CCR7 induced phosphorylation/inhibition of GSK3β, which was mediated by the prosurvival regulator Akt1, but it was independent of ERK1/2, a key regulator of chemotaxis. Stimulation of CCR7 also induced translocation of two transcription-factor targets of Akt, prosurvival NF-B and proapoptotic FOXO1, to the nucleus and cytosol, respectively, resulting in DCs with a phenotype more resistant to apoptotic stimuli. We analyzed if GSK3β was able to modulate the mobilizations of these transcription factors. Using pharmacological inhibitors, small interfering RNA, and a construct encoding constitutively active GSK3β, we show that active GSK3β fosters and hampers the translocations to the nucleus of FOXO and NF-B, respectively. Inhibition of GSK3β resulted in the degradation of the NF-B inhibitor IB, indicating a mechanism whereby GSK3 can control the translocation of NF-B to the nucleus. GSK3β and FOXO interacted in vivo, suggesting that this transcription factor could be a substrate of GSK3. The results provide a novel mechanism whereby active GSK3β contributes to regulate apoptosis in DCs. They also suggest that upon stimulation of CCR7, Akt-mediated phosphorylation/inhibition of GSK3β may be required to allow complete translocations of FOXO and NF-B that confer DCs an extended survival.

Localization of Kv1.3 Channels in the Immunological Synapse Modulates the Calcium Response to Antigen Stimulation in T Lymphocytes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

The immunological synapse (IS), a highly organized structure that forms at the point of contact between a T cell and an APC, is essential for the proper development of signaling events, including the Ca²⁺ response. Kv1.3 channels control Ca²⁺ homeostasis in human T cells and move into the IS upon Ag presentation. However, the process involved in channel accumulation in the IS and the functional implications of this localization are not yet known. Here we define the movement of Kv1.3 into the IS and study whether Kv1.3 localization into the IS influences Ca²⁺ signaling in Jurkat T cells. Crosslinking of the channel protein with an extracellular Ab limits Kv1.3 mobility and accumulation at the IS. Moreover, Kv1.3 recruitment to the IS does not involve the transport of newly synthesized channels and it does not occur through recycling of membrane channels. Kv1.3 localization in the IS modulates the Ca²⁺ response. Blockade of Kv1.3 movement into the IS by crosslinking significantly increases the amplitude of the Ca²⁺ response triggered by anti-CD3/anti-CD28-coated beads, which induce the formation of the IS. On the contrary, the Ca²⁺ response induced by TCR stimulation without the formation of the IS with soluble anti-CD3/anti-CD28 Abs is unaltered. The results presented herein indicate that, upon Ag presentation, membrane-incorporated Kv1.3 channels move along the plasma membrane to localize in the IS. This localization is important to control the amplitude of the Ca²⁺ response, and disruption of this process can account for alterations of downstream Ca²⁺-dependent signaling events.

IDO Mediates TLR9-Driven Protection from Experimental Autoimmune Diabetes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Originally predicated on the recognition of an increasing prevalence of allergy, the hygiene hypothesis was later found to accommodate the contrasting epidemiologic trends in developed countries for infectious vs autoimmune diseases. Experimentally, reduced exposure to infections will increase the risk of disease in several models of experimental autoimmunity. Although TLRs were initially considered as stimulatory molecules capable of activating early defense mechanisms against invading pathogens, emerging data suggest that they can also exert a regulatory function. In the present study, we evaluated whether TLR3 and TLR9, recognizing microbial dsDNA and CpG-containing DNA sequences, respectively, play a role in the protection from experimental autoimmune diabetes induced in C57BL/6 mice by streptozotocin. In wild-type animals, the disease was accompanied by up-regulation of IDO in pancreatic lymph nodes and would be greatly exacerbated by in vivo administration of an IDO inhibitor. Conversely, administration of a CpG-containing oligodeoxynucleotide greatly attenuated the disease in an IDO-dependent fashion. TLR9-, but not TLR3-deficient mice developed a more robust disease, an event accompanied by lack of IDO induction in pancreatic lymph nodes. Thus, our data suggest that the TLR9-IDO axis may represent a valuable target in the prevention/therapy of type 1 diabetes.

Mitochondrial Uncoupling Protein 2 Inhibits Mast Cell Activation and Reduces Histamine Content [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:39 PST

Mast cells are immune effector cells that are involved in allergies and inflammation through the release of mediators such as histamine, PGs, and cytokines. Uncoupling protein 2 (UCP2) is a mitochondrial protein that inhibits insulin secretion from β cells, possibly through down-regulation of reactive oxygen species production. We hypothesized that UCP2 could also regulate mast cell activation. In this study, we show that mouse bone marrow mast cells (BMMCs) and human leukemic LAD2 mast cells express UCP2. BMMCs from Ucp2^–^/^– mice exhibited greater histamine release, whereas overexpression of UCP2 in LAD2 cells reduced histamine release after both allergic and nonallergic triggers. Ucp2^–^/^– BMMCs also had elevated histamine content and histidine decarboxylase expression. Histamine content was reduced by overexpression of UCP2 or treatment with the mitochondrial-targeted superoxide dismutase-mimetic (TBAP) tetrakis(4-benzoic acid) porphyrin manganese(III). Furthermore, Ucp2^–^/^– BMMCs also had greater production of both IL-6 and PGD₂ as well as ERK phosphorylation, which is known to regulate PG synthesis. Intradermal administration of substance P, an activator of skin mast cells, and challenge with DNP-human serum albumin after passive sensitization induced significantly greater vascular permeability in the skin of Ucp2^–^/^– mice in vivo. Our results suggest that UCP2 can regulate mast cell activation.

Dec2 Promotes Th2 Cell Differentiation by Enhancing IL-2R Signaling [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Th cell differentiation is precisely regulated by thousands of genes at different stages. In the present study, we demonstrate that Dec2, a transcription factor belonging to the bHLH (basic helix-loop-helix) superfamily, is progressively induced during the course of Th2 differentiation, especially at the late stage. The up-regulated Dec2 can strongly promote Th2 development under Th2-inducing conditions, as evidenced by retrovirus-mediated gene transfer or transgenic manipulation. In addition, an enhancement of Th2 responses is also detectable in Dec2 transgenic mice in vivo. Conversely, RNA interference-mediated suppression of endogenous Dec2 could attenuate Th2 differentiation. Finally, we show that the enhanced Th2 development is at least in part due to substantial up-regulation of CD25 expression elicited by Dec2, thereby resulting in hyperresponsiveness to IL-2 stimulation.

Skin Melanoma Development in ret Transgenic Mice Despite the Depletion of CD25+Foxp3+ Regulatory T Cells in Lymphoid Organs [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) known to mediate self-tolerance were also shown to contribute to tumor progression. In mouse melanoma transplantation models, Treg depletion resulted in the stimulation of antitumor immune responses and tumor eradication. To study Treg in conditions close to the clinical situation, we used a ret transgenic mouse spontaneous melanoma model, which, in contrast to transplantation models, resembles human melanoma regarding clinical development. Significantly higher numbers of Treg were found in skin tumors and metastatic lymph nodes at early stages of melanoma progression compared with more advanced stages accompanied by the elevated CCR4 expression on Treg and higher production of its ligand CCL2 in tumor lesions. Numbers of tumor infiltrating Treg inversely correlated with Treg amounts in the bone marrow, suggesting their possible recruitment to melanoma lesions from this organ. The immunosuppressive function of Treg from transgenic tumor-bearing mice was similar to that from transgenic tumor-free mice or nontransgenic littermates. Although anti-CD25 mAb injections resulted in the efficient Treg depletion from lymphoid organs of transgenic mice, melanoma development was not significantly delayed. Furthermore, the treatment of mice with macroscopical tumors also failed to inhibit tumor progression, which correlated with the inability to deplete intratumoral Treg. We suggest that in the autochthonous melanoma genesis, other immunosuppressive cells could play an important role and replace immunosuppressive, tumor-promoting functions of Treg. Therefore, effective melanoma immunotherapy should include the inhibition of Treg migration into the tumor combined with neutralization of other immunosuppressive cells and factors in the tumor microenvironment.

Potent High-Affinity Antibodies for Treatment and Prophylaxis of Respiratory Syncytial Virus Derived from B Cells of Infected Patients [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Native human Abs represent attractive drug candidates; however, the low frequency of B cells expressing high-quality Abs has posed a barrier to discovery. Using a novel single-cell phenotyping technology, we have overcome this barrier to discover human Abs targeting the conserved but poorly immunogenic central motif of respiratory syncytial virus (RSV) G protein. For the entire cohort of 24 subjects with recent RSV infection, B cells producing Abs meeting these stringent specificity criteria were rare, <10 per million. Several of the newly cloned Abs bind to the RSV G protein central conserved motif with very high affinity (K_d 1–24 pM). Two of the Abs were characterized in detail and compared with palivizumab, a humanized mAb against the RSV F protein. Relative to palivizumab, the anti-G Abs showed improved viral neutralization potency in vitro and enhanced reduction of infectious virus in a prophylaxis mouse model. Furthermore, in a mouse model for postinfection treatment, both anti-G Abs were significantly more effective than palivizumab at reducing viral load. The combination of activity in mouse models for both prophylaxis and treatment makes these high-affinity human-derived Abs promising candidates for human clinical testing.

Regulatory T Cell (Treg) Subsets Return in Patients with Refractory Lupus following Stem Cell Transplantation, and TGF-{beta}-Producing CD8+ Treg Cells Are Associated with Immunological Remission of Lupus [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zhang, L., Bertucci, A. M., Ramsey-Goldman, R., Burt, R. K., Datta, S. K. — Wed, 04 Nov 2009 13:05:40 PST

Compared with conventional drug therapy, autologous hemopoietic stem cell transplantation (HSCT) can induce very-long-term remission in refractory lupus patients. Herein, we show that in posttransplant patients, both CD4⁺CD25^highFoxP3⁺ and an unusual CD8⁺FoxP3⁺ Treg subset return to levels seen in normal subjects; accompanied by almost complete inhibition of pathogenic T cell response to critical peptide autoepitopes from histones in nucleosomes, the major lupus autoantigen from apoptotic cells. In addition to a stably sustained elevation of FoxP3, posttransplant CD8 T cells also maintained markedly higher expression levels of latency-associated peptide (LAP), CD103, PD-1, PD-L1, and CTLA-4, as compared with pretransplant CD8 T cells that were identically treated by a one-time activation and rest in short-term culture. The posttransplant CD8 regulatory T cells (Treg) have autoantigen-specific and nonspecific suppressive activity, which is contact independent and predominantly TGF-β dependent. By contrast, the pretransplant CD8 T cells have helper activity, which is cell contact dependent. Although CD4⁺CD25^high Treg cells return during clinical remission of conventional drug-treated lupus, the posttransplant patient’s CD8 Treg cells are considerably more potent, and they are absent in drug-treated patients in whom CD4 T cell autoreactivity to nucleosomal epitopes persists even during clinical remission. Therefore, unlike conventional drug therapy, hemopoietic stem cell transplantation generates a newly differentiated population of LAP^highCD103^high CD8^TGF-β Treg cells, which repairs the Treg deficiency in human lupus to maintain patients in true immunological remission.

Generation of B Cell Memory to the Bacterial Polysaccharide {alpha}-1,3 Dextran [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Foote, J. B., Kearney, J. F. — Wed, 04 Nov 2009 13:05:40 PST

B1b B cells generate a novel form of memory and provide Ab mediated-protection to persisting bacterial pathogens. To understand how B1b B cells establish memory to polysaccharide Ags, we studied an oligoclonal B cell response to -1,3 dextran (DEX) expressed on Enterobacter cloacae. B cells specific for DEX enrich in the marginal zone (MZ) and B1b B cell populations. After E. cloacae immunization, MZ B cells were responsible for the generation of initial peak DEX-specific Ab titers, whereas, DEX-specific B1b B cells expanded and played an important role in boosted production of DEX-specific Ab titers upon E. cloacae rechallenge. Cell transfer experiments demonstrate that B1b B cells possess the capacity for both robust proliferation and plasma cell differentiation, thus distinguishing themselves from MZ B cells, which uniformly commit to plasma cell differentiation. These results define B1b B cells as the principal reservoir for memory to bacterial-associated polysaccharide Ags.

Mouse Mast Cell Protease 4 Is the Major Chymase in Murine Airways and Has a Protective Role in Allergic Airway Inflammation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

It is widely established that mast cells (MCs) have a harmful role in asthma, for example by secreting various proinflammatory substances stored within their secretory granule. However, in this study, we show that one of the substances stored within MC granule, chymase, in fact has a protective role in allergic airway inflammation, indicating that MCs may possess both harmful and protective activities in connection with this type of disease. Wild-type (WT) mice and mice lacking mouse MC protease 4 (mMCP-4), a chymase that is functionally homologous to human chymase, were sensitized and challenged with OVA, followed by the assessment of airway physiology and inflammatory parameters. Our results show that the airway hyperresponsiveness was significantly higher in mMCP-4^–/– as compared with WT mice. Moreover, the degree of lung tissue inflammation was markedly higher in mice lacking mMCP-4 than in WT controls. Histological analysis revealed that OVA sensitization/challenge resulted in a marked increased in the thickness of the smooth muscle cell (SMC) layer and, notably, that the degree of SMC layer thickening was more pronounced in mMCP-4^–/– animals than in WT controls, thus indicating that chymase may have an effect on airway SMCs. In support of this, mMCP-4-positive MCs were located in the close vicinity of the SMC layer, mainly in the upper airways, and mMCP-4 was shown to be the major chymase expressed in these MCs. Taken together, our results indicate that chymase present in the upper airways protects against allergic airway responses, possibly by regulating SMCs.

FoxP3+ Regulatory T Cells Restrain Splenic Extramedullary Myelopoiesis via Suppression of Hemopoietic Cytokine-Producing T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Lee, J. H., Wang, C., Kim, C. H. — Wed, 04 Nov 2009 13:05:40 PST

Extramedullary myelopoiesis occurs in peripheral organs such as spleen and produces many types of myeloid cells with diverse functions in response to inflammation and infection. It is increased during immune responses and chronic inflammation and is a significant factor in regulating inflammatory diseases and immunity. Increased myeloid cells are found in FoxP3-deficient mice but the mechanism has been unclear. We investigated the mechanism by which FoxP3⁺ regulatory T cells regulate the extramedullary myelopoiesis. We found that Ab or genetic depletion of FoxP3⁺ regulatory T cells greatly increased the number of the myeloid progenitors in spleen during immune responses. Consistently, the splenic myelopoiesis was effectively suppressed by increased numbers of natural or induced FoxP3⁺ regulatory T cells. We demonstrated that myelopoiesis is positively regulated by splenic CD4⁺ T cells that produce myelopoietic cytokines (GM-CSF and IL-3), and these effector CD4⁺ T cells are induced from naive CD4⁺ T cells in response to antigenic stimulation. FoxP3⁺ regulatory T cells were able to effectively suppress the differentiation of naive T cells into myelopoietic cytokine-producing T cells. This suppression was found to be dependent on cell contact but independent of TGFβ. Unlike splenic myelopoiesis, marrow myelopoiesis is not significantly affected by FoxP3⁺ regulatory T cells. We conclude that FoxP3⁺ T cells can negatively regulate splenic extramedullary myelopoiesis by suppressing the naive T cell differentiation into myelopoietic cytokine-producing CD4⁺ T cells. Our results provide new insights into regulation of extramedullary myelopoiesis.

FROUNT Is a Common Regulator of CCR2 and CCR5 Signaling to Control Directional Migration [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Toda, E., Terashima, Y., Sato, T., Hirose, K., Kanegasaki, S., Matsushima, K. — Wed, 04 Nov 2009 13:05:40 PST

FROUNT is a known CCR2-binding protein that facilitates monocyte/macrophage infiltration. Here we report that FROUNT also binds to the C-terminal region of CCR5 and enhances CCR5-mediated cellular chemotaxis. We show that FROUNT overexpression enhances the directionality of chemotaxis, while FROUNT suppression results in impaired responsiveness. Furthermore, we found an increase in consolidated pseudopodium formation in FROUNT-overexpressing cells (FNT cells) on uniform stimulation with CCL4 (MIP1-β), a specific ligand of CCR5. In most FNT cells, one to two pseudopodia directed toward higher chemokine concentration were found, whereas most FNT-suppressed cells had multiple pseudopodia. The data indicate that FROUNT is involved in sensing and amplifying a shallow extracellular chemokine gradient that leads to a limited number of accurate pseudopodia directed toward the chemokine concentration. In addition to its separate roles in CCR2- and CCR5-mediated chemotaxis, FROUNT, as a common regulator of these receptors, possibly plays a crucial role in the recruitment of immune cells expressing these receptors.

Lymph Node Stromal Cells Support Dendritic Cell-Induced Gut-Homing of T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

T cells are imprinted to express tissue-specific homing receptors upon activation in tissue-draining lymph nodes, resulting in their migration to the site of Ag entry. Expression of gut-homing molecules ₄β₇ and CCR9 is induced by retinoic acid, a vitamin A metabolite produced by retinal dehydrogenases, which are specifically expressed in dendritic cells as well as stromal cells in mucosa-draining lymph nodes. In this study, we demonstrate that mesenteric lymph node stromal cell-derived retinoic acid can directly induce the expression of gut-homing molecules on proliferating T cells, a process strongly enhanced by bone marrow-derived dendritic cells in vitro. Therefore, cooperation of sessile lymph node stromal cells with mobile dendritic cells warrants the imprinting of tissue specific homing receptors on activated T cells.

Reduced Diabetes in btk-Deficient Nonobese Diabetic Mice and Restoration of Diabetes with Provision of an Anti-Insulin IgH Chain Transgene [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Type 1 diabetes results from T cell-mediated destruction of insulin-producing β cells. Although elimination of B lymphocytes has proven successful at preventing disease, modulation of B cell function as a means to prevent type 1 diabetes has not been investigated. The development, fate, and function of B lymphocytes depend upon BCR signaling, which is mediated in part by Bruton’s tyrosine kinase (BTK). When introduced into NOD mice, btk deficiency only modestly reduces B cell numbers, but dramatically protects against diabetes. In NOD, btk deficiency mirrors changes in B cell subsets seen in other strains, but also improves B cell-related tolerance, as indicated by failure to generate insulin autoantibodies. Introduction of an anti-insulin BCR H chain transgene restores diabetes in btk-deficient NOD mice, indicating that btk-deficient B cells are functionally capable of promoting autoimmune diabetes if they have a critical autoimmune specificity. This suggests that the disease-protective effect of btk deficiency may reflect a lack of autoreactive specificities in the B cell repertoire. Thus, signaling via BTK can be modulated to improve B cell tolerance, and prevent T cell-mediated autoimmune diabetes.

Responsiveness of Stromal Fibroblasts to IFN-{gamma} Blocks Tumor Growth via Angiostasis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Lu, Y., Yang, W., Qin, C., Zhang, L., Deng, J., Liu, S., Qin, Z. — Wed, 04 Nov 2009 13:05:40 PST

The importance of stromal cells for tumor is akin to soil for seed. However, the interaction among these cells is far from understood. In this study, we show that stromal fibroblasts exist not only during tumor progression but also during regression stage, together with immune effector cells. Coinjection of stromal fibroblasts with tumor cells often promotes tumor growth. However, the presence of IFN- significantly impairs the ability of these cells to promote tumor growth due to a reduced angiogenesis. The mechanism relies mainly on the IFN--mediated down-regulation of vascular endothelial growth factor production by fibroblasts. The results reveal a novel link between immune cells and nonbone marrow-derived stromal cells, and define stromal fibroblasts as the main targets of IFN- in tumor immunity.

Histone Acetyltransferase Cofactor Trrap Is Essential for Maintaining the Hematopoietic Stem/Progenitor Cell Pool [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

The pool of hematopoietic stem/progenitor cells, which provide life-long reconstitution of all hematopoietic lineages, is tightly controlled and regulated by self-renewal and apoptosis. Histone modifiers and chromatin states are believed to govern establishment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In this study, we identified a role for the histone acetytransferase cofactor Trrap in the maintenance of hematopietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with induction of p53-independent apoptosis and deregulation of Myc transcription factors. Together, this study has identified a critical role for Trrap in the mechanism that maintains hematopoietic stem cells and hematopoietic system, and underscores the importance of Trrap and histone modifications in tissue homeostasis.

In Vitro Responses to Avian Influenza H5 by Human CD4 T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Cusick, M. F., Wang, S., Eckels, D. D. — Wed, 04 Nov 2009 13:05:40 PST

To address the question of whether human T cells are capable of recognizing novel isolates of influenza virus, in vitro responses to recombinant Ags and synthetic peptides derived from the sequences of H1, H3, and H5 were examined in a cohort of 64 individuals selected from a healthy blood donor population. Humans respond in vitro to H1 and H3 following exposure through natural infection and vaccination. Responses to H5 were well correlated with those to H1 or H3, and thus, a significant repertoire of H5-responsive T cells is present in many individuals; clear nonresponders to H1, H3, and H5, however, do exist. Differences were observed in the cytokine responses to H1, H3, and H5, whereas both IL-2 and IFN- production characteristic of memory responses were observed for H1 and H3, and H5-specific responses elicited primarily IL-2 and little or no IFN-, consistent with a naive T cell phenotype. Responses to all influenza HA were restricted by HLA-DR molecules. To address the structural basis for T cell recognition of H1 and H5, overlapping synthetic peptides were used to identify epitopes and to determine whether recognition of H5 was limited to homologous sequences in H1, the most closely related HA phylogenetically. Although responses were generally correlated, no complete structural overlap was observed. These results suggest that helper T cell cross reactivity between different influenza strains may impart cross-protection to H5N1 strain of influenza.

Chronic CD70-Driven Costimulation Impairs IgG Responses by Instructing T Cells to Inhibit Germinal Center B Cell Formation through FasL-Fas Interactions [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

CD70 provides costimulation that enhances effector T cell differentiation upon binding of its receptor, CD27. During chronic immune activation, CD70 is constitutively expressed on activated immune cells, and this induces T cell-driven disruption of neutralizing Ab responses via an unknown mechanism. We used CD70-transgenic mice to investigate the effect of constitutive expression of CD70 on T cell-dependent B cell responses. CD70 induced up-regulation of the B cell follicle homing chemokine receptor CXCR5 on T cells, enabling not only CD4 but also CD8 T cells to infiltrate the B cell follicles. CD70-transgenic mice failed to develop productive germinal center formation and displayed impaired IgG Ab responses. Defective germinal center B cell differentiation was critically dependent on CD70-mediated CD27 signaling in T cells, and involved Fas-dependent impairment of germinal center B cell differentiation. Thus, CD70-driven costimulation enables T cells to terminate B cell responses, thereby compromising durable Ab production. Our findings imply that the CD70- and CD27-driven costimulatory axis may be involved in shutdown of B cell responses before clearance of Ag. Because CD70 is expressed constitutively in chronic viral infections such as HIV-1 infection, this mechanism may also contribute to defects in humoral immunity associated with this disease.

CD36 and TLR Interactions in Inflammation and Phagocytosis: Implications for Malaria [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

CD36 participates in macrophage internalization of a variety of particles, and has been implicated in inflammatory responses to many of these ligands. To what extent CD36 cooperates with other receptors in mediating these processes remains unclear. Because CD36 has been shown to cooperate with TLR2, we investigated the roles and interactions of CD36 and TLRs in inflammation and phagocytosis. Using Ab-induced endocytosis of CD36 and phagocytosis of erythrocytes displaying Abs to CD36, we show that selective engagement and internalization of this receptor did not lead to proinflammatory cytokine production by primary human and murine macrophages. In addition, CD36-mediated phagocytosis of Plasmodium falciparum malaria-parasitized erythrocytes (PEs), which contain parasite components that activate TLRs, also failed to induce cytokine secretion from primary macrophages. Furthermore, we demonstrate that CD36-mediated internalization did not require TLR2 or the TLR-signaling molecule IRAK4. However, macrophage pretreatment with TLR agonists markedly stimulated particle uptake via CD36. Similarly, PE uptake was unaffected by TLR deficiency, but in wild-type cells was increased by pretreatment with purified P. falciparum glycosylphosphatidylinositols, which activate TLR2. Our findings indicate that CD36 must cooperate with other receptors such as TLRs to participate in cytokine responses. Although purified P. falciparum components activate TLRs, CD36-mediated internalization of intact PEs is not inflammatory. Further, CD36 mediates internalization of particles, including PEs, independently of TLR signaling, but can functionally cooperate with TLRs to enhance internalization.

Outside-In Signal Transmission by Conformational Changes in Integrin Mac-1 [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the _M and β₂ subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals.

IL-33 Amplifies the Polarization of Alternatively Activated Macrophages That Contribute to Airway Inflammation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 04 Nov 2009 13:05:40 PST

Alternatively activated macrophages (AAM) play a crucial role in type 2 immunity. Mice deficient in ST2, a receptor for the latest member of the IL-1 family, IL-33, have impaired type 2 immune responses. We therefore reasoned that IL-33/ST2 signaling may be involved in the differentiation and activation of AAM during airway inflammation. We report here that IL-33 changed the quiescent phenotype of alveolar macrophages toward an AAM phenotype that expressed mannose receptor, IL-4R, and produced high levels of CCL24 and CCL17 in an IL-13-dependent manner during IL-33-induced airway inflammation. Neutralization of AAM-derived CCL24 led to an amelioration of IL-33-induced eosinophilia in the lungs. Moreover, depletion of alveolar macrophages reduced IL-33-induced airway inflammation. Additionally, the attenuated OVA-induced airway inflammation in ST2^–/– mice was associated with a decrease in AAM differentiation. In vitro, IL-33 amplified IL-13-induced polarization of alveolar- and bone marrow-derived macrophage toward an AAM phenotype by increasing the expression of arginase I, Ym1, as well as the production of CCL24 and CCL17. IL-13/IL-4R signaling was crucial for IL-33-driven AAM amplification by inducing the expression of ST2L. Finally, we showed that IL-33 was more abundantly expressed in the lung epithelial cells of asthma patients than those from healthy controls, suggesting that IL-33 may be involved in lung macrophage activation in clinical asthma. Taken together, we demonstrate here that IL-33/ST2 plays a significant role in the amplification of AAM polarization and chemokine production which contribute to innate and Ag-induced airway inflammation.

Ca2+ Waves Initiate Antigen-Stimulated Ca2+ Responses in Mast Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Cohen, R., Torres, A., Ma, H.-T., Holowka, D., Baird, B. — Wed, 04 Nov 2009 13:05:41 PST

Ca²⁺ mobilization is central to many cellular processes, including stimulated exocytosis and cytokine production in mast cells. Using single cell stimulation by IgE-specific Ag and high-speed imaging of conventional or genetically encoded Ca²⁺ sensors in rat basophilic leukemia and bone marrow-derived rat mast cells, we observe Ca²⁺ waves that originate most frequently from the tips of extended cell protrusions, as well as Ca²⁺ oscillations throughout the cell that usually follow the initiating Ca²⁺ wave. In contrast, Ag conjugated to the tip of a micropipette stimulates local, repetitive Ca²⁺ puffs at the region of cell contact. Initiating Ca²⁺ waves are observed in most rat basophilic leukemia cells stimulated with soluble Ag and are sensitive to inhibitors of Ca²⁺ release from endoplasmic reticulum stores and to extracellular Ca²⁺, but they do not depend on store-operated Ca²⁺ entry. Knockdown of transient receptor potential channel (TRPC)1 and TRPC3 channel proteins by short hairpin RNA reduces the sensitivity of these cells to Ag and shifts the wave initiation site from protrusions to the cell body. Our results reveal spatially encoded Ca²⁺ signaling in response to immunoreceptor activation that utilizes TRPC channels to specify the initiation site of the Ca²⁺ response.

Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inflammatory Activity of Chemerin in a Lung Disease Model [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 04 Nov 2009 13:05:41 PST

Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

Discrete Domains of MARCH1 Mediate Its Localization, Functional Interactions, and Posttranscriptional Control of Expression [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Jabbour, M., Campbell, E. M., Fares, H., Lybarger, L. — Wed, 04 Nov 2009 13:05:41 PST

Within APCs, ubiquitination regulates the trafficking of immune modulators such as MHC class II and CD86 (B7.2) molecules. MARCH1 (membrane-associated RING-CH), a newly identified ubiquitin E3 ligase expressed in APCs, ubiquitinates MHC class II, thereby reducing its surface expression. Following LPS-induced maturation of dendritic cells, MARCH1 mRNA is down-regulated and MHC class II is redistributed to the cell surface from endosomal compartments. Here, we show that MARCH1 expression is also regulated at the posttranscriptional level. In primary dendritic cell and APC cell lines of murine origin, MARCH1 had a half-life of <30 min. MARCH1 degradation appears to occur partly in lysosomes, since inhibiting lysosomal activity stabilized MARCH1. Similar stabilization was observed when MARCH1-expressing cells were treated with cysteine protease inhibitors. Mutational analyses of MARCH1 defined discrete domains required for destabilization, proper localization, and functional interaction with substrates. Taken together, these data suggest that MARCH1 expression is regulated at a posttranscriptional level by trafficking within the endolysosomal pathway where MARCH1 is proteolyzed. The short half-life of MARCH1 permits very rapid changes in the levels of the protein in response to changes in the mRNA, resulting in efficient induction of Ag presentation once APCs receive maturational signals.

CD69 Gene Is Differentially Regulated in T and B Cells by Evolutionarily Conserved Promoter-Distal Elements [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Vazquez, B. N., Laguna, T., Carabana, J., Krangel, M. S., Lauzurica, P. — Wed, 04 Nov 2009 13:05:41 PST

CD69 is a type II C-type lectin involved in lymphocyte migration and cytokine secretion. CD69 expression represents one of the earliest available indicators of leukocyte activation and its rapid induction occurs through transcriptional activation. In this study we examined the molecular mechanism underlying mouse CD69 gene transcription in vivo in T and B cells. Analysis of the 45-kb region upstream of the CD69 gene revealed evolutionary conservation at the promoter and at four noncoding sequences (CNS) that were called CNS1, CNS2, CNS3, and CNS4. These regions were found to be hypersensitive sites in DNase I digestion experiments, and chromatin immunoprecipitation assays showed specific epigenetic modifications. CNS2 and CNS4 displayed constitutive and inducible enhancer activity in transient transfection assays in T cells. Using a transgenic approach to test CNS function, we found that the CD69 promoter conferred developmentally regulated expression during positive selection of thymocytes but could not support regulated expression in mature lymphocytes. Inclusion of CNS1 and CNS2 caused suppression of CD69 expression, whereas further addition of CNS3 and CNS4 supported developmental-stage and lineage-specific regulation in T cells but not in B cells. We concluded CNS1–4 are important cis-regulatory elements that interact both positively and negatively with the CD69 promoter and that differentially contribute to CD69 expression in T and B cells.

Epigenetic Regulation of TLR4 Gene Expression in Intestinal Epithelial Cells for the Maintenance of Intestinal Homeostasis [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Takahashi, K., Sugi, Y., Hosono, A., Kaminogawa, S. — Wed, 04 Nov 2009 13:05:41 PST

Intestinal epithelial cells (IECs) are continuously exposed to large numbers of commensal bacteria but are relatively insensitive to them, thereby averting an excessive inflammatory reaction. In this study, we show that the low responsiveness of human IEC lines to LPS was mainly brought about by a down-regulation of TLR4 gene transcription. Additionally, the presence of an IEC-specific repressor element in the 5' region of the TLR4 gene and binding of a NF to the element was shown. The transcription factor ZNF160, which was expressed more abundantly in a LPS-low responder IEC line than in a LPS-high responder IEC line, repressed TLR4 gene transcription. ZNF160 is known to interact with the scaffold protein KAP1 via its N terminus to recruit histone deacetylase. Histone deacetylation, as well as DNA methylation, at the 5' region of the TLR4 gene was significantly higher in LPS-low responder IEC lines than in a monocyte line or a LPS-high responder IEC line. It was demonstrated that TLR4 gene transcription was repressed by these epigenetic regulations, which were, at least in part, dependent on ZNF160. Down-regulaton of TLR4 gene expression by these mechanisms in IECs possibly contributes to the maintainance of homeostasis in the intestinal commensal system.

Defining the Turkey MHC: Sequence and Genes of the B Locus [IMMUNOGENETICS]

Chaves, L. D., Krueth, S. B., Reed, K. M. — Wed, 04 Nov 2009 13:05:41 PST

The MHC, the most polymorphic and gene dense region in the vertebrate genome, contains many loci essential to immunity. In mammals, this region spans ~4 Mb. Studies of avian species have found the MHC to be greatly reduced in size and gene content with an overall locus organization differing from that of mammals. The chicken MHC has been mapped to two distinct regions (MHC-B and -Y) of a single chromosome. MHC-B haplotypes possess tightly linked genes encoding the classical MHC molecules and few other disease resistance genes. Furthermore, chicken haplotypes possess a dominantly expressed class I and class II B locus that have a significant effect on the progression or regression of pathogenic disease. In this study, we present the MHC-B region of the turkey (Meleagris gallopavo) as a similarly constricted locus, with 34 genes identified within a 0.2-Mb region in near-perfect synteny with that of the chicken MHC-B. Notable differences between the two species are three BG and class II B loci in the turkey compared with one BG and two class II B loci in the chicken MHC-B. The relative size and high level of similarity of the turkey MHC in relation to that of the chicken suggest that similar associations with disease susceptibility and resistance may also be found in turkey.

A Rice-Based Oral Cholera Vaccine Induces Macaque-Specific Systemic Neutralizing Antibodies but Does Not Influence Pre-Existing Intestinal Immunity [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

We previously showed that oral immunization of mice with a rice-based vaccine expressing cholera toxin (CT) B subunit (MucoRice-CT-B) induced CT-specific immune responses with toxin-neutralizing activity in both systemic and mucosal compartments. In this study, we examined whether the vaccine can induce CT-specific Ab responses in nonhuman primates. Orally administered MucoRice-CT-B induced high levels of CT-neutralizing serum IgG Abs in the three cynomolgus macaques we immunized. Although the Ab level gradually decreased, detectable levels were maintained for at least 6 mo, and high titers were rapidly recovered after an oral booster dose of the rice-based vaccine. In contrast, no serum IgE Abs against rice storage protein were induced even after multiple immunizations. Additionally, before immunization the macaques harbored intestinal secretory IgA (SIgA) Abs that reacted with both CT and homologous heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and had toxin-neutralizing activity. The SIgA Abs were present in macaques 1 mo to 29 years old, and the level was not enhanced after oral vaccination with MucoRice-CT-B or after subsequent oral administration of the native form of CT. These results show that oral MucoRice-CT-B can effectively induce CT-specific, neutralizing, serum IgG Ab responses even in the presence of pre-existing CT- and heat-labile enterotoxin-reactive intestinal SIgA Abs in nonhuman primates.

Long Double-Stranded RNA Induces an Antiviral Response Independent of IFN Regulatory Factor 3, IFN-{beta} Promoter Stimulator 1, and IFN [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

Virus infection elicits a robust innate antiviral response dominated by the production of type 1 IFN. In nonprofessional innate immune cells such as fibroblasts, type 1 IFN is rapidly produced following the recognition of viral dsRNA and the subsequent activation of the constitutively expressed transcription factor IFN regulatory factor 3 (IRF3). Although origin, localization, and length are factors in mediating dsRNA recognition and binding by cellular dsRNA-binding proteins, the biological significance of differential dsRNA binding is unclear, since the subsequent signaling pathways converge on IRF3. In this study, we show a dsRNA length-dependent activation of IRFs, IFNs, and IFN-stimulated genes in mouse fibroblasts. The length dependence was exacerbated in fibroblasts deficient in the mitochondria-associated adaptor IFN-β promoter stimulator 1 and IRF3, suggesting that antiviral gene induction mediated by short and long dsRNA molecules is predominantly IFN-β promoter stimulator 1 and IRF3 dependent and independent, respectively. Furthermore, we provide evidence of an innate antiviral response in fibroblasts in the absence of both IRF3 and type 1 IFN induction. Even with these key modulators missing, a 60–90% inhibition of virus replication was observed following 24-h treatment with short or long dsRNA molecules, respectively. These data provide evidence of a novel antiviral pathway that is dependent on dsRNA length, but independent of the type 1 IFN system.

Resistance to Vaccinia Virus Is Less Dependent on TNF under Conditions of Heterologous Immunity [HOST DEFENSE]

Nie, S., Cornberg, M., Selin, L. K. — Wed, 04 Nov 2009 13:05:41 PST

TNF has been shown to be important for controlling many pathogens. Here, we directly demonstrate using wild-type TNF^–/– and TNFR1^–/– mice that TNF does play a role in protection against vaccinia virus (VV) infection in naive mice. Since VV replication is also partially controlled in lymphocytic choriomeningitis virus (LCMV)-immune C57BL/6J mice through the process of heterologous immunity, we questioned whether TNF was required in mediating this protection. VV-infected LCMV-immune mice that were TNF-deficient as a consequence of genetic deletion or receptor blockade demonstrated normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells and controlled VV infection similar to LCMV-immune mice having TNF function. This indicates that neither TNF nor lymphotoxin, which uses the same receptor, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV made the role of TNF in protection against VV infection much less important, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of patients with anti-TNF compounds is reasonably well tolerated with relatively few infectious complications.

Induction of Cross-Priming of Naive CD8+ T Lymphocytes by Recombinant Bacillus Calmette-Guerin That Secretes Heat Shock Protein 70-Major Membrane Protein-II Fusion Protein [HOST DEFENSE]

Mukai, T., Maeda, Y., Tamura, T., Matsuoka, M., Tsukamoto, Y., Makino, M. — Wed, 04 Nov 2009 13:05:41 PST

Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8⁺ T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8⁺ T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8⁺ T cells, but also CD4⁺ T cells of both types to produce IFN-. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8⁺ T cells by BCG-70M through DC. Thus, the CD8⁺ T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8⁺ T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4⁺ T cells, CD62L^lowCD8⁺ T cells and perforin-producing CD8⁺ T cells were efficiently produced. MMP-II-reactive CD4⁺ and CD8⁺ memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4⁺ T cells, and CD8⁺ T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

A Homolog of Formyl Peptide Receptor-Like 1 (FPRL1) Inhibitor from Staphylococcus aureus (FPRL1 Inhibitory Protein) That Inhibits FPRL1 and FPR [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

The members of the formyl peptide receptor (FPR) family are involved in the sensing of chemoattractant substances, including bacteria-derived N-formylated peptides and host-derived peptides and proteins. We have recently described two chemoattractant receptor inhibitors from Staphylococcus aureus. Chemotaxis inhibitory protein of S. aureus (CHIPS) blocks the formyl peptide receptor (FPR) and the receptor for complement C5a (C5aR), while FPR-like 1 (FPRL1) inhibitory protein (FLIPr) blocks the FPRL1. Here, we describe another staphylococcal chemoattractant-inhibiting protein with 73% overall homology to FLIPr and identical first 25 aa, which we termed FLIPr-like. This protein inhibits neutrophil calcium mobilization and chemotaxis induced by the FPRL1-ligand MMK-1 and FPR-ligand fMLP. While its FPRL1-inhibitory activity lies in the comparable nanomolar range of FLIPr, its antagonism of the FPR is ~100-fold more potent than that of FLIPr and comparable to that of CHIPS. The second N-terminal phenylalanine was required for its inhibition of the FPR, but it was dispensable for the FPRL1. Furthermore, the deletion of the first seven amino acids reduced its antagonism of the FPRL1, and the exchange of the first six amino acids with that of CHIPS-conferred receptor specificity. Finally, studies with cells transfected with several chemoattractant receptors confirmed that FLIPr-like specifically binds to the FPR and FPRL1. In conclusion, the newly described excreted protein from S. aureus, FLIPr-like, is a potent inhibitor of the FPR- and FPRL1-mediated neutrophil responses and may be used to selectively modulate these chemoattractant receptors.

The Natural Cytotoxicity Receptor NKp46 Is Dispensable for IL-22-Mediated Innate Intestinal Immune Defense against Citrobacter rodentium [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

Natural cytotoxicity receptors (including NKp30, NKp44, and NKp46 in humans and NKp46 in mice) are type I transmembrane proteins that signal NK cell activation via ITAM-containing adapter proteins in response to stress- and pathogen-induced ligands. Although murine NKp46 expression (encoded by Ncr1) was thought to be predominantly restricted to NK cells, the identification of distinct intestinal NKp46⁺ cell subsets that express the transcription factor Rorc and produce IL-22 suggests a broader function for NKp46 that could involve intestinal homeostasis and immune defense. Using mice carrying a GFP-modified Ncr1 allele, we found normal numbers of gut CD3^–GFP⁺ cells with a similar cell surface phenotype and subset distribution in the absence of Ncr1. Splenic and intestinal CD3^–NKp46⁺ cell subsets showed distinct patterns of cytokine secretion (IFN-, IL-22) following activation via NK1.1, NKp46, IL-12 plus IL-18, or IL-23. However, IL-22 production was sharply restricted to intestinal CD3^–GFP⁺ cells with the CD127⁺NK1.1^– phenotype and could be induced in an Ncr1-independent fashion. Because NKp46 ligands can trigger immune activation in the context of infectious pathogens, we assessed the response of wild-type and Ncr-1-deficient Rag2^–/– mice to the enteric pathogen Citrobacter rodentium. No differences in the survival or clinical score were observed in C. rodentium-infected Rag2^–/– mice lacking Ncr1, indicating that NKp46 plays a redundant role in the differentiation of intestinal IL-22⁺ cells that mediate innate defense against this pathogen. Our results provide further evidence for functional heterogeneity in intestinal NKp46⁺ cells that contrast with splenic NK cells.

Enterobacter sakazakii Targets DC-SIGN to Induce Immunosuppressive Responses in Dendritic Cells by Modulating MAPKs [HOST DEFENSE]

Mittal, R., Bulgheresi, S., Emami, C., Prasadarao, N. V. — Wed, 04 Nov 2009 13:05:41 PST

Enterobacter sakazakii (ES) is an emerging pathogen that causes meningitis and necrotizing enterocolitis in infants. Dendritic cells (DCs) are professional phagocytic cells that play an essential role in host defense against invading pathogens; however, the interaction of ES with DCs is not known. In this study, we demonstrate that ES targets DC-specific ICAM nonintegrin (DC-SIGN) to survive in myeloid DCs for which outer membrane protein A (OmpA) expression in ES is critical, although it is not required for uptake. In addition, DC-SIGN expression was sufficient to cause a significant invasion by ES in HeLa cells and intestinal epithelial cells, which are normally not invaded by ES. OmpA⁺ ES prevented the maturation of DCs by triggering the production of high levels of IL-10 and TGF-β and by suppressing the activation of MAPKs. Pretreatment of DCs with Abs to IL-10 and TGF-β or of bacteria with anti-OmpA Abs significantly enhanced the maturation markers on DCs. Furthermore, DCs pretreated with various inhibitors of MAPKs prohibited the increased production of proinflammatory cytokines stimulated by LPS or OmpA^– ES. LPS pretreatment followed by OmpA⁺ ES infection of DCs failed to induce maturation of DCs, indicating that OmpA⁺ ES renders the cells in immunosuppressive state to external stimuli. Similarly, OmpA⁺ ES-infected DCs failed to present Ag to T cells as indicated by the inability of T cells to proliferate in MLR. We conclude that ES interacts with DC-SIGN to subvert the host immune responses by disarming MAPK pathway in DCs.

Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces Up-Regulation of Galectin-1 Expression with Potential Anti-Inflammatory Activity [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1β, TNF-, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection.

Expansion of Functionally Skewed CD56-Negative NK Cells in Chronic Hepatitis C Virus Infection: Correlation with Outcome of Pegylated IFN-{alpha} and Ribavirin Treatment [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

NK cells are important innate immune effector cells, normally characterized as CD56⁺CD3^– lymphocytes. In this study, we report that CD56^–CD16⁺ NK cells expand in many patients with chronic hepatitis C virus infection. These CD56^– NK cells were functionally impaired with respect to cytokine production upon target cell recognition, in comparison to CD56^dim and CD56^bright NK cell subsets. In particular, CD56^– NK cells were strikingly defective in their polyfunctional response as measured by the coexpression of MIP-1β, IFN-, TNF-, and CD107a degranulation. The ability of these cells to mediate three or four of these functions was poor; expression of MIP-1β alone dominated their response. CD56^– NK cells retained expression of receptors such as the natural cytotoxicity receptors and NKG2D, whereas the expression of CD57 and perforin was lower when compared with CD56^dim NK cells. Interestingly, pretreatment levels of CD56^– NK cells correlated with the outcome of pegylated IFN- and ribavirin treatment. In patients with CD56^– NK cells in the range of healthy subjects, 80% reached a sustained virological response to treatment, whereas only 25% of patients with levels clearly above those in healthy subjects experienced a sustained virological response. Thus, chronic hepatitis C virus infection is associated with an expansion of CD56^– NK cells functionally skewed toward MIP-1β production only. Furthermore, high levels of these cells reveal a disturbance in innate cellular immunity that is associated with an impaired ability to respond to antiviral treatment with IFN- and ribavirin.

Adoptive Transfer of T Lymphocytes Sensitized against the Prion Protein Attenuates Prion Invasion in Scrapie-Infected Mice [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:41 PST

There is to date no effective way of preventing or curing neurodegenerative diseases such as Alzheimer disease or transmissible spongiform encephalopathies. The idea of treating those conditions by immunological approaches has progressively emerged over the last ten years. Encouraging results have been reported in Alzheimer disease and in peripheral forms of mouse prion diseases following passive injection of Abs or active immunization against the peptides or proteins presumably at the origin of those disorders. Still, major difficulties persist due to some characteristics of those conditions such as slow evolution, brain location, uncertainties regarding precise pathogenic pathways, and, above all, the fact that the target Ag is self, meaning that it is poorly immunogenic and potentially harmful if tolerance was transgressed. To analyze some of those difficulties, we are developing adoptive cell transfer approaches. In this study, lymphocytes sensitized against the prion protein in nontolerant Prnp^–/– mice were transferred into histocompatible wild-type recipients which were partly or totally devoid of their own lymphocytes. Under such conditions, we found that the engrafted T lymphocytes resisted peripheral tolerance, remained reactive for several months against epitopes of the prion protein, and significantly attenuated the progression of prions in secondary lymphoid organs with subsequent delay in the evolution of the neurological disease. Interestingly, those protective T lymphocytes secreted lymphokines and migrated more readily into the host CNS but did not appear to be engaged in cooperation with host B cells for Ab production.

Both TRIF- and MyD88-Dependent Signaling Contribute to Host Defense against Pulmonary Klebsiella Infection [HOST DEFENSE]

Cai, S., Batra, S., Shen, L., Wakamatsu, N., Jeyaseelan, S. — Wed, 04 Nov 2009 13:05:42 PST

Klebsiella pneumoniae causes extensive lung damage. TLR signaling involves adaptors TRIF and MyD88. However, the relative contribution of TRIF and MyD88 signaling in host defense against pulmonary K. pneumoniae infection has not been elucidated. Therefore, we investigated the role of TRIF and MyD88 in K. pneumoniae pneumonia. TRIF^–/– mice infected with K. pneumoniae showed impaired survival and reduced bacterial clearance, neutrophil influx, histopathologic evidence of inflammation, and TNF-, IL-6, KC, MIP-2, but not LIX, expression in the lungs. In addition, K. pneumoniae-induced late NF-B activation and phosphorylation of MAPKs was attenuated in the lungs of TRIF^–/– mice. However, MyD88^–/– mice infected with K. pneumoniae showed a much more remarkable phenotype, including impaired survival and reduced bacterial clearance, histopathology, and TNF-, IL-6, KC, MIP-2, and LIX expression with almost no neutrophil influx in the lungs. In MyD88^–/– mice, K. pneumoniae-induced early NF-B and MAPK activation in the lungs was also reduced. Furthermore, the role of MyD88 is dominant over TRIF because TRIF/MyD88 double knockout mice displayed a more pronounced phenotype than TRIF^–/– mice. Moreover, human alveolar macrophages pretreated with MyD88 blocking peptide showed attenuated TNF-, IL-6, and IL-8 expression. Also, C57BL/6 mice pretreated with MyD88 blocking peptide exhibited attenuation in K. pneumoniae-induced neutrophil influx and enhanced bacterial burden in the lungs and dissemination. Overall, this investigation provides new insights into the TRIF and MyD88 signaling triggered by pulmonary K. pneumoniae infection in the lungs and demonstrate the therapeutic potential of MyD88 in reducing excessive neutrophil influx in human disease during Gram-negative bacterial pneumonia.

IL-22 Produced by Human NK Cells Inhibits Growth of Mycobacterium tuberculosis by Enhancing Phagolysosomal Fusion [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:42 PST

We determined whether human NK cells could contribute to immune defenses against Mycobacterium tuberculosis through production of IL-22. CD3^–CD56⁺ NK cells produced IL-22 when exposed to autologous monocytes and -irradiated M. tuberculosis, and this depended on the presence of IL-15 and IL-23, but not IL-12 or IL-18. IL-15-stimulated NK cells expressed 10.6 times more DAP10 mRNA compared with control NK cells, and DAP10 siRNA inhibited IL-15-mediated IL-22 production by NK cells. Soluble factors produced by IL-15-activated NK cells inhibited growth of M. tuberculosis in macrophages, and this effect was reversed by anti-IL-22. Addition of rIL-22 to infected macrophages enhanced phagolysosomal fusion and reduced growth of M. tuberculosis. We conclude that NK cells can contribute to immune defenses against M. tuberculosis through production of IL-22, which inhibits intracellular mycobacterial growth by enhancing phagolysosomal fusion. IL-15 and DAP-10 elicit IL-22 production by NK cells in response to M. tuberculosis.

The Novel Lipopolysaccharide-Binding Protein CRISPLD2 Is a Critical Serum Protein to Regulate Endotoxin Function [HOST DEFENSE]

Wed, 04 Nov 2009 13:05:42 PST

LPS is an immunostimulatory component of Gram-negative bacteria. Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood samples are 607 µg/ml and 290 µg/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2–0.9 µg/ml) and enhance CRISPLD2 secretion (range, 1.5–4.2 µg/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF- and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum concentrations not only are indicators of the degree of a body’s exposure to LPS but also reflect an individual’s LPS sensitivity.

The Bile Acid Sensor Farnesoid X Receptor Is a Modulator of Liver Immunity in a Rodent Model of Acute Hepatitis [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. In this study, we report that activation of the farnesoid X receptor (FXR), a member of the ligand-activated nuclear receptor superfamily and bile sensor highly expressed in the liver, attenuates liver injury in a model of autoimmune hepatitis induced by Con A. We found that FXR gene ablation results in a time-dependent increase of liver expression (up to 20-fold in a 9-mo-old mouse) of osteopontin, a NKT cell-derived extracellular matrix protein and immunoregulatory cytokine. In comparison to wild-type, FXR^–/– mice are more susceptible to Con A-induced hepatitis and react to Con A administration by an unregulated production of osteopontin. Administering wild-type mice with a synthetic FXR agonist attenuated Con A-induced liver damage and liver expression of the osteopontin gene. By in vitro studies, we found that FXR is expressed by primarily isolated NKT cells and its ablation favors ostepontin production in response to Con A. Chromatin immunoprecipitation assay and coimmunoprecipitation experiments demonstrate that the short heterodimer partner (SHP), a nuclear receptor and FXR target, was expressed by NKT cell hybridomas and increased in response to FXR activation. FXR activates SHP that interacts with and inhibits c-Jun binding to the osteopontin promoter. These data indicate that in NKT cells, FXR activation causes a SHP-mediated inhibition of osteopontin production. These data support the notion that the bile acid sensor FXR regulates the activation of liver NKT cells.

Externally Triggered Egress Is the Major Fate of Toxoplasma gondii during Acute Infection [INFLAMMATION]

Tomita, T., Yamada, T., Weiss, L. M., Orlofsky, A. — Wed, 04 Nov 2009 13:05:42 PST

The apicomplexan parasite Toxoplasma gondii expands during acute infection via a cycle of invasion, intracellular replication, and lytic egress. Physiological regulation has not yet been demonstrated for either invasion or egress. We now report that, in contrast to cell culture systems, in which egress occurs only after five or more parasite divisions (2–3 days), intracellular residence is strikingly abbreviated in inflammatory cells in vivo, and early egress (after zero to two divisions) is the dominant parasite fate in acutely infected mice. Adoptive transfer experiments demonstrate rapid, reciprocal, kinetically uniform parasite transfer between donor and recipient compartments, with a t_1/2 of ~3 h. Inflammatory macrophages are major participants in this cycle of lytic egress and reinfection, which drives rapid macrophage turnover. Inflammatory triggering cells, principally macrophages, elicit egress in infected target macrophages, a process we term externally triggered egress (ETE). The mechanism of ETE does not require reactive oxygen or nitrogen species, the mitochondrial permeability transition pore, or a variety of signal transduction mediators, but is dependent on intracellular calcium and is highly sensitive to SB203580, an inhibitor of p38 MAPK as well as a related parasite-encoded kinase. SB203580 both inhibited the initiation of ETE and altered the progression of egress. Parasites recently completing a cycle of egress and reinfection were preferentially restricted in vivo, supporting a model in which ETE may favor host defense by a process of haven disruption. ETE represents a novel example of interaction between a parasite infectious cycle and host microenvironment.

Activation of the Cholinergic Anti-Inflammatory System by Nicotine Attenuates Neuroinflammation via Suppression of Th1 and Th17 Responses [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

The 7 nicotinic acetylcholine receptor (nAChR) was recently described as an anti-inflammatory target in both macrophages and T cells. Its expression by immune cells may explain the epidemiological data claiming a negative link between cigarette smoking and several inflammatory diseases. In this study, we determined the immunological effects of 7 nAChR activation by nicotine. Our results indicate that the 7 nAChR is expressed on the surface of CD4⁺ T cells and that this expression is up-regulated upon immune activation. Nicotine reduced T cell proliferation in response to an encephalitogenic Ag, as well as the production of Th1 (TNF- and IFN-) and Th17 cytokines (IL-17, IL-17F, IL-21, and IL-22). IL-4 production was increased in the same setting. Attenuation of the Th1 and Th17 lineages was accompanied by reduced T-bet (50%) and increased GATA-3 (350%) expression. Overall, nicotine induced a shift to the Th2 lineage. However, 7^–/–-derived T cells were unaffected by nicotine. Furthermore, nicotine reduced NF-B-mediated transcription as measured by IL-2 and IB transcription. In vivo, administration of nicotine (2 mg/kg s.c.) suppressed the severity of CD4⁺ T cell-mediated disease experimental autoimmune encephalomyelitis. 7^–/– mice were refractory to nicotine treatment, although disease severity in those animals was reduced, due to impairment in Ag presentation. Accordingly, CD4⁺ and CD11b⁺ cells infiltration into the CNS, demyelination, and axonal loss were reduced. Our data implicate a role for the 7 nAChR in immune modulation and suggest that 7 nAChR agonists may be effective in the treatment of inflammatory disorders.

Novel Role of Pin1 Induction in Type II Collagen-Mediated Rheumatoid Arthritis [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation in joints and subsequent destruction of cartilage and bone. Inflammatory mediators such as PGs and proinflammatory cytokines contribute to RA progress. Pin1, a peptidyl prolyl isomerase, plays important pathophysiological roles in several diseases, including cancer and neurodegeneration. We found that both Pin1 and cyclooxygenase-2 (COX-2) were highly expressed in ankle tissues of type II collagen-induced RA mice. HTB-94 cells overexpressing Pin1 and primary cultured human chondrocytes showed increased basal expression of proinflammatory proteins (COX-2, inducible NO synthase, TNF-, and IL-1β). Site-directed mutagenesis revealed that Pin1-mediated transcriptional activation of COX-2 was coordinately regulated by NF-B, CREB, and C/EBP. Gel shift, reporter gene, and Western blot analyses confirmed that NF-B, CREB, and C/EBP were consistently activated in chondrocytes overexpressing Pin1. Treatment of RA mice with juglone, a chemical inhibitor of Pin1, significantly reduced RA progress and COX-2 expression in the ankle tissues. Moreover, juglone dose dependently decreased the basal COX-2 expression in primary cultured chondrocytes from RA patients. These results demonstrate that Pin1 induction during RA progress stimulates proinflammatory protein expression by activating NF-B, CREB, and C/EBP, and suggest that Pin1 is a potential therapeutic target of RA.

CXCR2 Is Required for Neutrophilic Airway Inflammation and Hyperresponsiveness in a Mouse Model of Human Rhinovirus Infection [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Human rhinovirus (RV) infection is responsible for the majority of virus-induced asthma exacerbations. Using a mouse model of human RV infection, we sought to determine the requirement of CXCR2, the receptor for ELR-positive CXC chemokines, for RV-induced airway neutrophilia and hyperresponsiveness. Wild-type and CXCR2^–/– mice were inoculated intranasally with RV1B or sham HeLa cell supernatant. Following RV1B infection, CXCR2^–/– mice showed reduced airway and lung neutrophils and cholinergic responsiveness compared with wild-type mice. Similar results were obtained in mice treated with neutralizing Ab to Ly6G, a neutrophil-depleting Ab. Lungs from RV-infected, CXCR2^–/– mice showed significantly reduced production of TNF-, MIP-2/CXCL2, and KC/CXCL1 and lower expression of MUC5B compared with RV-treated wild-type mice. The requirement of TNF- for RV1B-induced airway responses was tested using TNFR1^–/– mice. TNFR1^–/– animals displayed reduced airway responsiveness to RV1B, even when exogenous MIP-2 was added to the airways. We conclude that CXCR2 is required for RV-induced neutrophilic airway inflammation and that neutrophil TNF- release is required for airway hyperresponsiveness.

Recognition of Fungal Protease Activities Induces Cellular Activation and Eosinophil-Derived Neurotoxin Release in Human Eosinophils [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils’ responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils’ innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.

Chemokine-Like Receptor-1 Expression by Central Nervous System-Infiltrating Leukocytes and Involvement in a Model of Autoimmune Demyelinating Disease [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

We examined the involvement of chemokine-like receptor-1 (CMKLR1) in experimental autoimmune encephalomyelitis (EAE), a model of human multiple sclerosis. Upon EAE induction by active immunization with myelin oligodendrocyte glycoprotein amino acids 35–55 (MOG_35–55), microglial cells and CNS-infiltrating myeloid dendritic cells expressed CMKLR1, as determined by flow cytometric analysis. In addition, chemerin, a natural ligand for CMKLR1, was up-regulated in the CNS of mice with EAE. We found that CMKLR1-deficient (CMKLR1 knockout (KO)) mice develop less severe clinical and histologic disease than their wild-type (WT) counterparts. CMKLR1 KO lymphocytes proliferate and produce proinflammatory cytokines in vitro, yet MOG_35–55-reactive CMKLR1 KO lymphocytes are deficient in their ability to induce EAE by adoptive transfer to WT or CMKLR1 KO recipients. Moreover, CMKLR1 KO recipients fail to fully support EAE induction by transferred MOG-reactive WT lymphocytes. The results imply involvement of CMKLR1 in both the induction and effector phases of disease. We conclude that CMKLR1 participates in the inflammatory mechanisms of EAE and represents a potential therapeutic target in multiple sclerosis.

Complement Activation by CpG in a Human Whole Blood Loop System: Mechanisms and Immunomodulatory Effects [INFLAMMATION]

Wed, 04 Nov 2009 13:05:42 PST

Phosphorothioate oligodeoxynucleotides can activate complement, and experimental murine studies have revealed differential effects upon simultaneous TLR stimulation and complement activation compared with either event alone. We set out to investigate the immune stimulatory effects of CpG 2006 in fresh non-anticoagulated human blood with or without presence of active complement. We also sought to elucidate the mechanism behind complement activation upon stimulation with phosphorothioate CpG 2006. In a human blood loop system, both backbone and sequence-specific effects by CpG were counteracted by selective inhibition of C3. Furthermore, DNA backbone-mediated CD40 and CD83 expression on monocytes and sequence-specific IL-6 and TNF production were reduced by complement inhibition. CpG-induced complement activation occurred via either the classical or the alternative pathway and deposits of both IgM and properdin, two activators of complement, were detected on CpG after incubation with EDTA plasma. Quartz crystal microbalance with dissipation monitoring demonstrated alternative pathway convertase build-up onto CpG as a likely pathway to initiate and sustain complement activation. Specific inhibition of C3 suppressed CpG 2006 uptake into monocytes indicating that C3 fragments are involved in CpG internalization. The interplay between complement and TLR9 signaling demonstrated herein warrants further investigation.

Bone Marrow Ly6Chigh Monocytes Are Selectively Recruited to Injured Kidney and Differentiate into Functionally Distinct Populations [INFLAMMATION]

Lin, S. L., Castano, A. P., Nowlin, B. T., Lupher, M. L., Duffield, J. S. — Wed, 04 Nov 2009 13:05:42 PST

Roles for monocyte/macrophages (M) in directing the development of tissue fibrosis are increasingly recognized. Macrophages form a heterogeneous group of inflammatory leukocytes, and the mechanisms by which they acquire heterogeneity and its functional significance are unclear. We used the unilateral ureteral obstruction model of progressive kidney fibrosis to explore macrophage heterogeneity and function further. Unilateral ureteral obstruction kidney Ms form three distinct subpopulations defined by the marker Ly6C, all of which are derived from a single Ly6C^high bone marrow monocyte population selectively recruited to the kidney. Conditional ablation of these Ms in vivo in CD11b-DTR mice is potently antifibrotic. The mRNA transcription profile of these populations is consistent with differential functional roles for each subpopulation, with Ly6C^low macrophages transcribing genes consistent with selective profibrotic or M2-type function. Furthermore, bone marrow chimerism studies indicate that although resident kidney macrophages proliferate markedly to comprise up to 40% of the inflammatory macrophage population, they do not contribute to fibrosis. Our data identify Ly6C as a marker of functionally discrete tissue macrophage subsets and support a model of selective recruitment of Ly6C^high bone marrow monocytes to the kidney that differentiate into three populations of kidney macrophages, including a profibrotic Ly6C^low population.

Nonhemopoietic Cell TLR4 Signaling Is Critical in Causing Early Lipopolysaccharide-Induced Ileus [INFLAMMATION]

Buchholz, B. M., Chanthaphavong, R. S., Bauer, A. J. M. — Wed, 04 Nov 2009 13:05:42 PST

Endotoxin-mediated ileus is poorly understood. Our objective was to mechanistically investigate the role of cell-specific TLR4 expression/signaling in causing gastrointestinal dysmotility. TLR4 chimeras and CSF-1-dependent macrophage-deficient mice were subjected to i.p. ultrapure (UP)-LPS (5 mg/kg). At 6 h, gastric emptying and gastrointestinal transit assessed in vivo motility, and jejunal circular muscle contractility was measured in vitro. Muscularis infiltration of neutrophils and monocytes were counted, and intestinal muscularis inflammatory mediators were quantified by quantitative PCR. Demonstrating TLR4 dependency, UP-LPS-induced gastric stasis and ileus of TLR4^WT mice were absent in mutant TLR4^LPS-d mice. Unexpectedly, engraftment of TLR4-mutant bone marrow into TLR4-competent mice (bmTLR4^LPS-d/TLR4^WT) exhibited a significant transit delay to UP-LPS similar to bmTLR4^WT/TLR4^WT mice. CSF-1^–/– mice were not protected from ileus. Contrary, UP-LPS-treated bmTLR4^WT/TLR4^LPS-d and bmTLR4^LPS-d/TLR4^LPS-d mice had normal transit. No leukocytic infiltration was detected at 6 h. Spontaneous jejunal contractions were markedly suppressed in UP-LPS-treated TLR4-competent mice, but bethanechol-stimulated contractions were not altered by UP-LPS in any group. UP-LPS-induced inflammatory mRNAs in a TLR4-dependent manner, but TLR4 mRNA itself was not significantly altered. In chimera mice, UP-LPS induction of IL-1β and IL-10 were hemopoietic dependent, and GM-CSF was nonhemopoietic dependent, whereas IL-6 and inducible NO synthase were derived from both cell types. Hemopoietic and nonhemopoietic cells contribute to TLR4-sensitive muscularis inflammatory signaling, but nonhemopoietic TLR4 signaling plays an exclusive primary role in causing functional UP-LPS-induced gastric stasis and ileus. Direct LPS suppression of spontaneous contractility participates in mediating early TLR4-transduced dysmotility.

Immunomodulatory Activity of Oenothein B Isolated from Epilobium angustifolium [INFLAMMATION]

Wed, 04 Nov 2009 13:05:43 PST

Epilobium angustifolium has been traditionally used to treat of a number of diseases; however, not much is known regarding its effect on innate immune cells. In this study, we report that extracts of E. angustifolium activated functional responses in neutrophils and monocyte/macrophages. Activity-guided fractionation, followed by mass spectroscopy and NMR analysis, resulted in the identification of oenothein B as the primary component responsible for phagocyte activation. Oenothein B, a dimeric hydrolysable tannin, dose-dependently induced a number of phagocyte functions in vitro, including intracellular Ca²⁺ flux, production of reactive oxygen species, chemotaxis, NF-B activation, and proinflammatory cytokine production. Furthermore, oenothein B was active in vivo, inducing keratinocyte chemoattractant production and neutrophil recruitment to the peritoneum after intraperitoneal administration. Biological activity required the full oenothein B structure, as substructures of oenothein B (pyrocatechol, gallic acid, pyrogallol, 3,4-dihydroxybenzoic acid) were all inactive. The ability of oenothein B to modulate phagocyte functions in vitro and in vivo suggests that this compound is responsible for at least part of the therapeutic properties of E. angustifolium extracts.

Adenosine Blocks IFN-{gamma}-Induced Phosphorylation of STAT1 on Serine 727 to Reduce Macrophage Activation [INFLAMMATION]

Barnholt, K. E., Kota, R. S., Aung, H. H., Rutledge, J. C. — Wed, 04 Nov 2009 13:05:43 PST

Macrophages are activated by IFN-, a proinflammatory and proatherogenic cytokine that mediates its downstream effects primarily through STAT1. IFN- signaling induces phosphorylation of two STAT1 residues: Tyr⁷⁰¹ (Y701), which facilitates dimerization, nuclear translocation, and DNA binding; and Ser⁷²⁷ (S727), which enables maximal STAT1 transcription activity. Immunosuppressive molecules such as adenosine in the cellular microenvironment can reduce macrophage inflammatory and atherogenic functions through receptor-mediated signaling pathways. We hypothesized that adenosine achieves these protective effects by interrupting IFN- signaling in activated macrophages. This investigation demonstrates that adding adenosine to IFN--stimulated murine RAW 264.7 and human THP-1 macrophages results in unique modulation of STAT1 serine and tyrosine phosphorylation events. We show that adenosine inhibits IFN--induced STAT1 S727 phosphorylation by >30% and phosphoserine-mediated transcriptional activity by 58% but has no effect on phosphorylation of Y701 or receptor-associated JAK tyrosine kinases. Inhibition of the adenosine A₃ receptor with a subtype-specific antagonist (MRS 1191 in RAW 264.7 cells and MRS 1220 in THP-1 cells) reverses this adenosine suppressive effect on STAT1 phosphoserine status by 25–50%. Further, RAW 264.7 A₃ receptor stimulation with Cl-IB-MECA reduces IFN--induced STAT1 transcriptional activity by 45% and STAT1-dependent gene expression by up to 80%. These data suggest that A₃ receptor signaling is key to adenosine-mediated STAT1 modulation and anti-inflammatory action in IFN--activated mouse and human macrophages. Because STAT1 plays a key role in IFN--induced inflammation and foam cell transformation, a better understanding of the mechanisms underlying STAT1 deactivation by adenosine may improve preventative and therapeutic approaches to vascular disease.

Cysteinyl-Leukotriene Receptor Type 1 Expression and Function Is Down-Regulated during Monocyte-Derived Dendritic Cell Maturation with Zymosan: Involvement of IL-10 and Prostaglandins [INFLAMMATION]

Thivierge, M., Stankova, J., Rola-Pleszczynski, M. — Wed, 04 Nov 2009 13:05:43 PST

TLRs sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). DCs have been shown to produce leukotrienes and, conversely, leukotrienes are known to modulate several DC functions. In this study, we examined the modulation of expression and function of cysteinyl-leukotriene receptor type 1 (CysLT1) on human monocyte-derived DCs during their differentiation and subsequent maturation with zymosan, a TLR2 agonist. Maturation of DCs with zymosan reduced CysLT1 mRNA levels and protein expression in a time-dependent fashion and was associated with a diminution of functional responsiveness to leukotriene D₄ as assessed by intracellular calcium mobilization, CCL2 and CCL3 production, and chemotaxis. The effect of zymosan was mediated by both TLR2 and dectin-1 activation. Zymosan also induced a rapid expression of cyclooxygenase-2 and the production of PGE₂ and IL-10. Addition of an anti-IL-10 neutralizing Ab or inhibitors of cyclooxygenase greatly reduced the ability of zymosan to down-regulate CysLT1 expression. Down-regulation of CysLT1 expression by zymosan could be reproduced by a combination of IL-10 and PGE₂, and was dependent on MAPK activation. Taken together, our findings indicate that zymosan down-regulates CysLT1 expression in DCs with consequently reduced functional responsiveness of the cells to leukotriene D₄ stimulation. This effect is partially dependent on an endogenous production of PGs and IL-10 by DCs.

Selective Prostacyclin Receptor Agonism Augments Glucocorticoid-Induced Gene Expression in Human Bronchial Epithelial Cells [INFLAMMATION]

Wed, 04 Nov 2009 13:05:43 PST

Prostacyclin receptor (IP-receptor) agonists display anti-inflammatory and antiviral activity in cell-based assays and in preclinical models of asthma and chronic obstructive pulmonary disease. In this study, we have extended these observations by demonstrating that IP-receptor activation also can enhance the ability of glucocorticoids to induce genes with anti-inflammatory activity. BEAS-2B bronchial epithelial cells stably transfected with a glucocorticoid response element (GRE) luciferase reporter were activated in a concentration-dependent manner by the glucocorticoid dexamethasone. An IP-receptor agonist, taprostene, increased cAMP in these cells and augmented luciferase expression at all concentrations of dexamethasone examined. Analysis of the concentration-response relationship that described this effect showed that taprostene increased the magnitude of transcription without affecting the potency of dexamethasone and was, thus, steroid-sparing in this simple system. RO3244794, an IP-receptor antagonist, and oligonucleotides that selectively silenced the IP-receptor gene, PTGIR, abolished these effects of taprostene. Infection of BEAS-2B GRE reporter cells with an adenovirus vector encoding a highly selective inhibitor of cAMP-dependent protein kinase (PKA) also prevented taprostene from enhancing GRE-dependent transcription. In BEAS-2B cells and primary cultures of human airway epithelial cells, taprostene and dexamethasone interacted either additively or cooperatively in the expression of three glucocorticoid-inducible genes (GILZ, MKP-1, and p57^kip2) that have anti-inflammatory potential. Collectively, these data show that IP-receptor agonists can augment the ability of glucocorticoids to induce anti-inflammatory genes in human airway epithelial cells by activating a cAMP/PKA-dependent mechanism. This observation may have clinical relevance in the treatment of airway inflammatory diseases that are either refractory or respond suboptimally to glucocorticoids.

Activated CD4+ T Cells Dramatically Enhance Chemotherapeutic Tumor Responses In Vitro and In Vivo [CLINICAL IMMUNOLOGY]

Radfar, S., Wang, Y., Khong, H. T. — Wed, 04 Nov 2009 13:05:43 PST

Chemoimmunotherapy has been widely studied in melanoma, with various degrees of success. One of the most common approaches is the so-called biochemotherapy, which is associated with increased toxicities, but without overall survival benefit. Another conventional strategy is the use of chemotherapy as an immunomodulator to enhance the effect of cancer vaccines or adoptive cell transfer therapy. Based on this approach, recent studies using chemotherapy to prepare the host before the infusion of ex vivo-activated, melanoma Ag-specific tumor-infiltrating lymphocytes and high dose IL-2 resulted in an impressive response rate. However, the development of immunotherapy for the treatment of a broad range of cancer type is still lacking. In this study, we report the development of a simple yet universal approach termed "chemocentric chemoimmunotherapy" that has potential application in the treatment of all cancer types. This technique uses nonspecifically activated CD4⁺ T cells as a chemosensitizer before the administration of chemotherapy. Dramatic enhancement of the cytotoxic effect of chemotherapeutic drugs, either active or nonactive as single agents, was observed both in in vitro and in vivo human tumor xenograft models. Soluble factors secreted from activated CD4⁺ T cells, likely acting on the tumor and its microenvironment, were responsible for the observed effect. Although IFN- played a major role in the therapeutic outcome, it was consistently found to be inferior to the use of activated CD4⁺ T cells in tumor chemosensitization. Our model may provide a plausible mechanism to facilitate further understanding, design and development of improved chemoimmunotherapy in the treatment of cancer.

Targeting a Mimotope Vaccine to Activating Fc{gamma} Receptors Empowers Dendritic Cells to Prime Specific CD8+ T Cell Responses in Tumor-Bearing Mice [CLINICAL IMMUNOLOGY]

Gil, M., Bieniasz, M., Wierzbicki, A., Bambach, B. J., Rokita, H., Kozbor, D. — Wed, 04 Nov 2009 13:05:43 PST

A major challenge for inducing antitumor immune responses with native or modified tumor/self-Ags in tumor-bearing hosts relates to achieving efficient uptake and processing by dendritic cells (DCs) to activate immune effector cells and limit the generation of regulatory T cell activity. We analyzed the ability of therapeutic DC vaccines expressing a CD166 cross-reactive mimotope of the GD2 ganglioside, 47-LDA, to selectively expand adoptively transferred, tumor-specific T cells in NXS2 neuroblastoma tumor-bearing syngeneic mice. Before the adoptive cell transfer and DC vaccination, the tumor-bearing mice were lymphodepleted by nonmyeloablative total body irradiation or a myeloablative regimen that required bone marrow transplantation. The 47-LDA mimotope was presented to DCs either as a linear polypeptide in conjunction with universal Th epitopes or as a fusion protein with the murine IgG2a Fc fragment (47-LDA-Fc2a) to deliver the antigenic cassette to the activating Fc receptors. We demonstrate that immunization of adoptively transferred T cells in tumor-bearing mice with the 47-LDA mimotope expressed in the context of the activating Fc fusion protein induced higher levels of antitumor immune responses and protection than the 47-LDA polypeptide-DC vaccine. The antitumor efficacy of the therapeutic 47-LDA-Fc2a-DC vaccine was comparable to that achieved by a virotherapy-associated cancer vaccine using a recombinant oncolytic vaccinia virus expressing the 47-LDA-Fc2a fusion protein. The latter treatment, however, did not require total body irradiation or adoptive cell transfer and resulted in induction of antitumor immune responses in the setting of established tolerance, paving the way for testing novel anticancer treatment strategies.

CMV-Specific TCR-Transgenic T Cells for Immunotherapy [CLINICAL IMMUNOLOGY]

Schub, A., Schuster, I. G., Hammerschmidt, W., Moosmann, A. — Wed, 04 Nov 2009 13:05:43 PST

Reactivation of CMV can cause severe disease after allogeneic hemopoietic stem cell transplantation. Adoptive T cell therapy was successfully used for patients who had received transplants from CMV-positive donors. However, patients with transplants from CMV-negative donors are at highest risk, and an adoptive therapy is missing because CMV-specific T cells are not available from such donors. To address this problem, we used retroviral transfer of CMV-specific TCR genes. We generated CMV-specific T cell clones of several HLA restrictions recognizing the endogenously processed Ag pp65. The genes of four TCRs were cloned and transferred to primary T cells from CMV-negative donors. These CMV-TCR-transgenic T cells displayed a broad spectrum of important effector functions (secretion of IFN- and IL-2, cytotoxicity, proliferation) in response to endogenously processed pp65 and could be enriched and expanded by strictly Ag-specific stimulation. Expansion of engineered T cells was accompanied by an increase in specific effector functions, indicating that the transferred specificity is stable and fully functional. Hence, we expect these CMV-TCR-transgenic T cells to be effective in controlling acute CMV disease and establishing an antiviral memory.

Type I Interferons Produced by Resident Renal Cells May Promote End-Organ Disease in Autoantibody-Mediated Glomerulonephritis [CLINICAL IMMUNOLOGY]

Wed, 04 Nov 2009 13:05:43 PST

Increased Type I IFNs or IFN-I have been associated with human systemic lupus erythematosus. Interestingly augmenting or negating IFN-I activity in murine lupus not only modulates systemic autoimmunity, but also impacts lupus nephritis, suggesting that IFN-I may be acting at the level of the end-organ. We find resident renal cells to be a dominant source of IFN-I in an experimental model of autoantibody-induced nephritis. In this model, augmenting IFN-I amplified antibody-triggered nephritis, whereas ablating IFN-I activity ameliorated disease. One mechanism through which increased IFN-I drives immune-mediated nephritis might be operative through increased recruitment of inflammatory monocytes and neutrophils, though this hypothesis needs further validation. Collectively, these studies indicate that an important contribution of IFN-I toward the disease pathology seen in systemic autoimmunity may be exercised at the level of the end-organ.

Apurinic/Apyrimidinic Endonuclease 1 Is a Key Modulator of Keratinocyte Inflammatory Responses [CLINICAL IMMUNOLOGY]

Wed, 04 Nov 2009 13:05:43 PST

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1) functions in both DNA repair and redox signaling, making it an attractive emerging therapeutic target. However, the role of APE1 in cutaneous inflammatory responses is largely unknown. In this study, we report that APE1 is a key upstream regulator in TLR2-dependent keratinocyte inflammatory responses. We found that nuclear expression of APE1 in epidermal layers was markedly up-regulated in psoriatic skin. APE1 was essential for the transcriptional activation and nuclear translocation of hypoxia-inducible factor-1 and NF-B, both of which are crucial for inflammatory signaling in keratinocytes. Moreover, APE1 played a crucial role in the expression of TLR2-mediated inflammatory mediators, including TNF-, CXCL8, and LL-37, in HaCaT cells and human primary keratinocytes. Silencing of APE1 attenuated cyclin D1/cyclin-dependent kinase 4 expression and phosphorylation of ERK1/2 and Akt, thereby affecting keratinocyte proliferation. Importantly, TLR2-induced generation of reactive oxygen species contributed to the nuclear translocation and expression of APE1, suggesting an autoregulatory circuit in which the subcellular localization of APE1 is associated with the production of APE1 per se through reactive oxygen species-dependent signaling. Taken together, these findings establish a role for APE1 as a master regulator of TLR2-dependent inflammatory responses in human keratinocytes.

Beneficial immunomodulation by Streptococcus mutans anti-P1 mAbs is Fc independent and correlates with increased exposure of a relevant target epitope [CORRECTIONS]

Robinette, R. A., Oli, M. W., McArthur, W. P., Brady, L. J. — Wed, 04 Nov 2009 13:05:43 PST

IN THIS ISSUE [IN THIS ISSUE]

Tue, 20 Oct 2009 13:05:18 PDT

Comment on "Critical Roles of NK and CD8+ T Cells in Central Nervous System Listeriosis" [LETTERS TO THE EDITOR]

Deckert, M., Schluter, D. — Tue, 20 Oct 2009 13:05:18 PDT

Response to Comment on "Critical Roles of NK and CD8+ T Cells in Central Nervous System Listeriosis" [LETTERS TO THE EDITOR]

Hayashi, T., Nagai, S., Koyasu, S. — Tue, 20 Oct 2009 13:05:18 PDT

Establishing Anergy as a Bona Fide In Vivo Mechanism of B Cell Tolerance [PILLARS OF IMMUNOLOGY]

Getahun, A., O'Neill, S. K., Cambier, J. C. — Tue, 20 Oct 2009 13:05:18 PDT

Pillars Article: Altered Immunoglobulin Expression and Functional Silencing of Self-Reactive B Lymphocytes in Transgenic Mice. Nature 1988. 334: 676-682 [PILLARS OF IMMUNOLOGY]

Tue, 20 Oct 2009 13:05:18 PDT

HOXB4-Transduced Embryonic Stem Cell-Derived Lin-c-kit+ and Lin-Sca-1+ Hematopoietic Progenitors Express H60 and Are Targeted by NK Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tabayoyong, W. B., Salas, J. G., Bonde, S., Zavazava, N. — Tue, 20 Oct 2009 13:05:18 PDT

Embryonic stem (ES) cells are a novel source of cells, especially hematopoietic progenitor cells that can be used to treat degenerative diseases in humans. However, there is a need to determine how ES cell-derived progenitors are regulated by both the adaptive and innate immune systems post transplantation. In this study, we demonstrate that hematopoietic progenitor cells (HPCs) derived from mouse ES cells ectopically expressing HOXB4 fail to engraft long-term in the presence of NK cells. In particular, the H60-expressing Lin^–c-kit⁺ and Lin^–Sca-1⁺ subpopulations were preferentially deleted in Rag2^–/–, but not in Rag2^–/–_c^–/– mice. Up-regulation of class I expression on HPCs prevented their lysis by NK cells, and Ab-mediated depletion of NK cells restored long-term HPC engraftment. In contrast to the notion that ES-derived cells are immune-privileged, we show in this study that NK cells form a formidable barrier to the long-term engraftment of ES cell-derived hematopoietic progenitors.

1,25-Dihydroxyvitamin D3 and IL-2 Combine to Inhibit T Cell Production of Inflammatory Cytokines and Promote Development of Regulatory T Cells Expressing CTLA-4 and FoxP3 [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tue, 20 Oct 2009 13:05:18 PDT

The active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious disease and autoimmune conditions. A variety of immune cells, including macrophages, dendritic cells, and activated T cells express the intracellular vitamin D receptor and are responsive to 1,25(OH)₂D_3. Despite this, how 1,25(OH)₂D₃ regulates adaptive immunity remains unclear and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue, we have assessed the effects of 1,25(OH)₂D₃ on human CD4⁺CD25^– T cells. We observed that stimulation of CD4⁺CD25^– T cells in the presence of 1,25(OH)₂D₃ inhibited production of proinflammatory cytokines including IFN- , IL-17, and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines, 1,25(OH)₂D₃ stimulated expression of high levels of CTLA-4 as well as FoxP3, the latter requiring the presence of IL-2. T cells treated with 1,25(OH)₂D₃ could suppress proliferation of normally responsive T cells, indicating that they possessed characteristics of adaptive regulatory T cells. Our results suggest that 1,25(OH)₂D₃ and IL-2 have direct synergistic effects on activated T cells, acting as potent anti-inflammatory agents and physiologic inducers of adaptive regulatory T cells.

The Adaptor Protein Shc Plays a Key Role during Early B Cell Development [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Giles, A. J., Bender, T. P., Ravichandran, K. S. — Tue, 20 Oct 2009 13:05:19 PDT

The adaptor protein Shc is phosphorylated downstream of many cell surface receptors, including Ag and cytokine receptors. However, the role of Shc in B cell development has not been addressed. Here, through conditional expression of a dominant negative Shc mutant and conditional loss of Shc protein expression, we tested a role for Shc during early B lymphopoiesis. We identified a requirement for Shc beginning at the transition from the pre-pro-B to pro-B stage, with a strong reduction in the number of pre-B cells. This developmental defect is due to increased cell death rather than impaired proliferation or commitment to the B lineage. Additional studies suggest a role for Shc in IL-7-dependent signaling in pro-B cells. Shc is phosphorylated in response to IL-7 stimulation in pro-B cells, and pro-B cells from mice with impaired Shc signaling display increased apoptosis. Together, these data demonstrate a critical role for Shc in early B lymphopoiesis with a requirement in early B cell survival. In addition, we also identify Shc as a required player in signaling downstream of the IL-7R in early B cells.

CD4+ T Cells and Lactobacillus casei Control Relapsing Colitis Mediated by CD8+ T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tue, 20 Oct 2009 13:05:19 PDT

Evidence that CD4⁺ regulatory T cells can control Ag-specific CD8⁺ T cell-mediated colitis in immunocompetent mice is poorly documented. To examine the potential of CD4⁺ T cells to control colitis, we used our model of CD8⁺ T cell-mediated colitis induced by intracolonic sensitization followed by challenge with the hapten 2,4-dinitrobenzene sulfonic acid. The defect of CD4⁺ T cells in MHC class II-deficient (Aβ°^/°) mice allowed priming of 2,4-dinitrobenzene sulfonic acid-specific IFN--producing CD8 colitogenic effectors and development of colitis in the otherwise resistant C57BL/6 strain. Cotransfer experiments in RAG2°^/° mice and ex vivo studies showed that CD4⁺CD25⁺ T cells completely prevented CD8⁺ T cell-mediated colitis and controlled CD8⁺ T cell activation, respectively. In the susceptible BALB/c strain, Ab depletion revealed that lack of CD4⁺ regulatory T cells resulted in 1) acute colitis elicited by a suboptimal dose of hapten challenge and 2) more severe relapsing episodes of colitis induced by effector/memory CD8⁺ T cell-mediated colitis at an optimal dose of hapten challenge, even when CD4 depletion was performed just before the second challenge. Oral administration of the probiotic strain Lactobacillus casei DN-114 001 alleviated colitis and increased the suppressive function of Foxp3⁺CD4⁺ regulatory T cells of colon lamina propria. These data demonstrate that CD4⁺ regulatory T cells exert a protective effect on colitis by controlling colitogenic effector/memory CD8⁺ T cells during the effector (symptomatic) phase of acute and relapsing colitis, respectively. Probiotics with natural adjuvant effects on mucosal regulatory T cells may represent a valuable approach to alleviate the colitogenic effect of Tc1-type CD8⁺ effectors.

A2A Adenosine Receptor May Allow Expansion of T Cells Lacking Effector Functions in Extracellular Adenosine-Rich Microenvironments [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tue, 20 Oct 2009 13:05:19 PDT

Immunosuppressive signaling via the A2A adenosine receptor (A2AR) provokes a mechanism that protects inflamed tissues from excessive damage by immune cells. This mechanism is desirable not only for preventing uncontrolled tissue destruction by overactive immune responses, but also for protecting tumor tissues from antitumor immune responses. In aforementioned circumstances, T cell priming may occur in an environment containing high concentrations of extracellular adenosine. To examine qualitative changes in T cells activated in the presence of adenosine, we asked whether different functional responses of T cells are equally susceptible to A2AR agonists. In this study, we demonstrate that A2AR signaling during T cell activation strongly inhibited development of cytotoxicity and cytokine-producing activity in T cells, whereas the inhibition of T cell proliferation was only marginal. Both CD8⁺ and CD4⁺ T cells proliferated well in the presence of A2AR agonists, but their IFN--producing activities were susceptible to inhibition by cAMP-elevating A2AR. Importantly, the impaired effector functions were maintained in T cells even after removal of the A2AR agonist, reflecting T cell memory of the immunoregulatory effect of adenosine. Thus, although the adenosine-rich environment may allow for the expansion of T cells, the functional activation of T cells may be critically impaired. This physiological mechanism could explain the inefficiency of antitumor T cells in the tumor microenvironment.

Phenotypical Characterization of Human Th17 Cells Unambiguously Identified by Surface IL-17A Expression [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tue, 20 Oct 2009 13:05:19 PDT

Th17 cells are involved in the defense against bacteria and fungi and play a prominent role in the pathogenesis of autoimmune diseases, but research on human Th17 cells is hindered due to the lack of a surface marker. In this study, we report that a subset of human and mouse CD4⁺ T cells as well as human Th17 T cell clones express IL-17A on their surface upon stimulation. Correlation of surface IL-17A expression with intracellular IL-17A production and with RORt mRNA expression identified surface IL-17A as a specific marker for human and mouse Th17 cells. Phenotype characterization of ex vivo CD4⁺ IL-17A⁺ cells showed that the chemokines CCR6 and CCR4, costimulatory molecules, as well as CD2 and CD49d were more prominently expressed on these cells than in surface IL-17A^– cells, supporting the concept of Th17 cells as a potent inflammatory effector subtype. In addition, we generated human Th1, Th1/17 (producing both IFN- and IL-17A), and Th17 T cell clones based on single cell sorting of surface IL-17A^–, IL-17A^int, and IL-17A^high CD4⁺ T cells, respectively, and showed the plasticity of the double producing clones to the cytokine milieu. The identification of surface IL-17A as a marker for Th17 cells should facilitate research on this subset.

FCRL3, an Autoimmune Susceptibility Gene, Has Inhibitory Potential on B-Cell Receptor-Mediated Signaling [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tue, 20 Oct 2009 13:05:19 PDT

A polymorphism that up-regulates the expression of Fc receptor-like 3 (FCRL3) gene has recently been described as predisposing for several human autoimmune diseases. FCRL3 is preferentially expressed on B cells and is unique in displaying both an ITAM and an ITIM in the cytosolic domain, suggesting signaling functions. Herein, we show that FCRL3 potentially inhibits BCR-mediated signaling, using murine FcRIIB/human FCRL3 chimeric protein. Coligation of the chimeric protein with BCR leads to phosphorylation of tyrosine residues in the cytosolic domain. This coligation inhibits cell tyrosine phosphorylation and calcium mobilization in addition to activation-induced cell death mediated by BCR signaling. Mutational analysis showed the tyrosine residues in two potential ITIMs at 662 and 692 offer the main contributions to this inhibition, which is further supported by strong associations of SH-2 domain-containing phosphatases with the following phosphotyrosine motifs: SHIP with the ITIM-like motif at 662; and SHP-1 and -2 with the canonical ITIM at 692. These results, together with previous genetic data, suggest that augmented inhibition of BCR-mediated signaling by FCRL3 with the disease-risk genotype alter the activation threshold and promote tolerance breakdown in B cells.

Functional Expression of Formyl Peptide Receptor Family in Human NK Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tue, 20 Oct 2009 13:05:19 PDT

We determined the expression of the formyl peptide receptor (FPR) family and the functional roles of the FPR family in NK cells. All tested human NK cells express two members of the FPR family (FPR1 and FPR2). The expression of FPR3 was noted to occur in a donor-specific manner. The stimulation of NK cells with FPR family-selective agonists (fMLF (N-formyl-Met-Leu-Phe), MMK-1, F2L, and WKYMVm (Trp-Lys-Tyr-Met-Val-d-Met)) elicited cytolytic activity in resting NK cells, but not in IL-2-activated NK cells; the cytolytic activity was not inhibited by pertussis toxin. The FPR family agonists also stimulated chemotactic migration of IL-2-activated NK cells, but not resting NK cells; the chemotactic migration was completely inhibited by pertussis toxin. WKYMVm stimulates ERK, p38 MAPK, and JNK activities in both resting and IL-2-activated NK cells. WKYMVm-induced chemotactic migration was partially inhibited by PD98059 (2'-amino-3'-methoxyflavone); however, the inhibition of JNK by its selective inhibitor (SP600125, anthra[1,9-cd]pyrazol-6(2H)-one) dramatically inhibited the WKYMVm-induced cytolytic activity. Furthermore, WKYMVm-induced chemotactic migration and cytolytic activity were partly inhibited by FPR family-selective antagonists (cyclosporin H and WRWWWW). Taken together, our findings indicate that human NK cells express functional members of the FPR family, and in turn the activation of the three members of the FPR receptor family elicit cytolytic activity in NK cells, thus suggesting that the receptors are potentially important therapeutic targets for the modulation of NK cell-mediated immune responses.

Ikaros Is a Regulator of Il10 Expression in CD4+ T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Umetsu, S. E., Winandy, S. — Tue, 20 Oct 2009 13:05:19 PDT

IL-10 is a regulatory cytokine critical for controlling inflammatory responses. Here we show that Ikaros, a zinc finger DNA-binding protein, plays an important role in the regulation of Il10 in murine CD4⁺ T cells. Upon initial stimulation of the TCR, T cells deficient in Ikaros express significantly lower levels of IL-10 compared with wild-type T cells. In addition, under Th2 skewing conditions, which induce IL-10 production by wild-type T cells, Ikaros null T cells are unable to properly differentiate, producing only low levels of IL-10. Expression of a dominant-negative isoform of Ikaros in wild-type Th2 cells represses IL-10 production but does not significantly alter expression levels of the genes encoding the transcription factors GATA-3 and T-bet. Furthermore, expression of Ikaros in Ikaros null T cells restores expression of the Th2 cytokines IL-10 and IL-4 while reducing production of the Th1 cytokine, IFN-. Coexpression of Ikaros and GATA-3 further increases IL-10 production, showing that these two factors have an additive effect on activating Il10 expression. Finally, we show that Ikaros binds to conserved regulatory regions of the Il10 gene locus in Th2 cells, supporting a direct role for Ikaros in Il10 expression. Thus, we provide evidence for Ikaros as a regulator of Il10 and Ifng gene expression and suggest a role for Ikaros in directing lineage-specific cytokine gene activation and repression.

The Specificity of Trimming of MHC Class I-Presented Peptides in the Endoplasmic Reticulum [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Hearn, A., York, I. A., Rock, K. L. — Tue, 20 Oct 2009 13:05:19 PDT

Aminopeptidases in the endoplasmic reticulum (ER) can cleave antigenic peptides and in so doing either create or destroy MHC class I-presented epitopes. However, the specificity of this trimming process overall and of the major ER aminopeptidase ERAP1 in particular is not well understood. This issue is important because peptide trimming influences the magnitude and specificity of CD8 T cell responses. By systematically varying the N-terminal flanking sequences of peptides in a cell-free biochemical system and in intact cells, we elucidated the specificity of ERAP1 and of ER trimming overall. ERAP1 can cleave after many amino acids on the N terminus of epitope precursors but does so at markedly different rates. The specificity seen with purified ERAP1 is similar to that observed for trimming and presentation of epitopes in the ER of intact cells. We define N-terminal sequences that are favorable or unfavorable for Ag presentation in ways that are independent from the epitopes core sequence. When databases of known presented peptides were analyzed, the residues that were preferred for the trimming of model peptide precursors were found to be overrepresented in N-terminal flanking sequences of epitopes generally. These data define key determinants in the specificity of Ag processing.

Microglial Activation by Citrobacter koseri Is Mediated by TLR4- and MyD88-Dependent Pathways [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Liu, S., Kielian, T. — Tue, 20 Oct 2009 13:05:19 PDT

Citrobacter koseri is a Gram-negative bacterium that can cause a highly aggressive form of neonatal meningitis, which often progresses to establish multifocal brain abscesses. Despite its tropism for the brain parenchyma, microglial responses to C. koseri have not yet been examined. Microglia use TLRs to recognize invading pathogens and elicit proinflammatory mediator expression important for infection containment. In this study, we investigated the importance of the LPS receptor TLR4 and MyD88, an adaptor molecule involved in the activation of the majority of TLRs in addition to the IL-1 and IL-18 receptors, for their roles in regulating microglial activation in response to C. koseri. Proinflammatory mediator release was significantly reduced in TLR4 mutant and MyD88 knockout microglia compared with wild-type cells following exposure to either live or heat-killed C. koseri, indicating a critical role for both TLR4- and MyD88-dependent pathways in microglial responses to this pathogen. However, residual proinflammatory mediator expression was still observed in TLR4 mutant and MyD88 KO microglia following C. koseri exposure, indicating a contribution of TLR4- and MyD88-independent pathway(s) for maximal pathogen recognition. Interestingly, C. koseri was capable of surviving intracellularly in both primary microglia and macrophages, suggesting that these cells may serve as a reservoir for the pathogen during CNS infections. These results demonstrate that microglia respond to C. koseri with the robust expression of proinflammatory molecules, which is dictated, in part, by TLR4- and MyD88-dependent signals.

Epidermal Langerhans Cells Are Not Required for UV-Induced Immunosuppression [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wang, L., Jameson, S. C., Hogquist, K. A. — Tue, 20 Oct 2009 13:05:19 PDT

UV light can be highly beneficial in the treatment of skin disorders such as psoriasis. It is thought to cause immunosuppression by depleting or altering the function of epidermal Langerhans cells (LC). Our previous studies identified a novel langerin⁺ dendritic cell in the dermis, distinct from LC in phenotype, circulation, and function. In this study, we determined the role of LC and dermal langerin⁺ cells in UV suppression. UV light suppressed the CD8 T cell response to both contact hypersensitivity and epicutaneous protein immunization, and resulted in a dramatically altered phenotype of LC. UV light did not alter early CD8 T cell activation in the lymph nodes, but rather reduced CD8 T cell expansion at later time points. We found that dermal langerin⁺ cells, but not LC, were essential for the CD8 T cell response. Furthermore, in the selective absence of LC, UV light still caused suppression of both CD8 T cell expansion and contact hypersensitivity.

The Activation Threshold of CD4+ T Cells Is Defined by TCR/Peptide-MHC Class II Interactions in the Thymic Medulla [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Stephen, T. L., Tikhonova, A., Riberdy, J. M., Laufer, T. M. — Tue, 20 Oct 2009 13:05:19 PDT

Immature thymocytes that are positively selected based upon their response to self-peptide-MHC complexes develop into mature T cells that are not overtly reactive to those same complexes. Developmental tuning is the active process through which TCR-associated signaling pathways of single-positive thymocytes are attenuated to respond appropriately to the peptide-MHC molecules that will be encountered in the periphery. In this study, we explore the mechanisms that regulate the tuning of CD4⁺ single-positive T cells to MHC class II encountered in the thymic medulla. Experiments with murine BM chimeras demonstrate that tuning can be mediated by MHC class II expressed by either thymic medullary epithelial cells or thymic dendritic cells. Tuning does not require the engagement of CD4 by MHC class II on stromal cells. Rather, it is mediated by interactions between MHC class II and the TCR. To understand the molecular changes that distinguish immature hyperactive T cells from tuned mature CD4⁺ T cells, we compared their responses to TCR stimulation. The altered response of mature CD4 single-positive thymocytes is characterized by the inhibition of ERK activation by low-affinity self-ligands and increased expression of the inhibitory tyrosine phosphatase SHP-1. Thus, persistent TCR engagement by peptide-MHC class II on thymic medullary stroma inhibits reactivity to self-Ags and prevents autoreactivity in the mature repertoire.

A Herceptin-Based Chimeric Antigen Receptor with Modified Signaling Domains Leads to Enhanced Survival of Transduced T Lymphocytes and Antitumor Activity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Tue, 20 Oct 2009 13:05:19 PDT

To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3 to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2⁺ tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3 signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3 signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3 ITAM mutations were less prone to apoptosis. By adding 4-1BB cytoplasmic domains to the CD28-CD3 signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4-1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.

Fas Apoptosis Inhibitory Molecule Expression in B Cells Is Regulated through IRF4 in a Feed-Forward Mechanism [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kaku, H., Rothstein, T. L. — Tue, 20 Oct 2009 13:05:19 PDT

Fas apoptosis inhibitory molecule (FAIM) was originally cloned as an inhibitor of Fas-mediated apoptosis in B cells that has been reported to affect multiple cell types. Recently, we found that FAIM enhances CD40L-mediated signal transduction, including induction of IFN regulatory factor (IRF)4, in vitro and augments plasma cell production in vivo. These results have keyed interest in the regulation of FAIM expression, about which little is known. Here, we show that Faim is regulated by IRF4. The Faim promoter contains three IRF binding sites, any two of which promote Faim expression. Faim promoter activity is lost following mutation of all three IRF binding sites, whereas activity of the full promoter is enhanced by concurrent expression of IRF4. In stimulated primary B cells, IRF4 expression precedes FAIM expression, IRF4 binds directly to the Faim promoter, and loss of IRF4 results in the failure of stimulated Faim up-regulation. Finally, FAIM is preferentially expressed in germinal center B cells. Taken together, these results indicate that FAIM expression is regulated through IRF4 and that this most likely occurs as part of germinal center formation. Because FAIM enhances CD40-induced IRF4 expression in B cells, these results suggest that induction of FAIM initiates a positive reinforcing (i.e., feed-forward) system in which IRF4 expression is both enhanced by FAIM and promotes FAIM expression.

c-Myb Is Required for Pro-B Cell Differentiation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Fahl, S. P., Crittenden, R. B., Allman, D., Bender, T. P. — Tue, 20 Oct 2009 13:05:19 PDT

The c-Myb transcription factor is required for normal adult hematopoiesis. However, the embryonic lethality of Myb-null mutations has been an impediment to identifying roles for c-Myb during lymphocyte development. We have used tissue-specific inactivation of the Myb locus in early progenitor cells to demonstrate that c-Myb is absolutely required for the differentiation of CD19⁺ B-lineage cells and B cell differentiation is profoundly blocked beyond the pre-pro-B cell stage in Myb^f/f Mb1-cre mice. We demonstrate that c-Myb is required for the intrinsic survival of CD19⁺ pro-B cells as well as the proper expression of the -chain of the IL-7 receptor (CD127) and Ebf1. However, survival of c-Myb-deficient CD19⁺ pro-B cells cannot be rescued by transduction with CD127-producing retrovirus, suggesting that c-Myb controls a survival pathway independent of CD127. Furthermore, c-Myb-deficient progenitor cells inefficiently generate CD19⁺ B-lineage cells during stromal cell culture but this process can be partially rescued with exogenous Ebf1. Thus, c-Myb does not appear to be required for commitment to B cell differentiation but is crucial for B cell differentiation to the CD19⁺ pro-B cell stage as well as survival of CD19⁺ pro-B cells. Surprisingly, forced c-Myb expression in lymphoid-primed multipotent progenitors favors differentiation toward the myeloid lineage, suggesting that proper c-Myb expression is crucial for B-lineage development.

Thalidomide Inhibits Activation of Caspase-1 [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Keller, M., Sollberger, G., Beer, H.-D. — Tue, 20 Oct 2009 13:05:19 PDT

Thalidomide is an efficient anti-inflammatory and anti-angiogenic drug, but its therapeutic use is problematic due to a strong teratogenic activity. Nevertheless, thalidomide was approved for the treatment of inflammatory skin diseases and certain types of cancer, and it is extensively tested for several other indications. Recently, we demonstrated that active caspase-1, whose activation is dependent on inflammasome complexes, is required for unconventional protein secretion of proinflammatory cytokines such as IL-1 and of the proangiogenic fibroblast growth factor 2. In this study, we show that pharmacological doses of thalidomide strongly reduced the secretion of both proteins. Thalidomide-treated cells also released less of other leaderless proteins, which require caspase-1 activity for their secretion. In line with these findings, the drug inhibited activation and activity of caspase-1 in cultured cells but not in vitro. The latter finding suggests that the pharmacological activity is exerted by a metabolite of the drug. The anti-inflammatory activity of thalidomide was also mediated via caspase-1 in mice. These findings represent a novel mechanism by which thalidomide exerts its pharmacological activity and suggest that inhibition of the activity of IL-1 might represent a novel strategy to substitute thalidomide.

Common {gamma}-Chain-Dependent Signals Confer Selective Survival of Eosinophils in the Murine Small Intestine [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Carlens, J., Wahl, B., Ballmaier, M., Bulfone-Paus, S., Forster, R., Pabst, O. — Tue, 20 Oct 2009 13:05:19 PDT

Eosinophils are potent effector cells that are recruited to sites of inflammation. However, in some tissues, in particular in the gastrointestinal tract, eosinophils constitute an abundant leukocyte population also under homeostatic conditions. The lack of suitable isolation protocols restricted the analysis of these cells to histological assessment of cell numbers while important aspects of their phenotype, turnover, and functions remain unresolved. In this study, we report a protocol that allows the quantitative isolation of intestinal eosinophils. We characterized small intestinal eosinophils by flow cytometry as SSC^highCD11b⁺CD11c⁺CCR3⁺Siglec-F⁺ cells. Intestinal eosinophils resembled eosinophils isolated from thymus and uterus but differed from eosinophils isolated from lung or blood. Eosinophils in intestine, thymus, and uterus showed in vivo a markedly higher life time compared with eosinophils present in lung and blood measured by incorporation of BrdU. This indicates that under steady-state conditions homeostasis of eosinophils is controlled by regulation of cell survival. Intestinal eosinophils are severely reduced in the intestines of Rag-2/common -chain double-deficient mice but not Rag-2-deficient mice, correlating with differential expression of GM-CSF and CCL11 in both mouse strains. Moreover, under steady-state conditions, intestinal eosinophils constitutively express high levels of the common -chain transcripts compared with lung eosinophils as well as eosinophils present under inflammatory conditions. These observations reveal a hitherto unrecognized diversity in phenotypic and functional properties of eosinophils and suggest that tissue-specific common -chain-dependent signals might profoundly affect eosinophil function and homeostasis.