Next in The JI

Cutting Edge: The Th1 Response Inhibits the Generation of Peripheral Regulatory T Cells [CUTTING EDGE]

Caretto, D., Katzman, S. D., Villarino, A. V., Gallo, E., Abbas, A. K. — Mon, 30 Nov 2009 14:05:48 PST

The possibility that effector T cells can be converted into forkhead box P3⁺ regulatory T cells (Tregs) has potential therapeutic implications. To analyze the relationship between Th1 effectors and Tregs, we have used a model of systemic autoimmunity in which both effector and Tregs arise from a single population specific for a transgene-encoded systemic protein. In vitro, the presence of IFN- inhibits Treg generation during activation. Using IFN- reporter mice, we demonstrate that IFN-–producing cells tend not to develop into Tregs, and Th1 priming of T cells prior to cell transfer limits the number of forkhead box P3⁺ T cells generated in vivo. Moreover, transfer of IFN-^–/– or STAT1^–/– T cells resulted in an increase in the number of Tregs. These data support a role for Th1 effector molecules and transcription factors in the control of peripheral Treg generation and demonstrates the limited plasticity of Th1 populations.

Cutting Edge: Resistance to Bacillus anthracis Infection Mediated by a Lethal Toxin Sensitive Allele of Nalp1b/Nlrp1b [CUTTING EDGE]

Mon, 30 Nov 2009 14:05:29 PST

Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase-1–dependent pyroptotic death in macrophages and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports, macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to infection by B. anthracis. Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.

Robust, Vaccine-Induced CD8+ T Lymphocyte Response against an Out-of-Frame Epitope [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:17 PST

Rational vaccines designed to engender T cell responses require intimate knowledge of how epitopes are generated and presented. Recently, we vaccinated 8 Mamu-A_*02⁺ rhesus macaques with every SIV protein except Envelope (Env). Surprisingly, one of the strongest T cell responses engendered was against the Env protein, the Mamu-A_*02–restricted epitope, Env_788–795RY8. In this paper, we show that translation from an alternate reading frame of both the Rev-encoding DNA plasmid and the rAd5 vector engendered Env_788–795RY8-specific CD8⁺ T cells of greater magnitude than "normal" SIV infection. Our data demonstrate both that the pathway from vaccination to immune response is not well understood and that products of alternate reading frames may be rich and untapped sources of T cell epitopes.

Mechanisms for Glycolipid Antigen-Driven Cytokine Polarization by V{alpha}14i NKT Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:48 PST

Certain glycolipid Ags for V14i NKT cells can direct the overall cytokine balance of the immune response. Th2-biasing OCH has a lower TCR avidity than the most potent agonist known, -galactosylceramide. Although the CD1d-exposed portions of OCH and -galactosylceramide are identical, structural analysis indicates that there are subtle CD1d conformational differences due to differences in the buried lipid portion of these two Ags, likely accounting for the difference in antigenic potency. Th1-biasing C-glycoside/CD1d has even weaker TCR interactions than OCH/CD1d. Despite this, C-glycoside caused a greater downstream activation of NK cells to produce IFN-, accounting for its promotion of Th1 responses. We found that this difference correlated with the finding that C-glycoside/CD1d complexes survive much longer in vivo. Therefore, we suggest that the pharmacokinetic properties of glycolipids are a major determinant of cytokine skewing, suggesting a pathway for designing therapeutic glycolipids for modulating invariant NKT cell responses.

Mitochondria Positioning Controls Local Calcium Influx in T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Schwindling, C., Quintana, A., Krause, E., Hoth, M. — Mon, 30 Nov 2009 14:05:28 PST

Formation of an immunological synapse (IS) between APC and T cells activates calcium entry through ORAI channels, which is indispensable for T cell activation. Successful proliferation and maturation of naive T cells is possible only if premature inactivation of ORAI channels is prevented. Although it is undisputed that calcium entry through ORAI channels is required for T cell function, it is not known if calcium influx is uniformly distributed over the plasma membrane or if preferential local calcium entry sites (for instance, at the IS) exist. In this study, we show that mitochondrial positioning determines the magnitude of local calcium entry anywhere in the plasma membrane by reducing local calcium-dependent channel inactivation: if mitochondria are close to any given local calcium entry site, calcium influx is large; if they are not close, calcium influx is small. Following formation of the IS, mitochondria are preferentially translocated to the IS in a calcium influx-dependent manner but independent of the exact calcium influx site. Mitochondrial enrichment at the IS favors local calcium entry at the IS without the necessity to enrich ORAI channels at the IS. We conclude that local calcium entry rather than global calcium entry is the preferential mechanism of calcium entry at stable ISs in Th cells.

Staphylococcal Complement Inhibitor Modulates Phagocyte Responses by Dimerization of Convertases [INFLAMMATION]

Mon, 30 Nov 2009 14:05:27 PST

The human pathogen Staphylococcus aureus produces several complement-evasion molecules that enable the bacterium to withstand the host immune response. The human-specific staphylococcal complement inhibitor (SCIN) blocks the central C3 convertase enzymes that trigger critical complement functions, such as C3b deposition, phagocytosis, and C5a generation. SCIN effectively blocks the conversion of C3 by alternative pathway C3 convertases (C3bBb), but also induces dimerization of these enzymes. In this study, we show that formation of dimeric convertases by SCIN is important for S. aureus immune evasion because it modulates complement recognition by phagocytic receptors. Dimeric, but not monomeric, SCIN convertases showed an impaired binding to complement receptor 1 and the complement receptor of the Ig superfamily. The dimerization site of SCIN is essential for its strong antiphagocytic properties. These studies provide critical insights into the unique immune-evasion strategies used by S. aureus.

Inhibitory TCR Coreceptor PD-1 Is a Sensitive Indicator of Low-Level Replication of SIV and HIV-1 [CLINICAL IMMUNOLOGY]

Mon, 30 Nov 2009 14:05:47 PST

Ongoing antigenic stimulation appears to be an important prerequisite for the persistent expression of programmed death 1 (PD-1), an inhibitory TCR coreceptor of the CD28 family. Although recent publications have emphasized the utility of PD-1 as a marker for dysfunctional T cells in chronic viral infections, its dependence on antigenic stimulation potentially renders it a sensitive indicator of low-level viral replication. To explore the antigenic threshold for the maintenance of PD-1 expression on virus-specific T cells, we compared PD-1 expression on virus-specific and memory T cell populations in controlled and uncontrolled SIV and HIV-1 infection. In both controlled live attenuated SIV infection in rhesus macaques and HIV-1 infection in elite controllers, elevated levels of PD-1 expression were observed on SIV- and HIV-1–specific CD8⁺ T cells. However, in contrast to chronic wild-type SIV infection and uncontrolled HIV-1 infection, controlled SIV/HIV-1 infection did not result in increased expression of PD-1 on total memory T cells. PD-1 expression on SIV-specific CD8⁺ T cells rapidly decreased after the emergence of CTL escape in cognate epitopes, but was maintained in the setting of low or undetectable levels of plasma viremia in live attenuated SIV-infected macaques. After inoculation of naive macaques with a single-cycle SIV, PD-1 expression on SIV-specific CD8⁺ T cells initially increased, but was rapidly downregulated. These results demonstrate that PD-1 can serve as a sensitive indicator of persistent, low-level virus replication and that generalized PD-1 expression on T lymphocytes is a distinguishing characteristic of uncontrolled lentiviral infections.

CD8+ T Cell Control of Hepatitis B Virus Replication: Direct Comparison between Cytolytic and Noncytolytic Functions [HOST DEFENSE]

Phillips, S., Chokshi, S., Riva, A., Evans, A., Williams, R., Naoumov, N. V. — Mon, 30 Nov 2009 14:05:26 PST

Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell–mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8⁺ T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8⁺ T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8⁺ T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8⁺ T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8⁺ T cells produce an array of cytokines, among which IFN- and TNF- are responsible for HBV inactivation in the target cells. Blockade of IFN- and TNF- abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN- and rTNF- resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8⁺ T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN- and TNF-.

Murine IgG1 and IgG3 Isotype Switch Variants Promote Phagocytosis of Cryptococcus neoformans through Different Receptors [HOST DEFENSE]

Saylor, C. A., Dadachova, E., Casadevall, A. — Mon, 30 Nov 2009 14:05:17 PST

Almost 3 decades ago, murine IgG3 was proposed to interact with a different receptor than the other IgG subclasses, but the issue remains unresolved. The question of whether a specific receptor exists for IgG3 is critically important for understanding Ab-mediated immunity against Cryptococcus neoformans, where the different isotypes manifest profound differences in protective efficacy. In this study, we revisited this question by analyzing IgG1- and IgG3-mediated phagocytosis with variable region-identical mAbs using mouse macrophages deficient in various receptors and in conditions of FcR and complement receptor blockage with specific Abs. IgG3 was an efficient opsonin for C. neoformans in FcR- and CD18-deficient cells and in the presence of blocking Abs to FcR and complement receptor. Like IgG1, IgG3-mediated phagocytosis was associated with fungal residence in a mature phagosome that was followed by intracellular replication and exocytosis events. We conclude that a specific receptor for IgG3 exists in mice that is structurally different from the known FcRs.

Diesel Exhaust Particles Stimulate Adaptive Immunity by Acting on Pulmonary Dendritic Cells [INFLAMMATION]

Mon, 30 Nov 2009 14:05:46 PST

Particulate matter, such as diesel exhaust particles (DEPs), modulate adaptive immune responses in the lung; however, their mechanism of action remains largely unclear. Pulmonary dendritic cells (DCs) are crucial mediators in regulating immune responses. We hypothesized that the immunomodulatory effects of DEPs are caused by alteration of DC function. To test this, we instilled mice with DEPs and examined the pulmonary DC recruitment and maturation, their migration to the mediastinal lymph node (MLN), and the subsequent T cell response. We demonstrated that exposure to DEPs increased DC numbers in the bronchoalveolar lavage and the lungs and that DEPs increased the maturation status of these DCs. DEP exposure also enhanced the DC migration to the MLN. Moreover, we showed that DEPs themselves were transported to the MLN in a CCR7- and DC-dependent manner. This resulted in an enhanced T cell recruitment and effector differentiation in the MLN. These data suggest that DEP inhalation modulates immune responses in the lung via stimulation of DC function.

The Pleckstrin Homology Domain Adaptor Protein Bam32/DAPP1 Is Required for Germinal Center Progression [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zhang, T.-t., Al-Alwan, M., Marshall, A. J. — Mon, 30 Nov 2009 14:05:26 PST

Ab affinity maturation within germinal centers (GCs) requires weeks to complete. Several signaling pathways in B cells have been shown to be required for initiation of the GC response; however, the signaling checkpoints controlling progression and eventual dissolution of the GC reaction are poorly understood. The adaptor protein Bam32/DAPP1 was originally isolated from human GCs and functions downstream of phosphoinositide 3-kinase enzymes, which are known to have critical roles in B cell activation and GC responses. In this study we identify a unique role of Bam32/DAPP1 in promoting GC progression. Bam32-deficient mice show normal GC initiation, but premature GC dissolution after immunization with protein Ag in alum or low doses of sheep red blood cells. Adoptive transfer studies confirmed that Bam32-deficient B cells have an intrinsic impairment in the ability to mount sustained GC responses. Bam32 deficiency was also associated with impaired Ab affinity maturation. Proliferation of Bam32-deficient GC B cells was not compromised; however, these cells show impaired switch to IgG1 and increased apoptosis in situ. GCs formed by Bam32-deficient B cells contain fewer T cells, indicating that Bam32 is required for B cell–dependent T cell accumulation within established GCs. Exogenous CD40 ligand restored GC B cell numbers and switch to IgG1, indicating that Bam32-deficient B cells are competent to respond to CD40 stimulation when ligand is available. These data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival.

Stored Fas Ligand (FasL), a Mediator of Rapid CTL-Mediated Killing, Has a Lower Threshold for Response Than Degranulation or Newly Synthesized FasL [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

He, J.-S., Gong, D.-E., Ostergaard, H. L. — Mon, 30 Nov 2009 14:05:46 PST

CTL lyse target cells through the release of cytolytic granule mediators and expression of the death receptor ligand Fas ligand (FasL). We previously demonstrated that FasL is stored in vesicles distinct from cytolytic granules and is translocated to the cell surface within 15 min of TCR stimulation, followed by a later wave of newly synthesized FasL cell surface expression at 2 h poststimulation. Initial studies suggested that the two FasL responses had different signaling thresholds. To test this possibility directly, we titrated Ag presented to murine CTL to measure FasL and degranulation response thresholds. Stored FasL translocation to the cell surface required substantially lower concentrations of peptide than was required for de novo expression of FasL and degranulation. Furthermore, a low-affinity agonist peptide stimulated strong stored FasL translocation but only limited de novo FasL expression and degranulation. These data imply that the two FasL populations may have distinct functions. We examined bystander killing and found that the rapidly expressed FasL triggered highly specific lysis of target cells, as did degranulation. In contrast, the newly synthesized later wave of FasL mediated extensive Fas-dependent bystander killing. Our data indicate that stored FasL is mobilized in response to low concentrations of Ag to mediate rapid, highly specific lysis of target cells, whereas the later, newly synthesized FasL requires higher concentrations of Ag and mediates indiscriminate lysis. These findings suggest that early and late FasL and degranulation represent nonredundant lytic mechanisms that have been selected for distinct situations, possibly for optimal pathogen clearance.

Trafficking, Persistence, and Activation State of Adoptively Transferred Allogeneic and Autologous Simian Immunodeficiency Virus-Specific CD8+ T Cell Clones during Acute and Chronic Infection of Rhesus Macaques [HOST DEFENSE]

Mon, 30 Nov 2009 14:05:25 PST

Despite multiple lines of evidence suggesting their involvement, the precise role of CD8⁺ T cells in controlling HIV replication remains unclear. To determine whether CD8⁺ T cells can limit retroviral replication in the absence of other immune responses, we transferred 1–13 x 10⁹ allogeneic in vitro expanded SIV-specific CD8⁺ T cell clones matched for the relevant restricting MHC-I allele into rhesus macaques near the time of i.v. SIV challenge. Additionally, in vitro expanded autologous SIV-specific CD8⁺ T cell clones were infused 4–9 mo postinfection. Infused cells did not appreciably impact acute or chronic viral replication. The partially MHC-matched allogeneic cells were not detected in the blood or most tissues after 3 d but persisted longer in the lungs as assessed by bronchoalveolar lavage (BAL). Autologous cells transferred i.v. or i.p. were found in BAL and blood samples for up to 8 wk postinfusion. Interestingly, despite having a nominally activated phenotype (CD69⁺HLA-DR⁺), many of these cells persisted in the BAL without dividing. This suggests that expression of such markers by T cells at mucosal sites may not reflect recent activation, but may instead identify stable resident memory T cells. The lack of impact following transfer of such a large number of functional Ag-specific CD8⁺ T cells on SIV replication may reflect the magnitude of the immune response required to contain the virus.

Distribution, Persistence, and Efficacy of Adoptively Transferred Central and Effector Memory-Derived Autologous Simian Immunodeficiency Virus-Specific CD8+ T Cell Clones in Rhesus Macaques during Acute Infection [HOST DEFENSE]

Mon, 30 Nov 2009 14:05:24 PST

Plasma viremia decreases coincident with the appearance of virus-specific CD8⁺ T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8⁺ T cell responses and transient increase in plasma viremia after depletion of CD8⁺ T cells in SIV-infected monkeys strongly suggest a role for CD8⁺ T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8⁺ T cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8⁺ T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9-, and Vif WY8-specific CD8⁺ T cell clones to acutely infected rhesus macaques. The cells persisted in PBMCs between 4 and 9 d, but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs, where they persisted at high frequency for >6 wk. Although persisting cells in the lungs were Ag reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8⁺ T cells demonstrated an escape mutation in this epitope <3 wk postinfection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8⁺ T cells for evaluating immune control during acute infection and demonstrate that infused cells retain function and persist for at least 2 mo in specific tissues.

CD4+ CD25+ Foxp3+ T Regulatory Cells with Limited TCR Diversity in Control of Autoimmunity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Adeegbe, D., Matsutani, T., Yang, J., Altman, N. H., Malek, T. R. — Mon, 30 Nov 2009 14:05:45 PST

The importance of high TCR diversity of T regulatory (Treg) cells for self-tolerance is poorly understood. To address this issue, TCR diversity was measured for Treg cells after transfer into IL-2Rβ^–/– mice, which develop lethal autoimmunity because of failed production of Treg cells. In this study, we show that high TCR diversity of pretransferred Treg cells led to selection of therapeutic Treg cells with lower TCR diversity that prevented autoimmunity. Pretransferred Treg cells with lower diversity led to selection of Treg cells through substantial peripheral reshaping with even more restricted TCR diversity that also suppressed autoimmune symptoms. Thus, in a setting of severe breakdown of immune tolerance because of failed production of Treg cells, control of autoimmunity is achieved by only a fraction of the Treg TCR repertoire, but the risk for disease increased. These data support a model in which high Treg TCR diversity is a mechanism to ensure establishing and maintaining self-tolerance.

Early CD4+ T Cell Help Prevents Partial CD8+ T Cell Exhaustion and Promotes Maintenance of Herpes Simplex Virus 1 Latency [HOST DEFENSE]

Mon, 30 Nov 2009 14:05:44 PST

HSV-specific CD8⁺ T cells provide constant immunosurveillance of HSV-1 latently infected neurons in sensory ganglia, and their functional properties are influenced by the presence of latent virus. In this study, we show that ganglionic HSV-specific CD8⁺ T cells exhibit a higher functional avidity (ability to respond to low epitope density) than their counterparts in noninfected lungs, satisfying a need for memory effector cells that can respond to low densities of viral epitopes on latently infected neurons. We further show that lack of CD4⁺ T cell help during priming leads to a transient inability to control latent virus, which was associated with a PD-1/PD-L1 mediated reduced functional avidity of ganglionic HSV-specific CD8⁺ T cells. CD4⁺ T cells are not needed to maintain CD8⁺ T cell memory through 34 d after infection, nor do they have a direct involvement in the maintenance of HSV-1 latency.

ATP-Binding Cassette Transporter G1 Negatively Regulates Thymocyte and Peripheral Lymphocyte Proliferation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Armstrong, A. J., Gebre, A. K., Parks, J. S., Hedrick, C. C. — Mon, 30 Nov 2009 14:05:23 PST

Cholesterol is a key component of cell membranes and is essential for cell growth and proliferation. How the accumulation of cellular cholesterol affects lymphocyte development and function is not well understood. We demonstrate that ATP-binding cassette transporter G1 (ABCG1) regulates cholesterol homeostasis in thymocytes and peripheral CD4 T cells. Our work is the first to describe a cell type in Abcg1-deficient mice with such a robust change in cholesterol content and the expression of cholesterol metabolism genes. Abcg1-deficient mice display increased thymocyte cellularity and enhanced proliferation of thymocytes and peripheral T lymphocytes in vivo. The absence of ABCG1 in CD4 T cells results in hyperproliferation in vitro, but only when cells are stimulated through the TCR. We hypothesize that cholesterol accumulation in Abcg1^–/– T cells alters the plasma membrane structure, resulting in enhanced TCR signaling for proliferation. Supporting this idea, we demonstrate that B6 T cells pretreated with soluble cholesterol have a significant increase in proliferation. Cholesterol accumulation in Abcg1^–/– CD4 T cells results in enhanced basal phosphorylation levels of ZAP70 and ERK1/2. Furthermore, inhibition of ERK phosphorylation in TCR-stimulated Abcg1^–/– T cells rescues the hyperproliferative phenotype. We describe a novel mechanism by which cholesterol can alter signaling from the plasma membrane to affect downstream signaling pathways and proliferation. These results implicate ABCG1 as an important negative regulator of lymphocyte proliferation through the maintenance of cellular cholesterol homeostasis.

Cutting Edge: TNF-Induced MicroRNAs Regulate TNF-Induced Expression of E-Selectin and Intercellular Adhesion Molecule-1 on Human Endothelial Cells: Feedback Control of Inflammation [CUTTING EDGE]

Suarez, Y., Wang, C., Manes, T. D., Pober, J. S. — Mon, 30 Nov 2009 14:05:44 PST

MicroRNAs (miRNAs) pair with target sequences in the 3' untranslated region of mRNAs to posttranscriptionally repress gene expression. In this study, we report that TNF-mediated induction of endothelial adhesion molecules can be regulated by miRNAs that are induced by TNF. Specifically, E-selectin and ICAM-1 are targets of TNF-induced miRNAs miR-31 and miR-17-3p, respectively. Specific antagonism of these TNF-induced miRNAs increased neutrophil adhesion to cultured endothelial cells. Conversely, transfections with mimics of these miRNAs decreased neutrophil adhesion to endothelial cells. These data suggest that miRNAs provide negative feedback control of inflammation.

Conserved Amino Acids within the Adenovirus 2 E3/19K Protein Differentially Affect Downregulation of MHC Class I and MICA/B Proteins [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Mon, 30 Nov 2009 14:05:43 PST

Successful establishment and persistence of adenovirus (Ad) infections are facilitated by immunosubversive functions encoded in the early transcription unit 3 (E3). The E3/19K protein has a dual role, preventing cell surface transport of MHC class I/HLA class I (MHC-I/HLA-I) Ags and the MHC-I–like molecules (MHC-I chain-related chain A and B [MICA/B]), thereby inhibiting both recognition by CD8 T cells and NK cells. Although some crucial functional elements in E3/19K have been identified, a systematic analysis of the functional importance of individual amino acids is missing. We now have substituted alanine for each of 21 aas in the luminal domain of Ad2 E3/19K conserved among Ads and investigated the effects on HLA-I downregulation by coimmunoprecipitation, pulse-chase analysis, and/or flow cytometry. Potential structural alterations were monitored using conformation-dependent E3/19K-specific mAbs. The results revealed that only a small number of mutations abrogated HLA-I complex formation (e.g., substitutions W52, M87, and W96). Mutants M87 and W96 were particularly interesting as they exhibited only minimal structural changes suggesting that these amino acids make direct contacts with HLA-I. The considerable number of substitutions with little functional defects implied that E3/19K may have additional cellular target molecules. Indeed, when assessing MICA/B cell-surface expression we found that mutation of T14 and M82 selectively compromised MICA/B downregulation with essentially no effect on HLA-I modulation. In general, downregulation of HLA-I was more severely affected than that of MICA/B; for example, substitutions W52, M87, and W96 essentially abrogated HLA-I modulation while largely retaining the ability to sequester MICA/B. Thus, distinct conserved amino acids seem preferentially important for a particular functional activity of E3/19K.

Lysine Acetylation Regulates Bruton's Tyrosine Kinase in B Cell Activation [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Liu, Z., Mai, A., Sun, J. — Mon, 30 Nov 2009 14:05:16 PST

Bruton's tyrosine kinase (Btk) is essential for BCR signal transduction and has diverse functions in B cells. Although Btk has been extensively studied, the role of lysine acetylation in Btk regulation has not been reported. In this study, we show that BCR cross-linking induces histone lysine acetylation at the Btk promoter, correlating with marked recruitment of histone acetyltransferase E1A-associated 300-kDa protein (p300) to the locus. These effects enhance Btk promoter activity and increase the expression of Btk mRNA and protein. Consistent with these results, activated B cells display increased p300 expression and total histone acetyltransferase activity in vitro and in vivo, resulting in global histone acetylation. Interestingly, we found that BCR signaling induces Btk lysine acetylation mediated by p300. Moreover, lysine acetylation of Btk promotes its phosphorylation. Together, our results indicate a novel regulatory mechanism for Btk transcription and reveal a previously unrecognized posttranslational modification of the Btk protein and its association with phosphorylation in B cell activation.

A Heat Shock Protein 70-Based Vaccine with Enhanced Immunogenicity for Clinical Use [CLINICAL IMMUNOLOGY]

Mon, 30 Nov 2009 14:05:42 PST

In previous studies, we have shown that heat shock protein 70-peptide complexes (HSP70.PCs) derived from the fusion of dendritic cells (DCs) to tumor cells (HSP70.PC-F) possess superior properties compared with HSP70.PCs from tumor cells. HSP70.PC-F are more effective in stimulation of DC maturation and induction of CTL that are able to provide protection of mice against challenge with tumor cells. To develop an improved formulation of HSP70.PC-based tumor vaccine for patient use, we extracted HSP70.PC-F from DCs fused to patient-derived ovarian cancer cells or established human breast cancer cells and examined their properties as tumor vaccines. HSP70.PC-F induced T cells that expressed higher levels of IFN- and exhibited increased levels of killing of tumor cells, compared with those induced by HSP70.PC derived from tumor cells. Enhanced immunogenicity of HSP70.PC-F was associated with improved composition of the vaccine, including increased content of tumor Ags and their processed intermediates, and the detection of other heat shock proteins (HSPs) such as HSP90 and HSP110. The present study has therefore provided an alternative approach to preparation of HSP-based vaccines using DC/tumor fusion technology and gentle and rapid isolation of HSP peptide complexes.

The Herpes Simplex Virus-1 Encoded Glycoprotein B Diverts HLA-DR into the Exosome Pathway [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Temme, S., Eis-Hubinger, A. M., McLellan, A. D., Koch, N. — Mon, 30 Nov 2009 14:05:23 PST

Neutralizing Abs play an important role for immunity against HSV-1 infection. This branch of the immune response is initiated by MHC class II Ag presentation and activation of T cell help. In this study, we show that the HSV-1 encoded glycoprotein B (gB) manipulates the class II processing pathway by perturbing endosomal sorting and trafficking of HLA-DR (DR) molecules. Expression of gB in the human melanoma cell line Mel JuSo results in formation of enlarged DR⁺ intracellular vesicles. Costaining of the vesicles revealed the presence of DR, gB, and the late endosomal marker CD63. The lumen of these late endosomal membranes shows a variable content, containing either gB or CD63, or both CD63 and gB. gB targets DR molecules on their biosynthetic route, after the MHC class II invariant chain is released from the DR heterodimer. gB-DR complexes were detected in a post-Golgi compartment and in exosomes, but not on the cell surface. Interestingly, increasing expression of gB strongly elevated the amount of DR and CD63 released into the exosome pathway. In conclusion, this is a previously undescribed mode of viral immune evasion involving hijacking of DR from its normal transport route to the cell surface, followed by viral-mediated release of DR into the exosome pathway.

Selective Mobilization of Cytotoxic Leukocytes by Epinephrine [CLINICAL IMMUNOLOGY]

Dimitrov, S., Lange, T., Born, J. — Mon, 30 Nov 2009 14:05:16 PST

It is well-known that acute stress, presumably as a first defense against pathogens, enhances PBMC counts by mobilizing these β2-adrenoceptor positive cells from the marginal pool. Yet, only select leukocyte subsets participate in this phenomenon of adrenergic leukocytosis and underlying mechanisms are obscure. In this study, we analyzed in human blood adhesion molecule and chemokine receptor profiles in 14 leukocyte subsets, and responsiveness of subsets to epinephrine in vivo and in vitro. Five subsets, namely, CCR7^–CD45RA⁺CD8⁺ effector T cells, CD4^–CD8^– / T cells, CD3⁺CD56⁺ NKT-like cells, CD16⁺CD56^dim cytotoxic NK cells, and CD14^dimCD16⁺ proinflammatory monocytes showed a rapid and transient increase after infusion of epinephrine at physiological concentrations. These cells were characterized by a CD62L^–CD11a^brightCX3CR^bright phenotype, whereby expression of both CD11a and CX3CR1 was strongly correlated with adrenergic leukocytosis in vivo (r = 0.86 and 0.78, p < 0.005). The same subsets showed highest adherence to activated endothelium in vitro, which (except for proinflammatory monocytes) was reversed by epinephrine. We conclude that these five cytotoxic effector leukocyte subsets comprise the marginal pool by a CD11a/CX3CR1-mediated attachment to the endothelium. Epinephrine rapidly attenuates this attachment to allow demargination and release of the cells into the circulation that, because of their cytotoxic effector function, provide immediate protection from invading pathogens.

Dissemination of Mycobacteria to the Thymus Renders Newly Generated T Cells Tolerant to the Invading Pathogen [HOST DEFENSE]

Mon, 30 Nov 2009 14:05:15 PST

The ability of the thymus to generate a population of T cells that is, for the most part, self-restricted and self-tolerant depends to a great extent on the Ags encountered during differentiation. We recently showed that mycobacteria disseminate to the thymus, which raised the questions of how mycobacteria within the thymus influence T cell differentiation and whether such an effect impacts host–pathogen interactions. Athymic nude mice were reconstituted with thymic grafts from Mycobacterium avium-infected or control noninfected donors. T cells generated from thymi of infected donors seemed generally normal, because they retained the ability to reconstitute the periphery and to respond to unspecific stimuli in vitro as well as to antigenic stimulation with third-party Ags, such as OVA, upon in vivo immunization. However, these cells were unable to mount a protective immune response against a challenge with M. avium. The observation that thymic infection interferes with T cell differentiation, generating T cells that are tolerant to pathogen-specific Ags, is of relevance to understand the immune response during chronic persistent infections. In addition, it has potential implications for the repertoire of T cells generated in patients with a mycobacterial infection recovering from severe lymphopenia, such as patients coinfected with HIV and receiving antiretroviral therapy.

The Host Defense Peptide Cathelicidin Is Required for NK Cell-Mediated Suppression of Tumor Growth [HOST DEFENSE]

Mon, 30 Nov 2009 14:05:42 PST

Tumor surveillance requires the interaction of multiple molecules and cells that participate in innate and the adaptive immunity. Cathelicidin was initially identified as an antimicrobial peptide, although it is now clear that it fulfills a variety of immune functions beyond microbial killing. Recent data have suggested contrasting roles for cathelicidin in tumor development. Because its role in tumor surveillance is not well understood, we investigated the requirement of cathelicidin in controlling transplantable tumors in mice. Cathelicidin was observed to be abundant in tumor-infiltrating NK1.1⁺ cells in mice. The importance of this finding was demonstrated by the fact that cathelicidin knockout mice (Camp^–/–) permitted faster tumor growth than wild type controls in two different xenograft tumor mouse models (B16.F10 and RMA-S). Functional in vitro analyses found that NK cells derived from Camp^–/– versus wild type mice showed impaired cytotoxic activity toward tumor targets. These findings could not be solely attributed to an observed perforin deficiency in freshly isolated Camp^–/– NK cells, because this deficiency could be partially restored by IL-2 treatment, whereas cytotoxic activity was still defective in IL-2-activated Camp^–/– NK cells. Thus, we demonstrate a previously unrecognized role of cathelicidin in NK cell antitumor function.

{gamma}{delta} T Lymphocytes Count Is Normal and Expandable in Peripheral Blood of Patients with Follicular Lymphoma, Whereas It Is Decreased in Tumor Lymph Nodes Compared with Inflammatory Lymph Nodes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:22 PST

T lymphocytes are attractive effector cells for immunotherapy. In vitro, they can be expanded and kill efficiently a variety of tumor cells. The frequency and distribution of T lymphocytes were compared in tumor lymph nodes of 51 patients with follicular lymphoma lymph nodes (FL-LNs) and 28 patients with inflammatory lymph nodes (I-LNs). and CD8 T lymphocytes were less abundant in FL-LNs than in I-LNs (p ≤ 10^–7). These lymphocytes were localized in the perifollicular zone outside of the tumor follicles. Perifollicular T lymphocytes expressed CCR7, in contrast to peripheral blood T lymphocytes and both perifollicular and peripheral blood T lymphocytes expressed CXCR4. The very low number of perifollicular T lymphocytes in FL-LNs could be explained in part by migratory problems because of absence of CCL19 expression in FL-LNs compared with I-LNs. Conversely, CCL21 and CXCL12 were similarly expressed in both FL-LNs and I-LNs. CCL19 and CCL21 were expressed in high endothelial venules and lymphatic vessels, whereas CXCL12 was expressed by stromal cells surrounding high endothelial venules and lymphatic vessels. Peripheral T lymphocytes from 34 patients with FL, expanded with Phosphostim and IL-2 in vitro, had the same expansion capacity as those from healthy individuals. Thus, T lymphocytes can be an attractive source for adoptive immunotherapy in patients with FL, providing they may home in tumor LNs.

Dynamics and Consequences of IL-21 Production in HIV-Infected Individuals: A Longitudinal and Cross-Sectional Study [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:41 PST

IL-21 is a relatively newly discovered immune-enhancing cytokine that plays an essential role in controlling chronic viral infections. It is produced mainly by CD4⁺ T cells, which are also the main targets of HIV-1 and are often depleted in HIV-infected individuals. Therefore, we sought to determine the dynamics of IL-21 production and its potential consequences for the survival of CD4⁺ T cells and frequencies of HIV-specific CTL. For this purpose, we conducted a series of cross-sectional and longitudinal studies on different groups of HIV-infected patients and show in this study that the cytokine production is compromised early in the course of the infection. The serum cytokine concentrations correlate with CD4⁺ T cell counts in the infected persons. Among different groups of HIV-infected individuals, only elite controllers maintain normal production of the cytokine. Highly active antiretroviral therapy only partially restores the production of this cytokine. Interestingly, HIV infection of human CD4⁺ T cells inhibits cytokine production by decreasing the expression of c-Maf in virus-infected cells, not in uninfected bystander cells. We also show that the frequencies of IL-21–producing HIV-specific, but not human CMV-specific, Ag-experienced CD4⁺ T cells are decreased in HIV-infected viremic patients. Furthermore, we demonstrate in this study that recombinant human IL-21 prevents enhanced spontaneous ex vivo death of CD4⁺ T cells from HIV-infected patients. Together, our results suggest that serum IL-21 concentrations may serve as a useful biomarker for monitoring HIV disease progression and the cytokine may be considered for immunotherapy in HIV-infected patients.

Anti-CD3 Antibody Decreases Inflammation and Improves Outcome in a Murine Model of Pneumocystis Pneumonia [CLINICAL IMMUNOLOGY]

Bhagwat, S. P., Wright, T. W., Gigliotti, F. — Mon, 30 Nov 2009 14:05:22 PST

The T cell–mediated immune response elicited by Pneumocystis plays a key role in pulmonary damage and dysfunction during Pneumocystis carinii pneumonia (PcP). Mice depleted of CD4⁺ and CD8⁺ T cells prior to infection are markedly protected from PcP-related respiratory deficit and death, despite progressive lung infection. However, the therapeutic effectiveness of Ab-mediated disruption of T cell function in mice already displaying clinical symptoms of disease has not been determined. Therefore, a murine model of PcP-related immune reconstitution inflammatory syndrome was used to assess whether Ab to the pan-T cell molecule CD3 is effective for reducing the severity of PcP when administered after the onset of disease. Mice that received anti-CD3 Ab exhibited a rapid and dramatic halt in the PcP-associated pulmonary function decline within 1 week after treatment, and a striking enhancement of survival rate compared with mice receiving the control Ab. Physiologic improvement in anti-CD3 treated mice was associated with a significant reduction in the number of CD4⁺ and CD8⁺ T cells recovered in lung lavage fluid. This effectiveness of anti-CD3 was noted whether the mice also received antibiotic therapy with trimethoprim-sulfamethoxazole. These data suggest that monoclonal Ab-mediated disruption of T cell function may represent a specific and effective adjunctive therapy to rapidly reverse the ongoing pathologic immune response occurring during active PcP. Thus, the anti-human CD3 monoclonal Ab OKT3, which is already in clinical use, has the potential to be developed as an adjunctive therapy for PcP.

Requisite Role of the Cholinergic {alpha}7 nAChR Pathway in Suppressing Gram-Negative Sepsis-Induced Acute Lung Inflammatory Injury [INFLAMMATION]

Su, X., Matthay, M. A., Malik, A. B. — Mon, 30 Nov 2009 14:05:40 PST

Although activation of the 7 nicotinic acetylcholine receptor (7 nAChR) modulates the response to sepsis, the role of this pathway in the development of sepsis-induced acute lung injury (ALI) is not known. In this study, we addressed the contribution of 7 nAChR in mediating endotoxin- and live Escherichia coli–induced ALI in mice. Because we found that 7 nAChR⁺ alveolar macrophages and neutrophils were present in bronchoalveolar lavage and injured lungs of mice, we tested whether acetylcholine released by lung vagal innervation stimulated these effector cells and thereby down-regulated proinflammatory chemokine/cytokine generation. Administration of 7 nAChR agonists reduced bronchoalveolar lavage MIP-2 production and transalveolar neutrophil migration and reduced mortality in E. coli pneumonia mice, whereas vagal denervation increased MIP-2 production and airway neutrophil accumulation and increased mortality. In addition, 7 nAChR^–/– mice developed severe lung injury and had higher mortality compared with 7 nAChR^+/+ mice. The immunomodulatory cholinergic 7 nAChR pathway of alveolar macrophages and neutrophils blocked LPS- and E. coli–induced ALI by reducing chemokine production and transalveolar neutrophil migration, suggesting that activation of 7 nAChR may be a promising strategy for treatment of sepsis-induced ALI.

Autoimmunity against Fibrinogen Mediates Inflammatory Arthritis in Mice [INFLAMMATION]

Mon, 30 Nov 2009 14:05:21 PST

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the presence of anticitrullinated protein Abs, although the exact targets and role of anticitrullinated protein autoimmunity in the pathogenesis of RA remain to be defined. Fibrinogen, which can be citrullinated, has recently emerged as a candidate autoantigen. To determine whether autoimmunity against fibrinogen can mediate inflammatory arthritis, we immunized a variety of common mouse strains with fibrinogen and found that DBA/1 and SJL mice developed an inflammatory and erosive arthritis. Mice with fibrinogen-induced arthritis (FIA) possess fibrinogen-reactive T cells that produce the proinflammatory cytokines IL-6, IL-17, TNF-, and IFN-. FIA can be adoptively transferred with either plasma or fibrinogen-specific T cells from diseased mice. Mice with FIA possess rheumatoid factor, circulating immune complexes, and anticyclic citrullinated peptide Abs, all of which are characteristic of human RA. These observations demonstrate that fibrinogen is arthritogenic in mice and that the pathogenesis of FIA is mediated by both autoantibodies and fibrinogen-reactive T cells.

A Minor Catalytic Activity of Src Family Kinases Is Sufficient for Maximal Activation of Mast Cells via the High-Affinity IgE Receptor [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:39 PST

Src family kinases (SFK) are critical for initiating and regulating the response of mast cells activated by engagement of the high-affinity IgE receptor, FcRI. Lyn is the predominant SFK in mast cells and has been ascribed both positive and negative roles in regulating mast cell activation. We analyzed the mast cell phenotype of WeeB, a recently described mouse mutant that expresses a Lyn protein with profoundly reduced catalytic activity. Surprisingly, we found that this residual activity is sufficient for wild-type levels of cytokine production and degranulation in bone marrow-derived mast cells after low-intensity stimulation with anti-IgE. High-intensity stimulation of lyn^–/– bone marrow-derived mast cells with highly multivalent Ag resulted in enhanced cytokine production as previously reported, and WeeB cells displayed an intermediate phenotype. Under this latter condition, SFK inhibition using PP2 increased cytokine production in wild-type and WeeB but not lyn^–/– cells, resulting in substantially higher levels in the PP2-treated WeeB than in lyn^–/– cells. Restoration of wild-type and WeeB lyn alleles in lyn^–/– cells generated activation phenotypes similar to those in nontransduced wild-type and WeeB cells, respectively, whereas a kinase-dead allele resulted in a phenotype similar to that of empty-vector–transduced cells. These data indicate that inhibition of Lyn and/or SFK activity can result in higher levels of mast cell activation than simple deletion of lyn and that only near-complete inhibition of Lyn can impair its positive regulatory functions. Furthermore, the data suggest that both positive and negative regulatory functions of Lyn are predominantly carried out by its catalytic activity and not an adaptor function.

Cutting Edge: Limited Specialization of Dendritic Cell Subsets for MHC Class II-Associated Presentation of Viral Particles [CUTTING EDGE]

Mon, 30 Nov 2009 14:05:39 PST

Dendritic cells (DCs) are the most important APC. It was recently reported that there is a dichotomy for Ag presentation by DC subsets; exogenous Ags reach the MHC class I pathway, but not the MHC class II pathway, in CD8⁺ DCs, whereas CD8^– DCs only process Ags for the MHC class II pathway. In this study, we used virus-like particles (VLPs) to show that CD8⁺ and CD8^– DCs efficiently capture and process VLPs for presentation in association with MHC class II in vivo. In contrast, CD8⁺ DCs, but not CD8^– DCs, cross presented VLP-derived peptides. This pattern was changed in an FcR-dependent fashion in the presence of VLP-specific Abs, because under those conditions both DC subsets failed to efficiently cross present. Thus, the presentation of viral particles to CD4⁺ T cells is not restricted to distinct DC subsets, whereas the presentation of viral particles to CD8⁺ T cells is limited to CD8⁺ DCs.

MART-1-Specific Melanoma Tumor-Infiltrating Lymphocytes Maintaining CD28 Expression Have Improved Survival and Expansion Capability Following Antigenic Restimulation In Vitro [CLINICAL IMMUNOLOGY]

Li, Y., Liu, S., Hernandez, J., Vence, L., Hwu, P., Radvanyi, L. — Mon, 30 Nov 2009 14:05:15 PST

We determined how CD8⁺ melanoma tumor–infiltrating lymphocytes (TILs) isolated from two distinct phases of expansion in preparation for adoptive T cell therapy respond to melanoma Ag restimulation. We found that TILs isolated after the rapid expansion protocol (REP) phase, used to generate the final patient TIL infusion product, were hyporesponsive to restimulation with MART-1 peptide-pulsed dendritic cells, with many CD8⁺ T cells undergoing apoptosis. Telomere length was shorter post-REP, but of sufficient length to support further cell division. Phenotypic analysis revealed that cell-surface CD28 expression was significantly reduced in post-REP TILs, whereas CD27 levels remained unchanged. Tracking post-REP TIL proliferation by CFSE dilution, as well as sorting for CD8⁺CD28⁺ and CD8⁺CD28^– post-REP subsets, revealed that the few CD28⁺ TILs remaining post-REP had superior survival capacity and proliferated after restimulation with MART-1 peptide. An analysis of different supportive cytokine mixtures during the REP found that a combination of IL-15 and IL-21 facilitated comparable expansion of CD8⁺ TILs as IL-2, but prevented the loss of CD28 expression with improved responsiveness to antigenic restimulation post-REP. These results suggest that current expansion protocols using IL-2 for melanoma adoptive T cell therapy yields largely CD8⁺ T cells unable to persist and divide in vivo following Ag contact. The few CD8⁺CD28⁺ T cells that remain may be the only CD8⁺ TILs that ultimately survive to repopulate the host and mediate long-term tumor control. A REP protocol using IL-15 and IL-21 may greatly increase the number of CD28⁺ TILs capable of long-term persistence.

Altered CD8+ T Cell Immunodominance after Vaccinia Virus Infection and the Naive Repertoire in Inbred and F1 Mice [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:14 PST

Previous studies of CD8⁺ T cell immunodominance after primary virus infection of F₁ mice compared with their inbred parents have generally concluded that no dramatic changes occur. In this study, we revisit this issue using vaccinia virus (VACV), which has a large genome, a recently defined immunodominance hierarchy in mice, and is a candidate vector for vaccines. We found that immunogenicity of VACV peptides defined using inbred mice was highly variable in F₁ progeny: some peptides were equally immunogenic in F₁ and inbred, whereas others elicited responses that were reduced by >90% in F₁ mice. Furthermore, the dominance of a peptide in the relevant inbred parent did not predict whether it would be poorly immunogenic in F₁ mice. This result held using F₁ hybrids of MHC-congenic mice, suggesting that MHC differences alone were responsible. It was also extended to foreign epitopes expressed by an rVACV vaccine. F₁ mice were less able to mount responses to the poorly immunogenic peptides when used as a sole immunogen, ruling out immunodomination. In addition, conserved TCR Vβ usage between inbred and F₁ mice did not always correlate with strong responses in F₁ mice. However, direct estimation of naive precursor numbers showed that these were reduced in F₁ compared with inbred mice for specificities that were poorly immunogenic in the hybrids. These data have implications for our understanding of the extent to which MHC diversity alters the range of epitopes that are immunogenic in outbred populations.

Marginal Zone Precursor B Cells as Cellular Agents for Type I IFN-Promoted Antigen Transport in Autoimmunity [CLINICAL IMMUNOLOGY]

Mon, 30 Nov 2009 14:05:38 PST

The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgM^hiCD1d^hiCD21^hiCD23^hi in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnr^–/–) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnr^–/– mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN–producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.

Human IgG2 Antibodies against Epidermal Growth Factor Receptor Effectively Trigger Antibody-Dependent Cellular Cytotoxicity but, in Contrast to IgG1, Only by Cells of Myeloid Lineage [CLINICAL IMMUNOLOGY]

Mon, 30 Nov 2009 14:05:37 PST

Ab-dependent cellular cytotoxicity (ADCC) is usually considered an important mechanism of action for immunotherapy with human IgG1 but not IgG2 Abs. The epidermal growth factor receptor (EGF-R) Ab panitumumab represents the only human IgG2 Ab approved for immunotherapy and inhibition of EGF-R signaling has been described as its principal mechanism of action. In this study, we investigated effector mechanisms of panitumumab compared with zalutumumab, an EGF-R Ab of the human IgG1 isotype. Notably, panitumumab was as effective as zalutumumab in recruiting ADCC by myeloid effector cells (i.e., neutrophils and monocytes) in contrast to NK cell-mediated ADCC, which was only induced by the IgG1 Ab. Neutrophil-mediated tumor cell killing could be stimulated by myeloid growth factors and was triggered via FcRIIa. Panitumumab-mediated ADCC was significantly affected by the functional FcRIIa-R131H polymorphism and was induced more effectively by neutrophils from FcRIIa-131H homozygous donors than from -131R individuals. This polymorphism did not affect neutrophil ADCC induced by the IgG1 Ab zalutumumab. The in vivo activity of both Abs was assessed in two animal models: a high-dose model, in which signaling inhibition is a dominant mechanism of action, and a low-dose model, in which effector cell recruitment plays a prominent role. Zalutumumab was more effective than panitumumab in the high-dose model, reflecting its stronger ability to induce EGF-R downmodulation and growth inhibition. In the low-dose model, zalutumumab and panitumumab similarly prevented tumor growth. Thus, our results identify myeloid cell-mediated ADCC as a potent and additional mechanism of action for EGF-R–directed immunotherapy.

CD4+CD25+ Regulatory T Cells Resist a Novel Form of CD28- and Fas-Dependent p53-Induced T Cell Apoptosis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:13 PST

Ag receptor stimulation of preactivated T cells causes rapid cell death in an IL-2– and Fas-dependent manner. This phenomenon, known as activation-induced cell death (AICD), plays a pivotal role in the removal of Ag-reactive T cells after initial expansion. In this study, we report a novel form of T cell apoptosis that is distinct from classic AICD. When peripheral T cells were activated with anti-CD3 and anti-CD28 Abs precoated onto plastic plates, CD4⁺CD25^– and CD8 T cells initially expanded but underwent massive apoptosis after 4 d. Unlike classic AICD, this type of T cell apoptosis pathway requires engagement of CD28 and expression of p53, a tumor-suppressor gene. The most striking feature of this form of apoptosis was regulatory T cell resistance. Under the same stimulating conditions, CD4⁺CD25⁺ T cells grew continuously beyond 4 d. Consequently, when the entire CD4 population was cultured with plate-bound anti-CD3 plus anti-CD28 Ab, CD4⁺CD25⁺FoxP3⁺ regulatory T cells outgrew nonregulatory T cells and expanded >7000-fold after 11 d. The data presented herein demonstrate a novel process of Ag-induced T cell death by sustained TCR and CD28 engagement and represent a simple and efficient procedure for the expansion of regulatory T cells in vitro.

Increased Killing of Liver NK Cells by Fas/Fas Ligand and NKG2D/NKG2D Ligand Contributes to Hepatocyte Necrosis in Virus-Induced Liver Failure [CLINICAL IMMUNOLOGY]

Mon, 30 Nov 2009 14:05:20 PST

The role of liver NK cells in virus-induced severe viral hepatitis and, subsequently, hepatic failure is not well defined. In this study, we investigated the role of liver NK cells in the development of hepatocyte necrosis in fulminant hepatic failure (FHF)and acute-on-chronic liver failure (ACLF) because of viral infection. A mouse model of FHF induced by murine hepatitis virus strain 3 (MHV-3) was used to study the role of liver NK cells. Samples from patients with hepatitis B virus-related ACLF (HBV-ACLF) were examined. After MHV-3 infection, the number of NK cells in livers of BALB/cJ mice increased markedly, peaked at 48 h postinfection, and remained at a high level until sacrifice. In peripheral blood, spleen, and bone marrow, this number decreased significantly. Expression of CD69, cytotoxic activity, and intracellular IFN- and TNF- production by liver NK cells at 48 h postinfection were all significantly upregulated. Depletion of NK cells 24 h post-MHV-3 infection increased the mice survival from 0 of 18 (0%) to 4 of 18 (22.2%). Highly activated liver NK cells were cytotoxic to MHV-3-infected hepatocytes and this effect was markedly inhibited by anti-Fas ligand (FasL) plus anti-NKG2D mAbs. Furthermore, the accumulation of hepatic NK cells and increased expression of FasL and natural cytotoxicity receptors (NKp30 and NKp46) on the peripheral NK cells from patients with HBV-ACLF were correlated with disease progression. These results indicate NK cells play a pivotal role in the pathogenesis of FHF and HBV-ACLF, in which process Fas/FasL and NKG2D/NKG2D ligand pathway contribute to the liver NK cell-mediated hepatocyte injury.

Caveolin-1 Modifies the Immunity to Pseudomonas aeruginosa [HOST DEFENSE]

Gadjeva, M., Paradis-Bleau, C., Priebe, G. P., Fichorova, R., Pier, G. B. — Mon, 30 Nov 2009 14:05:12 PST

The inflammatory response to Pseudomonas aeruginosa is not properly regulated in the lungs of patients with cystic fibrosis (CF). In the lung epithelium of individuals with wild-type CF transmembrane conductance regulator, lipid rafts containing CF transmembrance conductance regulator are rapidly formed in response to P. aeruginosa infection, and this response is closely linked to resistance to infection and disease. We found these rafts also contained high levels of caveolin-1 and thus examined the sensitivity of cav1 knockout (KO) mice to P. aeruginosa challenge in both acute and chronic P. aeruginosa infection models. We found that cav1 KO mice had increased sensitivity to P. aeruginosa infection, as represented by an increased mortality rate, elevated bacterial burdens recovered from lungs and spleens, and elevated inflammatory responses. These findings correlated with the decreased ability of cav1-deficient neutrophils to phagocytose P. aeruginosa. In addition, P. aeruginosa colonized cav1 KO mice much better compared with the wild-type controls in a model of chronic infection, indicting an important contribution of Cav-1 to innate host immunity to P. aeruginosa infection in the setting of both acute pneumonia and chronic infection typical of CF.

Phagocytosis of Apoptototic Cells by Neutrophil Granulocytes: Diminished Proinflammatory Neutrophil Functions in the Presence of Apoptotic Cells [INFLAMMATION]

Mon, 30 Nov 2009 14:05:36 PST

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN- results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF- and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-, which was independent of NF-B and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

Protective Roles of B and T Lymphocyte Attenuator in NKT Cell-Mediated Experimental Hepatitis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:35 PST

Although B and T lymphocyte attenuator (BTLA) was originally identified as an inhibitory coreceptor selectively expressed on Th1 cells and B cells, recent studies have revealed that BTLA is expressed on a variety of cells, including macrophages, dendritic cells, and NK cells, and modulates their functions. However, the role of BTLA in the regulation of NKT cell function remains unknown. In this study, we found that BTLA was expressed on NKT cells at the levels similar to those on T cells and that BTLA-deficient (BTLA^–/–) NKT cells produced larger amounts of IL-4 and IFN- upon -glactosylceramide stimulation as compared with wild-type (WT) NKT cells. In vivo, BTLA^–/– mice produced larger amounts of IL-4 and IFN- upon Con A injection and were more susceptible to Con A-induced hepatitis than WT mice. In addition, the augmentation of Con A-induced hepatitis in BTLA^–/– mice was not observed in BTLA/NKT-double deficient mice. Moreover, NKT^–/– mice reconstituted with BTLA^–/– NKT cells were significantly more susceptible to Con A-induced hepatitis as compared with NKT ^–/– mice reconstituted with WT NKT cells. These results suggest that BTLA functions as the inhibitory coreceptor of NKT cells and plays a critical role in the prevention of NKT cell-mediated liver injury.

IL-10 Mediates Resistance to Adoptive Transfer Experimental Autoimmune Encephalomyelitis in MyD88-/- Mice [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Cohen, S. J., Cohen, I. R., Nussbaum, G. — Mon, 30 Nov 2009 14:05:34 PST

MyD88 is an adaptor molecule that functions in the innate signaling induced by proinflammatory adjuvants that interact with TLRs. Mice lacking MyD88, for example, resist active experimental autoimmune encephalomyelitis (EAE) induced by immunization with an encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide in CFA. We reasoned that MyD88^–/– mice, nevertheless, should be susceptible to EAE mediated by adoptive transfer of activated encephalitogenic T cell lines, which do not require adjuvant signaling for their effector functions. We now report, however, that mice lacking MyD88 also resist adoptive EAE mediated by an anti-MOG T cell line that is strongly encephalitogenic in wild-type (WT) mice. The transferred anti-MOG T cells proliferated, secreted INF-, and migrated to the CNS in the MyD88^–/– mice, as they did in WT mice, but inflammatory infiltrates did not progress and clinical EAE did not develop. The resistance of the MyD88^–/– mice to adoptive EAE mediated by the otherwise encephalitogenic T cells was found to result from the secretion of IL-10 by recipient T cells of two different specificities: those specific for MOG and those responding to the T cell clone itself—both anticlonotypic and antiergotypic T regulators were detected. IL-10–producing anti-MOG T cells isolated from immunized MyD88^–/– mice suppressed the induction of active EAE in WT recipients. Moreover, the absence of IL-10 production in MyD88/IL-10 double-knockout mice rendered the mice susceptible to adoptive transfer of EAE. Thus, MyD88 signaling appears to be a key factor in determining the cytokine phenotype of T cells involved in autoimmune inflammation and regulation.

Mycobacterium smegmatis Expressing a Chimeric Protein MPT64-Proteolipid Protein (PLP) 139-151 Reorganizes the PLP-Specific T Cell Repertoire Favoring a CD8-Mediated Response and Induces a Relapsing Experimental Autoimmune Encephalomyelitis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:33 PST

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139–151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMS^p139). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMS^p139 was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMS^p139 modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4⁺ T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMS^p139 because lymph node APCs infected with rMS^p139 selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMS^p139 expanded p139-specific CD8⁺ cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross–reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

Sulforaphane Suppresses Oligomerization of TLR4 in a Thiol-Dependent Manner [INFLAMMATION]

Mon, 30 Nov 2009 14:05:32 PST

TLRs are pattern recognition receptors that detect invading microorganisms and nonmicrobial endogenous molecules to trigger immune and inflammatory responses during host defense and tissue repair. TLR activity is closely linked to the risk of many inflammatory diseases and immune disorders. Therefore, TLR signaling pathways can provide efficient therapeutic targets for chronic diseases. Sulforaphane (SFN), an isothiocyanate, has been well known for its anti-inflammatory activities. In this study, we investigated the modulation of TLR activity by SFN and the underlying mechanism. SFN suppressed ligand-induced and ligand-independent TLR4 activation because it prevented IL-1R–associated kinase-1 degradation, activation of NF-B and IFN regulatory factor 3, and cyclooxygenase-2 expression induced by LPS or overexpression of TLR4. Receptor oligomerization, which is one of the initial and critical events of TLR4 activation, was suppressed by SFN, resulting in the downregulation of NF-B activation. SFN formed adducts with cysteine residues in the extracellular domain of TLR4 as confirmed by liquid chromatography-tandem mass spectrometry analysis and the inhibitory effects of SFN on oligomerization and NF-B activation were reversed by thiol donors (DTT and N-acetyl-l-cysteine). These suggest that the reactivity of SFN to sulfhydryl moiety contributes to its inhibitory activities. Blockade of TLR4 signaling by SFN resulted in the reduced production of inflammatory cytokines and the decreased dermal inflammation and edema in vivo in experimental inflammatory animal models. Collectively, our results demonstrated that SFN downregulated TLR4 signaling through the suppression of oligomerization process in a thiol-dependent manner. These present a novel mechanism for beneficial effects of SFN and a novel anti-inflammatory target in TLR4 signaling.

Biodegradable Polyelectrolyte Microcapsules: Antigen Delivery Tools with Th17 Skewing Activity after Pulmonary Delivery [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:20 PST

Because of their large surface area, the lungs appear an attractive route for noninvasive vaccine delivery, harboring the potential to induce local mucosal immune responses in addition to systemic immunity. To evoke adaptive immunity, Ags require the addition of adjuvants that not only enhance the strength of the immune response but also determine the type of response elicited. In this study, we evaluate the adjuvant characteristics of polyelectrolyte microcapsules (PEMs) consisting of the biopolymers dextran-sulfate and poly-l-arginine. PEMs form an entirely new class of microcapsules that are generated by the sequential adsorption of oppositely charged polymers (polyelectrolytes) onto a sacrificial colloidal template, which is subsequently dissolved leaving a hollow microcapsule surrounded by a thin shell. Following intratracheal instillation, PEMs were not only efficiently taken up by APCs but also enhanced their activation status. Pulmonary adaptive immune responses were characterized by the induction of a strongly Th17-polarized response. When compared with a mixture of soluble Ag with empty microcapsules, Ag encapsulation significantly enhanced the strength of this local mucosal response. Given their unique property to selectively generate Th17-polarized immune responses, PEMs may become of significant interest in the development of effective vaccines against fungal and bacterial species.

Absence of Tapasin Alters Immunodominance against a Lymphocytic Choriomeningitis Virus Polytope [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:31 PST

Tapasin edits the peptide repertoire presented to CD8⁺ T cells by favoring loading of slow off-rate peptides on MHC I molecules. To investigate the role of tapasin on T cell immunodominance we used poxvirus viral vectors expressing a polytope of lymphocytic choriomeningitis virus epitopes with different off-rates. In tapasin-deficient mice, responses to subdominant fast off-rate peptides were clearly favored. This alteration of the CD8⁺ T cell hierarchy was a consequence of tapasin editing and not a consequence of the alteration of the T cell repertoire in tapasin-deficient mice, because bone marrow chimeric mice (wild-type recipients reconstituted with tapasin knockout bone marrow) showed the same hierarchy as the tapasin knockout mice. Tapasin editing is therefore a contributing factor to the phenomenon of immunodominance. Although tapasin knockout cells have low MHC I surface expression, Ag presentation was efficient and resulted in strong T cell responses involving T cells with increased functional avidity. Therefore, in this model, tapasin-deficient mice do not have a reduced but rather have an altered immune response.

IL-15 Regulates Both Quantitative and Qualitative Features of the Memory CD8 T Cell Pool [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Sandau, M. M., Kohlmeier, J. E., Woodland, D. L., Jameson, S. C. — Mon, 30 Nov 2009 14:05:19 PST

Memory T cells are critical for immunity to various intracellular pathogens. Recent studies have indicated that CD8 secondary memory cells, induced by prime-boost approaches, show enhanced protective function compared with primary memory cells and exhibit phenotypic and functional characteristics that distinguish them from primary memory cells. However, little is known about the cytokine requirements for generation and maintenance of boosted memory CD8 T cells. We studied the role of IL-15 in determining the size and composition of the secondary (2°) memory CD8 T cell pool induced by Listeria monocytogenes infection in mice. Following boosting, IL-15–deficient animals failed to generate a subset of CD8 effector memory cells, including a population of IL-7R^low cells, which were prominent among secondary memory cells in normal mice. IL-15 deficiency also resulted in changes within the IL-7R^highCD62L^low subset of 2° memory CD8 T cells, which expressed high levels of CD27 but minimal granzyme B. In addition to these qualitative changes, IL-15 deficiency resulted in reduced cell cycle and impaired Bcl-2 expression by 2° memory CD8 T cells, suggesting a role for IL-15 in supporting both basal proliferation and survival of the pool. Analogous qualitative differences in memory CD8 T cell populations were observed following a primary response to Sendai virus in IL-15^–/– animals. Collectively, these findings demonstrate that IL-15 plays an important role in dictating the composition rather than simply the maintenance of the CD8 memory pool.

Aldosterone Promotes Autoimmune Damage by Enhancing Th17-Mediated Immunity [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:19 PST

Excessive production of aldosterone leads to the development of hypertension and cardiovascular disease by generating an inflammatory state that can be promoted by T cell immunity. Because nature and intensity of T cell responses is controlled by dendritic cells (DCs), it is important to evaluate whether the function of these cells can be modulated by aldosterone. In this study we show that aldosterone augmented the activation of CD8⁺ T cells in a DC-dependent fashion. Consistently, the mineralocorticoid receptor was expressed by DCs, which showed activation of MAPK pathway and secreted IL-6 and TGF-β in response to aldosterone. In addition, DCs stimulated with aldosterone impose a Th17 phenotype to CD4⁺ T cells, which have recently been associated with the promotion of inflammatory and autoimmune diseases. Accordingly, we observed that aldosterone enhances the progression of experimental autoimmune encephalomyelitis, an autoimmune disease promoted by Th17 cells. In addition, blockade of the mineralocorticoid receptor prevented all aldosterone effects on DCs and attenuated experimental autoimmune encephalomyelitis development in aldosterone-treated mice. Our data suggest that modulation of DC function by aldosterone enhances CD8⁺ T cell activation and promotes Th17-polarized immune responses, which might contribute to the inflammatory damage leading to hypertension and cardiovascular disease.

Direct CD1d-Mediated Stimulation of APC IL-12 Production and Protective Immune Response to Virus Infection In Vivo [HOST DEFENSE]

Mon, 30 Nov 2009 14:05:30 PST

CD1d-restricted NKT cells rapidly stimulate innate and adaptive immunity through production of Th1 and/or Th2 cytokines and induction of CD1d⁺ APC maturation. However, therapeutic exploitation of NKT cells has been hampered by their paucity and defects in human disease. NKT cell–APC interactions can be modeled by direct stimulation of human APCs through CD1d in vitro. We have now found that direct ligation with multiple CD1d mAbs also stimulated bioactive IL-12 release from CD1d⁺ but not CD1d knockout murine splenocytes in vitro. Moreover, all of the CD1d mAbs tested also induced IL-12 as well as both IFN- and IFN- in vivo from CD1d⁺ but not CD1d-deficient recipients. Unlike IFN-, CD1d-induced IFN- was at least partially dependent on invariant NKT cells. Optimal resistance to infection with picornavirus encephalomyocarditis virus is known to require CD1d-dependent APC IL-12–induced IFN- as well as IFN-. CD1d ligation in vivo enhanced systemic IL-12, IFN-, and IFN- and was protective against infection by encephalomyocarditis virus, suggesting an alternative interpretation for previous results involving CD1d "blocking" in other systems. Such protective responses, including elevations in Th1 cytokines, were also seen with CD1d F(ab’)₂s in vivo, whereas an IgM mAb (with presumably minimal tissue penetration) was comparably effective at protection in vivo as well as cytokine induction both in vivo and in vitro. Although presumably acting immediately "downstream," CD1d mAbs were protective later during infection than the invariant NKT cell agonist -galactosylceramide. These data indicate that NKT cells can be bypassed with CD1d-mediated induction of robust Th1 immunity, which may have therapeutic potential both directly and as an adjuvant.

Differentiation Stage-Specific Requirement in Hypoxia-Inducible Factor-1{alpha}-Regulated Glycolytic Pathway during Murine B Cell Development in Bone Marrow [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 30 Nov 2009 14:05:18 PST

Hypoxia-inducible factor (HIF)-1 plays a central role in oxygen homeostasis and energy supply by glycolysis in many cell types. We previously reported that an HIF-1 gene deficiency caused abnormal B cell development and autoimmunity. In this study we show that HIF-1–enabled glycolysis during B cell development is required in a developmental stage-specific manner. Supporting this conclusion are observations that the glycolytic pathway in HIF-1–deficient B220⁺ bone marrow cells is much less functionally effective than in wild-type control cells. The expression of genes encoding the glucose transporters and the key glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bishosphatase 3, was greatly reduced in HIF-1–deficient cells. The compensatory adaptation to the defect of glycolysis was reflected in higher levels of expression of respiratory chain-related genes and TCA cycle-related genes in HIF-1–deficient cells than in wild-type cells. In agreement with these findings, HIF-1–deficient cells used pyruvate more efficiently than wild-type cells. The key role of HIF-1–enabled glycolysis in bone marrow B cells was also demonstrated by glucose deprivation during in vitro bone marrow cell culture and by using a glycolysis inhibitor in the bone marrow cell culture. Taken together, these findings indicate that glucose dependency differs at different B cell developmental stages and that HIF-1 plays an important role in B cell development.

Tracking the Total CD8 T Cell Response to Infection Reveals Substantial Discordance in Magnitude and Kinetics between Inbred and Outbred Hosts [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Rai, D., Pham, N.-L. L., Harty, J. T., Badovinac, V. P. — Mon, 23 Nov 2009 09:06:35 PST

Determining the magnitude and kinetics, together with the phenotypic and functional characteristics of responding CD8 T cells, is critical for understanding the regulation of adaptive immunity as well as in evaluating vaccine candidates. Recent technical advances have allowed tracking of some CD8 T cells responding to infection, and a body of information now exists describing phenotypic changes that occur in CD8 T cells of known Ag-specificity during their activation, expansion, and memory generation in inbred mice. In this study, we demonstrate that Ag but not inflammation-driven changes in expression of CD11a and CD8 can be used to distinguish naive from Ag-experienced (effector and memory) CD8 T cells after infection or vaccination. Interestingly and in contrast to inbred mice, tracking polyclonal CD8 T cell responses with this approach after bacterial and viral infections revealed substantial discordance in the magnitude and kinetics of CD8 T cell responses in outbred hosts. These data reveal limitations to the use of inbred mouse strains as preclinical models at vaccine development and suggest the same dose of infection or vaccination can lead to substantial differences in the magnitude and timing of Ag-specific CD8 expansion as well in differences in protective memory CD8 T cell numbers in outbred individuals. This concept has direct relevance to development of vaccines in outbred humans.

Accumulation of CD11b+ Lung Dendritic Cells in Response to Fungal Infection Results from the CCR2-Mediated Recruitment and Differentiation of Ly-6Chigh Monocytes [HOST DEFENSE]

Mon, 23 Nov 2009 09:06:35 PST

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans is associated with the CCR2-mediated accumulation of lung dendritic cells (DC) and the development of a T1 adaptive immune response. The objective of this study was to identify the circulating DC precursor(s) responsible for this large increase in lung DC numbers. An established murine model was used to evaluate putative DC precursors in the blood, bone marrow, and lungs of CCR2^+/+ mice and CCR2^–/– mice throughout a time course following infection with C. neoformans. Results demonstrate that numbers of Ly-6C^high monocytes increased in parallel in the peripheral blood and lungs of CCR^+/+ mice, whereas CD11c⁺ MHC class II⁺ pre-DC were 10-fold less prevalent in the peripheral blood and did not differ between the two strains. Accumulation of Ly-6C^high monocytes correlated with a substantial increase in the numbers of CD11b⁺ DC in the lungs of infected CCR2^+/+ mice. Comparative phenotypic analysis of lung cells recovered in vivo suggests that Ly-6C^high monocytes differentiate into CD11b⁺ DC in the lung; differentiation is associated with up-regulation of costimulatory molecules and decreased Ly-6C expression. Furthermore, in vitro experiments confirmed that Ly-6C^high monocytes differentiate into CD11b⁺ DC. Accumulation of Ly-6C^high monocytes and CD11b⁺ DC was not attributable to their proliferation in situ. We conclude that the CCR2-mediated accumulation of CD11b⁺ DC in the lungs of Cryptococcus-infected mice is primarily attributable to the continuous recruitment and differentiation of Ly-6C^high monocytes.

Siglec-E Is Up-Regulated and Phosphorylated Following Lipopolysaccharide Stimulation in Order to Limit TLR-Driven Cytokine Production [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:34 PST

Although production of cytokines by TLR is essential for viral and bacterial clearance, overproduction can be detrimental, thus controlling these responses is essential. CD33-related sialic acid binding Ig-like lectin receptors (Siglecs) have been implicated in the control of leukocyte responses. In this study, we report that murine Siglec-E is induced by TLRs in a MyD88-specific manner, is tyrosine phosphorylated following LPS stimulation, and negatively regulates TLR responses. Specifically, we demonstrate the Siglec-E expression inhibits TLR-induced NF-B and more importantly, the induction of the antiviral cytokines IFN-β and RANTES. Siglec-E mediates its inhibitory effects on TIR domain containing adaptor inducing IFN-β (TRIF)-dependent cytokine production via recruitment of the serine/threonine phosphatase SHP2 and subsequent inhibition of TBK1 activity as evidenced by enhanced TBK1 phosphorylation in cells following knockdown of Siglec-E expression. Taken together, our results demonstrate a novel role for Siglec-E in controlling the antiviral response to TLRs and thus helping to maintain a healthy cytokine balance following infection.

Thymus-Blood Protein Interactions Are Highly Effective in Negative Selection and Regulatory T Cell Induction [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Atibalentja, D. F., Byersdorfer, C. A., Unanue, E. R. — Mon, 23 Nov 2009 09:06:33 PST

Using hen egg-white lysozyme, the effect of blood proteins on CD4 thymic cells was examined. A small fraction of i.v. injected hen egg-white lysozyme rapidly entered the thymus into the medulla. There it was captured and presented by dendritic cells (DCs) to thymocytes from two TCR transgenic mice, one directed to a dominant peptide and a second to a poorly displayed peptide, both presented by MHC class II molecules I-A^k. Presentation by DC led to negative selection and induction of regulatory T cells, independent of epithelial cells. Presentation took place at very low levels, less than 100 peptide-MHC complexes per DC. Such low levels could induce negative selection, but even lower levels could induce regulatory T cells. The anatomy of the thymus-blood barrier, the highly efficient presentation by DC, together with the high sensitivity of thymic T cells to peptide-MHC complexes, results in blood protein Ags having a profound effect on thymic T cells.

Bim-Mediated Apoptosis Is Not Necessary for Thymic Negative Selection to Ubiquitous Self-Antigens [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Hu, Q., Sader, A., Parkman, J. C., Baldwin, T. A. — Mon, 23 Nov 2009 09:06:32 PST

T cell education in the thymus is critical for establishing a functional, yet self-tolerant, T cell repertoire. Negative selection is a key process in enforcing self-tolerance. There are many questions that surround the mechanism of negative selection, but it is currently held that apoptosis initiated by Bim and/or Nur77 is critical for negative selection. Recent studies, however, have questioned the necessity of Bim in maintaining both central and peripheral T cell tolerance. To reconcile these apparently contradictory findings, we examined the role of Bim in negative selection in the well-characterized, physiological HY^cd4 mouse model. We found that while Bim expression was required for CD4⁺CD8⁺ double-positive thymocyte apoptosis, it was not required for negative selection. Furthermore, Bim deficiency did not alter the frequency or affinity of male reactive cells that escape negative selection in an oligoclonal repertoire. Collectively, these studies indicate that negative selection occurs efficiently in the absence of apoptosis and suggest that the current paradigm of negative selection requiring apoptosis be revisited.

{beta}-Defensins 2 and 3 Together Promote Resistance to P. aeruginosa Keratitis [HOST DEFENSE]

Wu, M., McClellan, S. A., Barrett, R. P., Zhang, Y., Hazlett, L. D. — Mon, 23 Nov 2009 09:06:31 PST

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine β-defensins (mBD) 3 and 4, the murine homologs of human β-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-, MIP-2, IL-1β, TNF-, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-B. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

Signal-Transducing Adaptor Protein-2 Regulates Stromal Cell-Derived Factor-1{alpha}-Induced Chemotaxis in T Cells [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Mon, 23 Nov 2009 09:06:31 PST

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a YXXQ motif in its C-terminal region. Our previous studies revealed that STAP-2 regulates integrin-mediated T cell adhesion. In the present study, we find that STAP-2 expression affects Jurkat T cell migration after stromal cell-derived factor-1 (SDF-1)-treatment. Furthermore, STAP-2-deficient T cells exhibit reduced cell migration after SDF-1-treatment. Importantly, overexpression of STAP-2 in Jurkat T cells induces activation of small guanine triphosphatases, such as Rac1 and Cdc42. Regarding the mechanism for this effect, we found that STAP-2 associates with Vav1, the guanine-nucleotide exchanging factor for Rac1, and enhances downstream Vav1/Rac1 signaling. These results reveal a novel STAP-2-mediated mechanism for the regulation of SDF-1-induced chemotaxis of T cells via activation of Vav1/Rac1 signaling.

RasGRP1 Is Required for Human NK Cell Function [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:30 PST

Cross-linking of NK activating receptors activates phospholipase- and subsequently induces diacylglycerol and Ca²⁺ as second messengers of signal transduction. Previous studies reported that Ras guanyl nucleotide-releasing protein (RasGRP) 1, which is activated by diacylglycerol and Ca²⁺, is crucial for TCR-mediated Ras-ERK activation. We now report that RasGRP1, which can also be detected in human NK cells, plays an essential role in NK cell effector functions. To examine the role of RasGRP1 in NK cell functions, the expression of RasGRP1 was suppressed using RNA interference. Knockdown of RasGRP1 significantly blocked ITAM-dependent cytokine production as well as NK cytotoxicity. Biochemically, RasGRP1-knockdown NK cells showed markedly decreased ability to activate Ras, ERK, and JNK. Activation of the Ras-MAPK pathway was independently shown to be indispensable for NK cell effector functions via the use of specific pharmacological inhibitors. Our results reveal that RasGRP1 is required for the activation of the Ras-MAPK pathway leading to NK cell effector functions. Moreover, our data suggest that RasGRP1 might act as an important bridge between phospholipase- activation and NK cell effector functions via the Ras-MAPK pathway.

In a Murine Tuberculosis Model, the Absence of Homeostatic Chemokines Delays Granuloma Formation and Protective Immunity [HOST DEFENSE]

Mon, 23 Nov 2009 09:06:29 PST

Mycobacterium tuberculosis infection (Mtb) results in the generation of protective cellular immunity and formation of granulomatous structures in the lung. CXCL13, CCL21, and CCL19 are constitutively expressed in the secondary lymphoid organs and play a dominant role in the homing of lymphocytes and dendritic cells. Although it is known that dendritic cell transport of Mtb from the lung to the draining lymph node is dependent on CCL19/CCL21, we show in this study that CCL19/CCL21 is also important for the accumulation of Ag-specific IFN--producing T cells in the lung, development of the granuloma, and control of mycobacteria. Importantly, we also show that CXCL13 is not required for generation of IFN- responses, but is essential for the spatial arrangement of lymphocytes within granulomas, optimal activation of phagocytes, and subsequent control of mycobacterial growth. Furthermore, we show that these chemokines are also induced in the lung during the early immune responses following pulmonary Mtb infection. These results demonstrate that homeostatic chemokines perform distinct functions that cooperate to mediate effective expression of immunity against Mtb infection.

TLR2 Engagement on Dendritic Cells Promotes High Frequency Effector and Memory CD4 T Cell Responses [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Chandran, S. S., Verhoeven, D., Teijaro, J. R., Fenton, M. J., Farber, D. L. — Mon, 23 Nov 2009 09:06:28 PST

Ligation of TLR by distinct pathogen components provides essential signals for T cell priming, although how individual TLR engagement affects primary and memory T cell responses is not well defined. In this study, we demonstrate distinct effects of TLR2 vs TLR4 engagement on primary and memory CD4 T cell responses due to differential effects on APC. Priming of influenza hemagglutinin (HA)-specific naive CD4 T cells with HA peptide and the TLR2 agonist Pam3CysK in vivo resulted in a high frequency of activated HA-specific CD4 T cells that predominantly produced IL-2 and IL-17, whereas priming with HA peptide and the TLR4 agonist LPS yielded a lower frequency of HA-specific CD4 T cells and predominant IFN- producers. TLR2 agonist priming depended on TLR2 expression by APC, as wild-type CD4 T cells did not expand in response to peptide and Pam3CysK in TLR2-deficient hosts. TLR2-mediated priming also led to an increased frequency of Ag-specific memory CD4 T cells compared with TLR4 priming and mediated enhanced secondary responses to influenza challenge. Our results show that TLR engagement on APC influences both primary and secondary CD4 T cell responses, and suggest that long-term functional capacities of T cells are set by innate signals during early phases of an infection.

The IL-27 p28 Subunit Binds Cytokine-Like Factor 1 to Form a Cytokine Regulating NK and T Cell Activities Requiring IL-6R for Signaling [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:27 PST

IL-27 is formed by the association of a cytokine subunit, p28, with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3^–/– and WSX-1^–/– mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27, p28/CLF is produced by dendritic cells and is biologically active on human NK cells, increasing IL-12- and IL-2-induced IFN- production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6R in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6R-dependent STAT1 and STAT3 phosphorylation. Furthermore, p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors.

Tumor Progression Locus 2 (Map3k8) Is Critical for Host Defense against Listeria monocytogenes and IL-1{beta} Production [HOST DEFENSE]

Mon, 23 Nov 2009 09:06:26 PST

Tumor progression locus 2 (Tpl2, also known as Map3k8 and Cot) is a serine-threonine kinase critical in innate immunity, linking toll-like receptors (TLRs) to TNF production through its activation of ERK. Tpl2^–/– macrophages have abrogated TNF production but overproduce IL-12 in response to TLR ligands. Despite enhanced IL-12 production, Tpl2^–/– T cells have impaired IFN- production. Therefore, the role of Tpl2 in a bona fide bacterial infection where all of these cytokines are important in host defense is unclear. To address this issue, we infected Tpl2^–/– mice with the model pathogen Listeria monocytogenes. We found that Tpl2^–/– mice infected i.v. with L. monocytogenes had increased pathogen burdens compared with wild-type mice and rapidly succumbed to infection. Enhanced susceptibility correlated with impaired signaling through TLR2 and nucleotide-binding oligomerization domain 2, two receptors previously shown to mediate Listeria recognition. Surprisingly, TNF production in response to infection was not significantly impaired, even though Tpl2 has been implicated in the regulation of TNF. We found that the role of Tpl2 has cell-type specific effects in regulating TNF and transduces signals from some, but not all, pattern recognition receptors (PRR). In contrast to the cell-type- and receptor-specific regulation of TNF, we found that Tpl2 is essential for IL-1β production from both macrophages and dendritic cells. These studies implicate Tpl2 as an important mediator for collaboration of pattern recognition receptors with danger-associated molecular patterns to induce TNF and IL-1β production and optimal host defense.

Human Activated T Lymphocytes Modulate IDO Expression in Tumors through Th1/Th2 Balance [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:25 PST

Previous cancer vaccination approaches have shown some efficiency in generating measurable immune responses, but they have rarely led to tumor regression. It is therefore possible that tumors emerge with the capacity to down-regulate immune counterparts, through the local production of immunosuppressive molecules, such as IDO. Although it is known that IDO exerts suppressive effects on T cell functions, the mechanisms of IDO regulation in tumor cells remain to be characterized. Here, we demonstrate that activated T cells can induce functional IDO expression in breast and kidney tumor cell lines, and that this is partly attributable to IFN-. Moreover, we found that IL-13, a Th2 cytokine, has a negative modulatory effect on IDO expression. Furthermore, we report IDO expression in the majority of breast and kidney carcinoma samples, with infiltration of activated Th1-polarized T cells in human tumors. These findings demonstrate complex control of immune activity within tumors. Future immune therapeutic interventions should thus include strategies to counteract these negative mechanisms.

Activation, Immune Polarization, and Graft-versus-Leukemia Activity of Donor T Cells Are Regulated by Specific Subsets of Donor Bone Marrow Antigen-Presenting Cells in Allogeneic Hemopoietic Stem Cell Transplantation [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:24 PST

We investigated the roles of specific subsets of donor APCs purified from bone marrow in donor T cell activation and graft-vs-leukemia (GvL) activity in murine models of hemopoietic stem cell transplantation. Lineage^–CD11c⁺ APC precursors were separated from donor bone marrow based on expression of CD11b. Transplanting lineage^–CD11c⁺CD11b^– APC (CD11b^– APC) in combination with c-kit⁺Sca-1⁺lineage^– hemopoietic stem cells (HSC) and congenic donor T cells led to increased donor CD4⁺ and CD8⁺ T cell proliferation and higher donor T cell chimerism than with transplanting grafts containing HSC, T cells, and lineage^–CD11c⁺CD11b⁺ APCs (CD11b⁺ APC), or grafts containing only HSC and T cells. Transplanting CD11b^– APCs induced Th1/type 1 cytotoxic T lymphocyte donor T cell immune polarization and enhanced GvL activity of donor T cells without increased graft-vs-host disease in both MHC- and minor histocompatibility Ag-mismatched murine hemopoietic stem cell transplantation models, whereas CD11b⁺ APCs led to Th2/type 2 cytotoxic T lymphocyte donor T cell immune polarization. Donor CD11b^– APCs were plasmacytoid dendritic cell progenitors (>90% CD317; PDCA-1⁺) and up-regulated CD80, CD86, and IL-12 during alloantigen presentation, whereas CD11b⁺ APCs expressed Gr-1 and up-regulated expression of programmed death ligands-1 and 2 after activation. These results are the first to show that manipulation of the content of donor APCs in allogeneic HSC grafts can regulate donor T cell immunity and enhance GvL without increasing graft-vs-host disease activity.

Ubiquitin Conjugation of ORFF DNA Vaccine Leads to Enhanced Cell-Mediated Immune Response and Induces Protection against Both Antimony-Susceptible and -Resistant Strains of Leishmania donovani [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Sharma, A., Madhubala, R. — Mon, 23 Nov 2009 09:06:23 PST

Resistance of Leishmania donovani to sodium antimony gluconate has become a critical issue in the current, prolonged epidemic in India. Hence, there is an urgent need for a vaccine that is protective against both antimony-susceptible and -resistant strains of L. donovani. The multigene LD1 locus located on chromosome 35 of Leishmania is amplified in ~15% of the isolates examined. The open reading frame F (ORFF), a potential vaccine candidate against visceral leishmaniasis, is part of the multigene LD1 locus. ORFF was expressed as a chimeric conjugate of ubiquitin to elicit an Ag-specific cell-mediated immune response. Analysis of the cellular immune responses of ubiquitin-conjugated ORFF (UBQ-ORFF) DNA-immunized, uninfected BALB/c mice demonstrated that the vaccine induced enhanced IFN--producing CD4⁺ and CD8⁺ T cells compared with nonubiquitinated ORFF DNA vaccine. Higher levels of IL-12 and IFN- and the low levels of IL-4 and IL-10 further indicated that the immune responses with UBQ-ORFF were mediated toward the Th1 rather than Th2 type. Infection of immunized mice with either the antimony-susceptible (AG83) or -resistant (GE1F8R) L. donovani strain showed that UBQ-ORFF DNA vaccine induced higher protection when compared with ORFF DNA. UBQ-ORFF DNA-immunized and -infected mice showed a significant increase in IL-12 and IFN- and significant down-regulation of IL-10. High levels of production of nitrite and superoxide, two macrophage-derived oxidants that are critical in controlling Leishmania infection, were observed in protected mice. The feasibility of using ubiquitinated-conjugated ORFF DNA vaccine as a promising immune enhancer for vaccination against both antimony-susceptible and -resistant strains of L. donovani is reported.

The Yellow Fever Virus Vaccine Induces a Broad and Polyfunctional Human Memory CD8+ T Cell Response [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:23 PST

The live yellow fever vaccine (YF-17D) offers a unique opportunity to study memory CD8⁺ T cell differentiation in humans following an acute viral infection. We have performed a comprehensive analysis of the virus-specific CD8⁺ T cell response using overlapping peptides spanning the entire viral genome. Our results showed that the YF-17D vaccine induces a broad CD8⁺ T cell response targeting several epitopes within each viral protein. We identified a dominant HLA-A2-restricted epitope in the NS4B protein and used tetramers specific for this epitope to track the CD8⁺ T cell response over a 2 year period. This longitudinal analysis showed the following. 1) Memory CD8⁺ T cells appear to pass through an effector phase and then gradually down-regulate expression of activation markers and effector molecules. 2) This effector phase was characterized by down-regulation of CD127, Bcl-2, CCR7, and CD45RA and was followed by a substantial contraction resulting in a pool of memory T cells that re-expressed CD127, Bcl-2, and CD45RA. 3) These memory cells were polyfunctional in terms of degranulation and production of the cytokines IFN-, TNF-, IL-2, and MIP-1β. 4) The YF-17D-specific memory CD8⁺ T cells had a phenotype (CCR7^–CD45RA⁺) that is typically associated with terminally differentiated cells with limited proliferative capacity (T_EMRA). However, these cells exhibited robust proliferative potential showing that expression of CD45RA may not always associate with terminal differentiation and, in fact, may be an indicator of highly functional memory CD8⁺ T cells generated after acute viral infections.

B7-1/2 (CD80/CD86) Direct Signaling to B Cells Enhances IgG Secretion [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Rau, F. C., Dieter, J., Luo, Z., Priest, S. O., Baumgarth, N. — Mon, 23 Nov 2009 09:06:22 PST

B cell responses are regulated by Ag recognition, costimulatory signals provided by interaction with helper T cells, and by innate signals. We recently provided evidence for a link between the effects of innate and costimulatory signals on B cells during influenza virus infection, by demonstrating that most B cells in the regional lymph nodes of the respiratory tract enhance surface expression of the costimulator B7-2 (CD86) within 24–48 h following infection via a type I IFNR-dependent mechanisms, a finding we are confirming here. While the role of B7-1/2 for helper T cell activation is well documented, its role in direct B cell regulation is poorly understood. Here, our in vivo studies with mixed bone marrow irradiation chimeric mice, lacking B7-1/2 only on B cells, demonstrated that B7-1/2 expression is crucial for induction of maximal local, but to a lesser extent systemic, IgG Ab responses following influenza virus infection. In contrast to mice that completely lack B7-1/2 expression, loss of B7-1/2 on B cells alone did not significantly affect germinal center formation or the extent of CD4⁺ T cell activation and IFN- secretion. Instead, our in vitro studies identify a dramatic effect of B7-2 engagement on IgG, but not IgM secretion by already class-switched B cells. Concomitantly, B7-2 engagement induced expression of X-box binding protein 1 (XBP-1) and spliced XBP1, evidence for increased protein synthesis by these cells. Taken together, these results identify direct signaling through B7-1/2 as a potent regulator of IgG secretion by previously activated B cells.

Sublingual Immunization with Nonreplicating Antigens Induces Antibody-Forming Cells and Cytotoxic T Cells in the Female Genital Tract Mucosa and Protects against Genital Papillomavirus Infection [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:21 PST

We have recently reported that the sublingual (s.l.) mucosa is an efficient site for inducing systemic and mucosal immune responses. In this study, the potential of s.l. immunization to induce remote Ab responses and CD8⁺ cytotoxic responses in the female genital tract was examined in mice by using a nonreplicating Ag, OVA, and cholera toxin (CT) as an adjuvant. Sublingual administration of OVA and CT induced Ag-specific IgA and IgG Abs in blood and in cervicovaginal secretions. These responses were associated with large numbers of IgA Ab-secreting cells (ASCs) in the genital mucosa. Genital ASC responses were similar in magnitude and isotype distribution after s.l., intranasal, or vaginal immunization and were superior to those seen after intragastric immunization. Genital, but not blood or spleen, IgA ASC responses were inhibited by treatment with anti-CCL28 Abs, suggesting that the chemokine CCL28 plays a major role in the migration of IgA ASC progenitors to the reproductive tract mucosa. Furthermore, s.l. immunization with OVA induced OVA-specific effector CD8⁺ cytolytic T cells in the genital mucosa, and these responses required coadministration of the CT adjuvant. Furthermore, s.l. administration of human papillomavirus virus-like particles with or without the CT adjuvant conferred protection against genital challenge with human papillomavirus pseudovirions. Taken together, these findings underscore the potential of s.l. immunization as an efficient vaccination strategy for inducing genital immune responses and should impact on the development of vaccines against sexually transmitted diseases.

The Runx3 Transcription Factor Augments Th1 and Down-Modulates Th2 Phenotypes by Interacting with and Attenuating GATA3 [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:20 PST

Recently, it was reported that the expression of Runt-related transcription factor 3 (Runx3) is up-regulated in CD4⁺ helper T cells during Th1 cell differentiation, and that Runx3 functions in a positive feed-forward manner with the T-box family transcription factor, T-bet, which is a master regulator of Th1 cell differentiation. The relative expression levels of IFN- and IL-4 are also regulated by the Th2-associated transcription factor, GATA3. Here, we demonstrate that Runx3 was induced in Th2 as well as Th1 cells and that Runx3 interacted with GATA3 and attenuated GATA3 transcriptional activity. Ectopic expression of Runx3 in vitro in cultured cells or transgenic expression of Runx3 in mice accelerated CD4⁺ cells to a Th1-biased population or down-modulated Th2 responses, in part by neutralizing GATA3. Our results suggest that the balance of Runx3 and GATA3 is one factor that influences the manifestation of CD4⁺ cells as the Th1 or Th2 phenotypes.

Caspase-1, Caspase-8, and Calpain Are Dispensable for IL-33 Release by Macrophages [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Mon, 23 Nov 2009 09:06:19 PST

In addition to IL-1 and IL-18, IL-33 was recently identified as a member of the IL-1 cytokine family. rIL-33 can promote production of Th2-type cytokines by Th2 cells and mast cells in vitro. Administration of rIL-33 to mice results in increases in IgE secretion and eosinophilic inflammation. However, the precise immune cell source of IL-33 remains unclear. Moreover, although recombinant pro-IL-33 is cleaved by recombinant caspase-1 in vitro, as are pro-IL-1β and pro-IL-18, the involvement of caspase-1 in pro-IL-33 cleavage remains controversial. In this study, we show that mouse peritoneal macrophages, but not splenic dendritic cells, produced IL-33 upon stimulation with LPS. Likewise, mouse bone marrow cell-derived cultured mast cells also produced a small, but significant amount of IL-33 via FcRI cross-linking, but not in response to stimulation with LPS. To our surprise, IL-33 release was found even in caspase-1-deficient, caspase-8 inhibitor-treated, and calpain inhibitor-treated macrophages. These observations suggest that caspase-1-, caspase-8-, and calpain-independent IL-33 production by macrophages and/or mast cells may contribute to the pathogenesis of Th2-type allergic inflammation.

The Heavy Chain Variable Segment Gene Repertoire in Chronic Chagas' Heart Disease [HOST DEFENSE]

Mon, 23 Nov 2009 09:06:19 PST

Patients chronically infected with Trypanosoma cruzi develop chronic Chagas` heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas` disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.

Modulation of Allergenicity of Major House Dust Mite Allergens Der f 1 and Der p 1 by Interaction with an Endogenous Ligand [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Mon, 23 Nov 2009 09:06:18 PST

Although many allergens bind endogenous molecules other than Abs in the human body, whether the interaction can modulate allergenicity has been unknown. Here, we investigated the effect of the interaction of recombinant major mite group 1 allergens (Der f 1 and Der p 1), which belong to the papain-like cysteine protease family, with an endogenous protease inhibitor, cystatin A, on their allergenicity. Cystatin A bound reduced forms of the allergens, in which the cysteine residue at the catalytic center of the protease activity was reduced by treatment with l-cysteine, but did not bind oxidized forms. Cystatin A partially inhibited the binding of IgE in mite-allergic volunteers' sera to the reduced forms, but unexpectedly enhanced the basophil histamine-releasing activity. A catalytic site-mutant of Der f 1 behaved in terms of histamine release, similarly to the reduced form. Molecular modeling showed that cystatin A interacts with the allergens within a narrow area. The results indicate that interaction with cystatin A reduces the limited number of IgE epitopes of the allergens but enhances their biological activity to release histamine, suggesting a new concept, that interaction between allergens and their endogenous ligands modulates the allergenicity even toward enhancement in the effector phase. On the other hand, i.p. immunization without alum of mice with cystatin A-treated reduced Der f 1 induced less serum Der f 1-specific IgE than immunization with reduced Der f 1 alone, suggesting that endogenous protease inhibitors suppress the induction of allergen-specific IgE, which is dependent on the enzymatic activity of cysteine protease-allergens, in the sensitization process.

Distinct Antiviral Roles for Human 2',5'-Oligoadenylate Synthetase Family Members against Dengue Virus Infection [HOST DEFENSE]

Lin, R.-J., Yu, H.-P., Chang, B.-L., Tang, W.-C., Liao, C.-L., Lin, Y.-L. — Wed, 18 Nov 2009 12:21:50 PST

The 2`,5`-oligoadenylate synthetase (OAS) and its downstream effector RNase L play important roles in host defense against virus infection. Oas1b, one of the eight Oas1 genes in the mouse genome, has been identified as a murine flavivirus-resistance gene. Four genes, OAS1, OAS2, OAS3, and OAS-like (OASL), have been identified in the human OAS gene family, and 10 isoforms, including OAS1 (p42, p44, p46, p48, and p52), OAS2 (p69 and p71), OAS3 (p100), and OASL (p30 and p59) can be generated by alternative splicing. In this study, we determined the role of the human OAS/RNase L pathway in host defense against dengue virus (DEN) infection and assessed the antiviral potential of each isoform in the human OAS family. DEN replication was reduced by overexpression and enhanced by knockdown of RNase L expression, indicating a protective role for RNase L against DEN replication in human cells. The human OAS1 p42, OAS1 p46, and OAS3 p100, but not the other OAS isoforms, blocked DEN replication via an RNase L-dependent mechanism. Furthermore, the anti-DEN activities of these three OAS isoforms correlated with their ability to trigger RNase L activation in DEN-infected cells. Thus, OAS1 p42/p46 and OAS3 p100 are likely to contribute to host defense against DEN infection and play a role in determining the outcomes of DEN disease severity.

The Tpl2 Mutation Sluggish Impairs Type I IFN Production and Increases Susceptibility to Group B Streptococcal Disease [IMMUNOGENETICS]

Wed, 18 Nov 2009 12:21:50 PST

Sluggish was identified in a population of third generation mice descended from N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes exhibited impaired TNF- production in response to all TLR ligands tested and displayed impaired type I IFN production in response to TLR7 and TLR9 stimulations. The phenotype was confined to a critical region on mouse chromosome 18 and then ascribed to a T to A transversion in the acceptor splice site of intron 4 at position 13346 of the Map3k8 gene, resulting in defective splicing. The Map3k8^Sluggish mutation does not result in susceptibility to viral infections, but Sluggish mice displayed high susceptibility to group B streptococcus infection, with impaired TNF- and type I IFN production in infected macrophages. Our data demonstrate that the encoded protein kinase Tpl2 plays an essential role in cell signaling in the immune response to certain pathogens.

Langerin+CD8{alpha}+ Dendritic Cells Are Critical for Cross-Priming and IL-12 Production in Response to Systemic Antigens [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 12:21:49 PST

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8⁺ DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8⁺ T cells, while CD8^– DC are more likely to present extracellular Ags on MHC class II molecules to CD4⁺ T cells. As a proportion of CD8⁺ DC have been shown to express langerin (CD207), we investigated the role of langerin⁺CD8⁺ DC in presenting Ag and priming T cell responses to soluble Ags. When splenic DC populations were sorted from animals administered protein i.v., the ability to cross-present Ag was restricted to the langerin⁺ compartment of the CD8⁺ DC population. The langerin⁺CD8⁺ DC population was also susceptible to depletion following administration of cytochrome c, which is known to trigger apoptosis if diverted to the cytosol. Cross-priming of CTL in the presence of the adjuvant activity of the TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride or the invariant NKT cell ligand -galactosylceramide was severely impaired in animals selectively depleted of langerin⁺ cells in vivo. The production of IL-12p40 in response to these systemic activation stimuli was restricted to langerin⁺CD8⁺ DC, and the release of IL-12p70 into the serum following invariant NKT cell activation was ablated in the absence of langerin⁺ cells. These data suggest a critical role for the langerin⁺ compartment of the CD8⁺ DC population in cross-priming and IL-12 production.

Activated CD8+ T-Effector/Memory Cells Eliminate CD4+ CD25+ Foxp3+ T-Suppressor Cells from Tumors via FasL Mediated Apoptosis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Kilinc, M. O., Rowswell-Turner, R. B., Gu, T., Virtuoso, L. P., Egilmez, N. K. — Wed, 18 Nov 2009 12:21:48 PST

Tumor-resident CD8⁺ T cells display a quiescent effector/memory phenotype that is maintained in part by infiltrating CD4⁺ CD25⁺ Foxp3⁺ T-suppressor cells. Intratumoral delivery of IL-12, in contrast, can restore cytotoxic function to tumor-associated CD8⁺ T cells and induce the apoptotic death of T-suppressor cells. Depletion of CD8⁺ T cells from tumors before IL-12 treatment resulted in the abrogation of treatment-mediated T-suppressor cell apoptosis revealing a link between CD8⁺ T cell activation and T-suppressor elimination. Furthermore, IL-12 failed to induce T-suppressor cell loss in IFN-- or FasL-deficient mice demonstrating a requirement for IFN- and FasL in this process. Adoptive transfer of wild-type CD8⁺ T cells to FasL-knockout mice restored posttherapy T-suppressor cell elimination from tumors establishing that expression of FasL on CD8⁺ T cells was sufficient to promote T-suppressor cell death. IL-12 failed to induce FasL on T-effectors in IFN--knockout mice demonstrating a requirement for IFN- in FasL up-regulation. Adoptive transfer of wild-type CD8⁺ T cells induced T-suppressor cell death in IFN--knockout mice confirming that autocrine IFN- was sufficient for CD8⁺ T cell FasL expression. These findings reveal a mechanism by which cytotoxic T cells can abrogate regulatory cell activity.

New Design of MHC Class II Tetramers to Accommodate Fundamental Principles of Antigen Presentation [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 18 Nov 2009 12:21:47 PST

Direct identification and isolation of Ag-specific T cells became possible with the development of MHC tetramers, based on fluorescent avidins displaying biotinylated peptide-MHC complexes. This approach, extensively used for MHC class I-restricted T cells, has met very limited success with class II peptide-MHC complex tetramers (pMHCT-2) for the detection of CD4⁺-specific T cells. In addition, a very large number of these reagents, although capable of specifically activating T cells after being coated on solid support, is still unable to stain. To try to understand this puzzle and design usable tetramers, we examined each parameter critical for the production of pMHCT-2 using the I-A^d-OVA system as a model. Through this process, the geometry of peptide-MHC display by avidin tetramers was examined, as well as the stability of rMHC molecules. However, we discovered that the most important factor limiting the reactivity of pMHCT-2 was the display of peptides. Indeed, long peptides, as presented by MHC class II molecules, can be bound to I-A/HLA-DQ molecules in more than one register, as suggested by structural studies. This mode of anchorless peptide binding allows the selection of a broader repertoire on single peptides and should favor anti-infectious immune responses. Thus, beyond the technical improvements that we propose, the redesign of pMHCT-2 will give us the tools to evaluate the real size of the CD4 T cell repertoire and help us in the production and testing of new vaccines.

Mesenchymal Stem Cells Derived from Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zhang, Q., Shi, S., Liu, Y., Uyanne, J., Shi, Y., Shi, S., Le, A. D. — Wed, 18 Nov 2009 12:21:46 PST

Aside from the well-established self-renewal and multipotent differentiation properties, mesenchymal stem cells exhibit both immunomodulatory and anti-inflammatory roles in several experimental autoimmune and inflammatory diseases. In this study, we isolated a new population of stem cells from human gingiva, a tissue source easily accessible from the oral cavity, namely, gingiva-derived mesenchymal stem cells (GMSCs), which exhibited clonogenicity, self-renewal, and multipotent differentiation capacities. Most importantly, GMSCs were capable of immunomodulatory functions, specifically suppressed peripheral blood lymphocyte proliferation, induced expression of a wide panel of immunosuppressive factors including IL-10, IDO, inducible NO synthase (iNOS), and cyclooxygenase 2 (COX-2) in response to the inflammatory cytokine, IFN-. Cell-based therapy using systemic infusion of GMSCs in experimental colitis significantly ameliorated both clinical and histopathological severity of the colonic inflammation, restored the injured gastrointestinal mucosal tissues, reversed diarrhea and weight loss, and suppressed the overall disease activity in mice. The therapeutic effect of GMSCs was mediated, in part, by the suppression of inflammatory infiltrates and inflammatory cytokines/mediators and the increased infiltration of regulatory T cells and the expression of anti-inflammatory cytokine IL-10 at the colonic sites. Taken together, GMSCs can function as an immunomodulatory and anti-inflammatory component of the immune system in vivo and is a promising cell source for cell-based treatment in experimental inflammatory diseases.

Viruses within the Flaviviridae Decrease CD4 Expression and Inhibit HIV Replication in Human CD4+ Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 12:21:45 PST

Viral infections alter host cell homeostasis and this may lead to immune evasion and/or interfere with the replication of other microbes in coinfected hosts. Two flaviviruses are associated with a reduction in HIV replication or improved survival in HIV-infected people (dengue virus (DV) and GB virus type C (GBV-C)). GBV-C infection and expression of the GBV-C nonstructural protein 5A (NS5A) and the DV NS5 protein in CD4⁺ T cells inhibit HIV replication in vitro. To determine whether the inhibitory effect on HIV replication is conserved among other flaviviruses and to characterize mechanism(s) of HIV inhibition, the NS5 proteins of GBV-C, DV, hepatitis C virus, West Nile virus, and yellow fever virus (YFV; vaccine strain 17D) were expressed in CD4⁺ T cells. All NS5 proteins inhibited HIV replication. This correlated with decreased steady-state CD4 mRNA levels and reduced cell surface CD4 protein expression. Infection of CD4⁺ T cells and macrophages with YFV (17D vaccine strain) also inhibited HIV replication and decreased CD4 gene expression. In contrast, mumps virus was not inhibited by the expression of flavivirus NS5 protein or by YFV infection, and mumps infection did not alter CD4 mRNA or protein levels. In summary, CD4 gene expression is decreased by all human flavivirus NS5 proteins studied. CD4 regulation by flaviviruses may interfere with innate and adaptive immunity and contribute to in vitro HIV replication inhibition. Characterization of the mechanisms by which flaviviruses regulate CD4 expression may lead to novel therapeutic strategies for HIV and immunological diseases.

An Innate Response to Allogeneic Nonself Mediated by Monocytes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Zecher, D., van Rooijen, N., Rothstein, D. M., Shlomchik, W. D., Lakkis, F. G. — Wed, 18 Nov 2009 12:21:44 PST

The mammalian innate immune system has evolved diverse strategies to distinguish self from microbial nonself. How the innate immune system distinguishes self-tissues from those of other members of the same species (allogeneic nonself) is less clear. To address this question, we studied the cutaneous hypersensitivity response of lymphocyte-deficient RAG^–/– mice to spleen cells transplanted from either allogeneic or syngeneic RAG^–/– donors. We found that RAG^–/– mice mount a specific response to allogeneic cells characterized by swelling and infiltration of the skin with host monocytes/macrophages and neutrophils. The response required prior priming with allogeneic splenocytes or skin grafts and exhibited features of memory as it could be elicited at least 4 wk after immunization. Neither depletion of host NK cells nor rechallenging immunized mice with F₁ hybrid splenocytes inhibited the response, indicating that the response is not mediated by NK cells. Depletion of host monocytes/macrophages or neutrophils at the time of rechallenge significantly diminished the response and, importantly, the adoptive transfer of monocytes from alloimmunized RAG^–/– mice conferred alloimmunity to naive RAG^–/– hosts. Unlike NK- and T cell-dependent alloresponses, monocyte-mediated alloimmunity could be elicited only when donor and responder mice differed at non-MHC loci. These observations indicate that monocytes mount a response to allogeneic nonself, a function not previously attributed to them, and suggest the existence of mammalian innate allorecognition strategies distinct from detection of missing self-MHC molecules by NK cells.

Duality of Enhancer Functioning Mode Revealed in a Reduced TCR{beta} Gene Enhancer Knockin Mouse Model [MOLECULAR AND STRUCTURAL IMMUNOLOGY]

Wed, 18 Nov 2009 12:21:43 PST

The TCRβ gene enhancer (Eβ) commands TCRβ gene expression through the lifespan of T lymphocytes. Genetic and molecular studies have implied that in early thymocytes, Eβ directs chromatin opening over the Dβ-Jβ-Cβ domains and triggers initial Dβ-Jβ recombination. In mature T cells, Eβ is required for expression of the assembled TCRβ gene. Whether these separate activities rely on distinct Eβ regulatory sequences and involve differing modes of activation is unclear. Using gene targeting in mouse embryonic stem cells, we replaced Eβ by a conserved core fragment (Eβ169). We found that Eβ169-carrying alleles were capable of sustaining β gene expression and the development of mature T cells in homozygous knockin mice. Surprisingly, these procedures and underlying molecular transactions were affected to a wide range of degrees depending on the developmental stage. Early thymocytes barely achieved Dβ-Jβ germline transcription and recombination. In contrast, T cells displayed substantial though heterogeneous levels of VDJ-rearranged TCRβ gene expression. Our results have implications regarding enhancer function in cells of the adaptive immune system and, potentially, TCRβ gene recombination and allelic exclusion.

IL-7 Enhances Thymic Human T Cell Development in "Human Immune System" Rag2-/-IL-2R{gamma}c-/- Mice without Affecting Peripheral T Cell Homeostasis [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 12:21:39 PST

IL-7 is a central cytokine in the development of hematopoietic cells, although interspecies discrepancies have been reported. By coculturing human postnatal thymus hematopoietic progenitors and OP9-huDL1 stromal cells, we found that murine IL-7 is ~100-fold less potent than human IL-7 for supporting human T cell development in vitro. We investigated the role of human IL-7 in newborn BALB/c Rag2^–/–_c^–/– mice transplanted with human hematopoietic stem cells (HSC) as an in vivo model of human hematopoiesis using three approaches to improve IL-7 signaling: administration of human IL-7, ectopic expression of human IL-7 by the transplanted human HSC, or enforced expression of a murine/human chimeric IL-7 receptor binding murine IL-7. We show that premature IL-7 signaling at the HSC stage, before entrance in the thymus, impeded T cell development, whereas increased intrathymic IL-7 signaling significantly enhanced the maintenance of immature thymocytes. Increased thymopoiesis was also observed when we transplanted BCL-2- or BCL-x_L-transduced human HSC. Homeostasis of peripheral mature T cells in this humanized mouse model was not improved by any of these strategies. Overall, our results provide evidence for an important role of IL-7 in human T cell development in vivo and highlight the notion that IL-7 availability is but one of many signals that condition peripheral T cell homeostasis.

CCR7 Modulates Pulmonary and Lymph Node Inflammatory Responses in Cigarette Smoke-Exposed Mice [INFLAMMATION]

Wed, 18 Nov 2009 12:21:37 PST

Peribronchial lymphoid follicles have recently been identified as one of the hallmark features of (severe) chronic obstructive pulmonary disease (COPD). However, little is known about the relative contribution of peribronchial lymphoid follicles vs mediastinal lymph nodes in inflammatory responses in COPD patients and animal models. In a murine model of COPD, we studied inflammatory responses in airways, lungs, and mediastinal lymph nodes of wild-type (WT) vs CCR7 knockout (CCR7^–/–) mice upon subacute or chronic exposure to cigarette smoke (CS). Although crucial for the organization of the secondary lymphoid organs, CCR7 was not required for the development of chronic CS-induced pulmonary lymphoid follicles. Moreover, T cell numbers were significantly increased in airways and lungs of air-exposed CCR7^–/– mice, and they continued to increase upon chronic CS exposure. Unexpectedly, subacute CS-induced inflammation in airways and lungs, including airway neutrophilia and the recruitment of inflammatory-type CD11b⁺ dendritic cells, depended greatly on CCR7. In the draining lymph nodes, chronic CS exposure induced CCR7-dependent recruitment of airway-derived dendritic cells, accompanied by increases in CD4⁺ and CD8⁺ T cells. Correspondingly, CS exposure up-regulated mRNA expression of CCR7 ligands CCL19 and CCL21-Ser in lymph nodes of WT mice, but not CCR7^–/– mice. In the lungs of WT mice, chronic CS exposure significantly increased CCL19 mRNA and protein. Furthermore, double staining for CCL19 and pro-surfactant protein C showed that alveolar type II cells express high levels of CCL19. These data unveil a so far unappreciated role for CCR7 in modulating inflammatory responses in airways and lungs.

Matrix Metalloproteinase (MMP)-1 and MMP-3 Induce Macrophage MMP-9: Evidence for the Role of TNF-{alpha} and Cyclooxygenase-2 [INFLAMMATION]

Wed, 18 Nov 2009 12:21:36 PST

Matrix metalloproteinase (MMP)-9 (gelatinase B) participates in a variety of diverse physiologic and pathologic processes. We recently characterized a cyclooxygenase-2 (COX-2)->PGE₂->EP4 receptor axis that regulates macrophage MMP-9 expression. In the present studies, we determined whether MMPs, commonly found in inflamed and neoplastic tissues, regulate this prostanoid-EP receptor axis leading to enhanced MMP-9 expression. Results demonstrate that exposure of murine peritoneal macrophages and RAW264.7 macrophages to MMP-1 (collagenase-1) or MMP-3 (stromelysin-1) lead to a marked increase in COX-2 expression, PGE₂ secretion, and subsequent induction of MMP-9 expression. Proteinase-induced MMP-9 expression was blocked in macrophages preincubated with the selective COX-2 inhibitor celecoxib or transfected with COX-2 small interfering RNA (siRNA). Likewise, proteinase-induced MMP-9 was blocked in macrophages preincubated with the EP4 antagonist ONO-AE3-208 or transfected with EP4 siRNA. Exposure of macrophages to MMP-1 and MMP-3 triggered the rapid release of TNF-, which was blocked by MMP inhibitors. Furthermore, both COX-2 and MMP-9 expression were inhibited in macrophages preincubated with anti-TNF- IgG or transfected with TNF- siRNA. Thus, proteinase-induced MMP-9 expression by macrophages is dependent on the release of TNF-, induction of COX-2 expression, and PGE₂ engagement of EP4. The ability of MMP-1 and MMP-3 to regulate macrophage secretion of PGE₂ and expression of MMP-9 defines a nexus between MMPs and prostanoids that is likely to play a role in the pathogenesis of chronic inflammatory diseases and cancer. These data also suggest that this nexus is targetable utilizing anti-TNF- therapies and/or selective EP4 antagonists.

Cutting Edge: Inhibitory Effects of CD4 and CD8 on T Cell Activation Induced by High-Affinity, Noncognate Ligands [CUTTING EDGE]

Chervin, A. S., Stone, J. D., Bowerman, N. A., Kranz, D. M. — Wed, 18 Nov 2009 12:21:35 PST

It has been proposed that MHC restriction during thymocyte selection is controlled by coreceptor (CD4 or CD8) sequestration of the signaling molecule Lck. We explored this model as a mechanism for preventing peripheral T cell activation due to non-MHC ligand cross-reactivities of TCRs. TCRs that have a range of affinities for a class I MHC ligand were transduced into a T cell hybridoma in the absence or presence of coreceptors. High and intermediate affinity TCRs (K_D=17 and 540 nM) did not require CD8 for T cell activity, but CD4 acted as a potent inhibitor of the intermediate affinity TCR. These and other findings support the view that even high-affinity TCR:ligand interactions can be influenced by coreceptor sequestration of Lck. Thus, CD4 and CD8 act as "coreceptor inhibitors" to maintain appropriate TCR-mediated MHCrestriction in peripheral T cell activity.

IL-23 Is Required for Protection against Systemic Infection with Listeria monocytogenes [HOST DEFENSE]

Meeks, K. D., Sieve, A. N., Kolls, J. K., Ghilardi, N., Berg, R. E. — Wed, 18 Nov 2009 12:21:34 PST

Listeria monocytogenes (LM) is a Gram-positive, intracellular bacterium that can induce spontaneous abortion, septicemia, and meningitis. Although it is known that neutrophils are required for elimination of the bacteria and for survival of the host, the mechanisms governing the recruitment of neutrophils to LM-infected tissues are not fully understood. We demonstrate here that IL-23 and the IL-17 receptor A (IL-17RA), which mediates both IL-17A and IL-17F signaling, are necessary for resistance against systemic LM infection. LM-infected IL-23p19 knockout (KO) mice have decreased production of IL-17A and IL-17F, while IFN- production is not altered by the lack of IL-23. LM induces the production of IL-17A from T cells, but not CD4, CD8, or NK cells. Furthermore, a lack of efficient neutrophil recruitment to the liver is evident in both IL-23p19 KO and IL-17RA KO mice during LM infection. Immunocytochemical analysis of infected livers revealed that neutrophils were able to localize with LM in IL-23p19 KO and IL-17RA KO mice, indicating that IL-23 and IL-17RA do not regulate the precise localization of neutrophils with LM. The importance of IL-23-induced IL-17A was demonstrated by injecting IL-23p19 KO mice with recombinant IL-17A. These mice had reduced LM bacterial burdens compared with IL-23p19 KO mice that did not receive IL-17A. These results indicate that during LM infection, IL-23 regulates the production of IL-17A and IL-17F from T cells, resulting in optimal liver neutrophil recruitment and enhanced bacterial clearance.

Brain-Derived Neurotrophic Factor (BDNF) Induces Sustained Elevation of Intracellular Ca2+ in Rodent Microglia [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 12:21:33 PST

Microglia are intrinsic immune cells that release factors, including proinflammatory cytokines, NO, and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]i) is important for microglial functions, such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we observed that BDNF induced a sustained increase in [Ca²⁺]i through binding with the truncated tropomyosin-related kinase B receptor, resulting in activation of the PLC pathway and store-operated calcium entry in rodent microglial cells. RT-PCR and immunocytochemical techniques revealed that truncated tropomyosin-related kinase B-T1 receptors were highly expressed in rodent microglial cells. Sustained activation of store-operated calcium entry occurred after brief BDNF application and contributed to the maintenance of sustained [Ca²⁺]i elevation. Pretreatment with BDNF significantly suppressed the release of NO from activated microglia. Additionally, pretreatment of BDNF suppressed the IFN--induced increase in [Ca²⁺]i, along with a rise in basal levels of [Ca²⁺]i in rodent microglial cells. We show direct evidence that rodent microglial cells are able to respond to BDNF, which may be important for the regulation of inflammatory responses, and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

IFN-{gamma} Expressed by T Cells Regulates the Persistence of Antigen Presentation by Limiting the Survival of Dendritic Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Russell, M. S., Dudani, R., Krishnan, L., Sad, S. — Wed, 18 Nov 2009 12:21:32 PST

Ag presentation to T cells orchestrates the development of acquired immune response. Although it is considered that Ag presentation may persist at high levels during chronic infections, we have previously reported that in mice infected with bacillus Calmette-Guérin, Ag presentation gets drastically curtailed during the chronic stage of infection despite antigenic persistence. In this report we evaluated the mechanism of this curtailment. Ag presentation declined precipitously as the T cell response developed, and Ag presentation was not curtailed in mice that were deficient in CD8⁺ T cells or MHC class II, suggesting that T cells regulate Ag presentation. Curtailment of Ag presentation was reduced in IFN--deficient mice, but not in mice with a deficiency/mutation in inducible NOS2, perforin, or Fas ligand. In hosts with no T cells (Rag1^–/–), Ag presentation was not curtailed during the chronic stage of infection. However, adoptive transfer of wild-type, but not IFN-^–/–, CD4⁺ and CD8⁺ T cells into Rag1-deficient hosts strongly curtailed Ag presentation. Increased persistence of Ag presentation in IFN--deficient hosts correlated to increased survival of dendritic cells, but not of macrophages, and was not due to increased stimulatory capacity of IFN--deficient dendritic cells. These results reveal a novel mechanism indicating how IFN- prevents the persistence of Ag presentation, thereby preventing memory T cells from going into exhaustion.

Signaling Crosstalk during Sequential TLR4 and TLR9 Activation Amplifies the Inflammatory Response of Mouse Macrophages [INFLAMMATION]

De Nardo, D., De Nardo, C. M., Nguyen, T., Hamilton, J. A., Scholz, G. M. — Wed, 18 Nov 2009 12:21:31 PST

The TLR family of pattern recognition receptors is largely responsible for meditating the activation of macrophages by pathogens. Because macrophages may encounter multiple TLR ligands during an infection, signaling crosstalk between TLR pathways is likely to be important for the tailoring of inflammatory reactions to pathogens. Here, we show that rather than inducing tolerance, LPS pretreatment primed the inflammatory response (e.g., TNF production) of mouse bone marrow-derived macrophages (BMM) to the TLR9 ligand, CpG DNA. The priming effects of LPS, which correlated with enhanced Erk1/2, JNK, and p38 MAPK activation, appeared to be mediated via both c-Fms-dependent and -independent mechanisms. LPS pretreatment and inhibition of the M-CSF receptor, c-Fms, with GW2580 had comparable effects on CpG DNA-induced Erk1/2 and p38 MAPK activation. However, c-Fms inhibition did not enhance CpG DNA-induced JNK activation; also, the levels of TNF produced were significantly lower than those from LPS-primed BMM. Thus, the priming effects of LPS on TLR9 responses appear to be largely mediated via the c-Fms-independent potentiation of JNK activity. Indeed, inhibition of JNK abrogated the enhanced production of TNF by LPS-pretreated BMM. The c-Fms-dependent priming effects of LPS are unlikely to be a consequence of the inhibitory constraints of M-CSF signaling on TLR9 expression being relieved by LPS; instead, LPS may exert its priming effects via signaling molecules downstream of TLR9. In summary, our findings highlight the importance of signaling crosstalk between TLRs, as well as between TLRs and c-Fms, in regulating the inflammatory reaction to pathogens.

Enhanced Susceptibility to Leishmania Infection in Resistant Mice in the Absence of Immediate Early Response Gene X-1 [HOST DEFENSE]

Akilov, O. E., Ustyugova, I. V., Zhi, L., Hasan, T., Wu, M. X. — Wed, 18 Nov 2009 12:21:30 PST

Immediate early response gene X-1 (IEX-1) is a stress-inducible gene abundantly expressed in macrophages and T cells following various stimuli. To explore a potential role for IEX-1 in control of the susceptibility to Leishmania major infection, the inflammatory response during cutaneous leishmaniasis was evaluated in 129Sv/C57BL/6-resistant mice in the presence or absence of IEX-1. Null mutation of IEX-1 enhanced the susceptibility of the mice to L. major infection, and aggravated inflammatory responses in comparison with wild-type control mice. The excessive inflammation was not ascribed to a Th2-biased immune response or a defect in Th1 polarization, but rather to an elevated level of IL-17 production by both T and CD4⁺ cells, concomitant with an increase of the neutrophil recruitment early in the infection. The lack of IEX-1 also suppressed TNF- production in both macrophages and T cells, resulting in a high intralesional load of parasites and delayed healing of the lesion, both of which were reversed by TNF- treatment. These findings indicate the crucial role of IL-17 and TNF- in determining the outcome of L. major infection beyond a balance between Th1- and Th2-mediated immune responses.

CD69 Controls the Pathogenesis of Allergic Airway Inflammation [INFLAMMATION]

Wed, 18 Nov 2009 12:21:29 PST

Airway inflammation and airway hyperresponsiveness are central issues in the pathogenesis of asthma. CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In CD69-deficient mice, OVA-induced eosinophilic airway inflammation, mucus hyperproduction, and airway hyperresponsiveness were attenuated. Cell transfer of Ag-primed wild-type but not CD69-deficient CD4 T cells restored the induction of allergic inflammation in CD69-deficient mice, indicating a critical role of CD69 expressed on CD4 T cells. Th2 responses induced by CD69-deficient CD4 T cells in the lung were attenuated, and the migration of CD4 T cells into the asthmatic lung was severely compromised. The expression of VCAM-1 was also substantially altered, suggesting the involvement of VCAM-1 in the CD69-dependent migration of Th2 cells into the asthmatic lung. Interestingly, the administration of anti-CD69 Ab inhibited the induction of the OVA-induced airway inflammation and hyperresponsiveness. This inhibitory effect induced by the CD69 mAb was observed even after the airway challenge with OVA. These results indicate that CD69 plays a crucial role in the pathogenesis of allergen-induced eosinophilic airway inflammation and hyperresponsiveness and that CD69 could be a possible therapeutic target for asthmatic patients.

Soluble B and T Lymphocyte Attenuator Possesses Antitumor Effects and Facilitates Heat Shock Protein 70 Vaccine-Triggered Antitumor Immunity against a Murine TC-1 Cervical Cancer Model In Vivo [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 12:21:28 PST

B and T lymphocyte attenuator (BTLA)-herpesvirus entry mediator (HVEM) signaling coinhibitory pathway is believed to impair antitumor immune competences. An intriguing unresolved question is whether blockade of BTLA-HVEM guides an effective therapeutic tool against established tumors. To address this issue, we constructed a eukaryotic expression plasmid (psBTLA) that expressed the extracellular domain of murine BTLA (soluble form of BTLA), which could bind HVEM, the ligand of BTLA, and block BTLA-HVEM interactions. The data in this study showed that treatment by injection of psBTLA resulted in down-regulation of IL-10 and TGF-β and promotion of dendritic cell function by increasing the expression of B7-1 and IL-12, but the adaptive antitumor immune responses achieved by psBTLA administration alone were limited and could not eradicate the tumor effectively. Next, we evaluated the immunotherapeutic efficacy and mechanism of combination therapy of heat shock protein 70 (HSP70) vaccine/psBTLA by using murine TC-1 cervical cancer mice as an ectopic tumor model. Our in vivo studies revealed that treatment with HSP70 vaccine alone did not lead to satisfactory tumor growth inhibition, whereas cotreatment with psBTLA significantly improved antitumor immunity and compensated the deficiency of HSP70 vaccine by increasing the expression of Th1 cytokines, IL-2, and IFN- and decreasing transcription levels of IL-10, TGF-β, and Foxp3 in the tumor microenvironment. Taken together, our findings indicate that blocking the BTLA-HVEM interaction with sBTLA enhances antitumor efficacy and results in a significant synergistic effect against existent tumor cells in vivo when combined with the HSP70 vaccine.

Cutting Edge: Depletion of Foxp3+ Cells Leads to Induction of Autoimmunity by Specific Ablation of Regulatory T Cells in Genetically Targeted Mice1 [CUTTING EDGE]

Wed, 18 Nov 2009 12:21:27 PST

We have recently described two independent mouse models in which the administration of diphtheria toxin (DT) leads to specific depletion of regulatory T cells (Tregs) due to expression of DT receptor-enhanced GFP under the control of the Foxp3 promoter. Both mouse models develop severe autoimmune disorders when Foxp3⁺ Tregs are depleted. Those findings were challenged in a recent study published in this journal suggesting the expression of Foxp3 in epithelial cells as the cause for disease development. By using genetic, cellular, and immunohistochemical approaches, we do not find evidence for Foxp3-expression in nonhematopoietic cells. DT injection does not lead to a loss of epithelial integrity in our Foxp3-DTR models. Instead, Foxp3 expression is Treg-specific and ablation of Foxp3⁺ Tregs leads to the induction of fatal autoimmune disorders. Autoimmunity can be reversed by the adoptive transfer of Tregs into depleted hosts, and the transfer of Foxp3-deficient bone marrow into T cell-deficient irradiated recipients leads to full-blown disease development.

Cutting Edge: Normal Regional Lymph Node Enrichment of Antigen-Specific Regulatory T Cells with Autoimmune Disease-Suppressive Capacity [CUTTING EDGE]

Wheeler, K. M., Samy, E. T., Tung, K. S. K. — Wed, 18 Nov 2009 12:21:26 PST

Natural CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) effectively prevent autoimmune disease development, but their role in maintaining physiological tolerance against self-Ag of internal organs is not yet defined. In this study, we quantified disease-specific Treg (DS-Treg) as Treg that preferentially suppress one autoimmune disease over another in day 3 thymectomized recipients. A striking difference was found among individual lymph nodes (LN) of normal mice; Treg from draining LN were 15-50 times more efficient than those of nondraining LN at suppressing autoimmune diseases of ovary, prostate, and lacrimal glands. The difference disappeared upon auto-Ag ablation and returned upon auto-Ag re-expression. In contrast, the CD4⁺CD25^– effector T cells from different individual LN induced multiorgan inflammation with comparable organ distribution. We propose that peripheral tolerance for internal organs relies on the control of autoreactive effector Tcells by strategic enrichment of Ag-specific Treg in the regional LN.

Autoimmune Regulator Deficiency Results in Decreased Expression of CCR4 and CCR7 Ligands and in Delayed Migration of CD4+ Thymocytes [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Laan, M., Kisand, K., Kont, V., Moll, K., Tserel, L., Scott, H. S., Peterson, P. — Wed, 18 Nov 2009 12:21:25 PST

Autoimmune regulator (Aire) has been viewed as a central player in the induction of tolerance. This study examines whether Aire can modulate the production of the thymic chemokines involved in corticomedullary migration and thus play a role in intrathymic thymocyte migration and maturation. Aire deficiency resulted in reduced gene expression and protein levels of the CCR4 and CCR7 ligands in whole thymi of mice, as determined by quantitative PCR analysis and ELISA. The expression of the CCR4 ligands coincided with Aire expression in the CD80^high medullary thymic epithelial cells, whereas the expression of the CCR7 ligands was detected in other cell populations. Also, the expression pattern of the CCR4 and CCR7 ligands follows that of Aire during postnatal but not during embryonic development. In vitro, overexpression of Aire resulted in an up-regulation of selected CCR4 and CCR7 ligands, which induced selective migration of double-positive and single-positive CD4⁺ cells. In vivo, Aire deficiency resulted in a diminished emigration of mature CD4⁺ T cells from the thymi of 5-day-old mice. In conclusion, Aire regulates the production of CCR4 and CCR7 ligands in medullary thymic epithelial cells and alters the coordinated maturation and migration of thymocytes. These results suggest a novel mechanism behind the Aire-dependent induction of central tolerance.

Infection with Arginase-Deficient Leishmania major Reveals a Parasite Number-Dependent and Cytokine-Independent Regulation of Host Cellular Arginase Activity and Disease Pathogenesis [HOST DEFENSE]

Wed, 18 Nov 2009 12:21:24 PST

The balance between the products of l-arginine metabolism in macrophages regulates the outcome of Leishmania major infection. l-arginine can be oxidized by host inducible NO synthase to produce NO, which contributes to parasite killing. In contrast, l-arginine hydrolysis by host arginase blocks NO generation and provides polyamines, which can support parasite proliferation. Additionally, Leishmania encode their own arginase which has considerable potential to modulate infectivity and disease pathogenesis. In this study, we compared the infectivity and impact on host cellular immune response in vitro and in vivo of wild-type (WT) L. major with that of a parasite arginase null mutant (arg^–) L. major. We found that arg^– L. major are impaired in their macrophage infectivity in vitro independent of host inducible NO synthase activities. As with in vitro results, the proliferation of arg^– L. major in animal infections was also significantly impaired in vivo, resulting in delayed onset of lesion development, attenuated pathology, and low parasite burden. Despite this attenuated pathology, the production of cytokines by cells from the draining lymph node of mice infected with WT and arg^– L. major was similar at all times tested. Interestingly, in vitro and in vivo arginase levels were significantly lower in arg^– than in WT-infected cases and were directly correlated with parasite numbers inside infected cells. These results suggest that Leishmania-encoded arginase enhances disease pathogenesis by augmenting host cellular arginase activities and that contrary to previous in vitro studies, the host cytokine response does not influence host arginase activity.

Integration of Distinct Intracellular Signaling Pathways at Distal Regulatory Elements Directs T-bet Expression in Human CD4+ T Cells [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 12:21:23 PST

T-bet is a key regulator controlling Th1 cell development. This factor is not expressed in naive CD4⁺ T cells, and the mechanisms controlling expression of T-bet are incompletely understood. In this study, we defined regulatory elements at the human T-bet locus and determined how signals originating at the TCR and at cytokine receptors are integrated to induce chromatin modifications and expression of this gene during human Th1 cell differentiation. We found that T cell activation induced two strong DNase I-hypersensitive sites (HS) and rapid histone acetylation at these elements in CD4⁺ T cells. Histone acetylation and T-bet expression were strongly inhibited by cyclosporine A, and we detected binding of NF-AT to a HS in vivo. IL-12 and IFN- signaling alone were not sufficient to induce T-bet expression in naive CD4⁺ T cells, but enhanced T-bet expression in TCR/CD28-stimulated cells. We detected a third HS 12 kb upstream of the mRNA start site only in developing Th1 cells, which was bound by IL-12-induced STAT4. Our data suggest that T-bet locus remodeling and gene expression are initiated by TCR-induced NF-AT recruitment and amplified by IL-12-mediated STAT4 binding to distinct distal regulatory elements during human Th1 cell differentiation.

Neutrophils and Macrophages Cooperate in Host Resistance against Leishmania braziliensis Infection [HOST DEFENSE]

Wed, 18 Nov 2009 12:21:22 PST

Neutrophils play an active role in the control of infections caused by intracellular pathogens such as Leishmania. In the present study, we investigated the effect of neutrophil depletion at the time of Leishmania braziliensis infection of BALB/c mice and how neutrophils interact with the infected macrophage to promote parasite elimination. The in vivo depletion of neutrophils led to a significant increase in parasite load and enhanced the Th1-Th2 immune response in this experimental model of infection. BALB/c mice coinoculated with both parasites and live neutrophils displayed lower parasite burdens at the site of infection and in the draining lymph nodes. In vitro, we observed that live neutrophils significantly reduced the parasite load in L. braziliensis-infected murine macrophages, an effect not observed with Leishmania major. L. braziliensis elimination was dependent on the interaction between neutrophils and macrophages and was associated with TNF- as well as superoxide production. Furthermore, cooperation between neutrophils and macrophages toward parasite elimination was also observed in experiments performed with L. braziliensis-infected human cells and, importantly, with two other New World Leishmania species. These results indicate that neutrophils play an important and previously unappreciated role in L. braziliensis infection, favoring the induction of a protective immune response.

Role of CX3CL1/Fractalkine in Osteoclast Differentiation and Bone Resorption [CELLULAR IMMUNOLOGY AND IMMUNE REGULATION]

Wed, 18 Nov 2009 12:21:21 PST

The recruitment of osteoclast precursors toward osteoblasts and subsequent cell-cell interactions are critical for osteoclast differentiation. Chemokines are known to regulate cell migration and adhesion. CX3CL1 (also called fractalkine) is a unique membrane-bound chemokine that has dual functions for cells expressing its receptor CX3CR1: a potent chemotactic factor in its soluble form and a type of efficient cell adhesion molecule in its membrane-bound form. In this paper, we demonstrate a novel role of CX3CL1 in osteoblast-induced osteoclast differentiation. We found that osteoclast precursors selectively expressed CX3CR1, whereas CX3CL1 is expressed by osteoblasts. We confirmed that soluble CX3CL1 induced migration of bone marrow cells containing osteoclast precursors, whereas immobilized CX3CL1 mediated firm adhesion of osteoclast precursors. Furthermore, a blocking mAb against CX3CL1 efficiently inhibited osteoclast differentiation in mouse bone marrow cells cocultured with osteoblasts. Anti-CX3CL1 also significantly suppressed bone resorption in neonatal mice by reducing the number of bone-resorbing mature osteoclasts. Collectively, CX3CL1 expressed by osteoblasts plays an important role in osteoclast differentiation, possibly through its dual functions as a chemotactic factor and adhesion molecule for osteoclast precursors expressing CX3CR1. The CX3CL1-CX3CR1 axis may be a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis, osteoporosis, and cancer bone metastasis.