The Journal of Immunology current issue

[PRESIDENTIAL ADDRESS] Immunological Weapons Acquired Early in Life Win Battles with Cancer Late in Life

Finn, O. J. — 2008-07-18

[IN THIS ISSUE] IN THIS ISSUE

2008-07-18

[PILLARS OF IMMUNOLOGY] PU.1, a Shared Transcriptional Regulator of Innate and Adaptive Immune Cell Fates

Singh, H. — 2008-07-18

[PILLARS OF IMMUNOLOGY] Pillars Article: The Macrophage and B Cell-Specific Transcription Factor PU.1 Is Related to the ets Oncogene. Cell, 1990. 61: 113-124.

Klemsz, M. J., McKercher, S. R., Celada, A., Van Beveren, C., Maki, R. A. — 2008-07-18

[BRIEF REVIEWS] Auditing Protein Therapeutics Management by Professional APCs: Toward Prevention of Immune Responses against Therapeutic Proteins

2008-07-18

Alloimmunization is a crippling concern in the management of patients undergoing administration of protein therapeutics as evidenced in replacement therapy and other treatment procedures. Several issues in the genesis and modulation of such deleterious immune responses have been studied. While authors have focused on the downstream events of the specific immune response and suggested modification of protein therapeutics to eliminate epitopes that interact with B cell receptors, T cell receptors, or MHCII molecules, the mechanisms underlying Ag interaction with APCs, a step upstream of immune effectors, have been grossly neglected. We wish to emphasize that the recent knowledge in understanding the capacities of an APC to handle an Ag and the importance of the surrounding microenvironment in this process are crucial for designing novel protein therapeutics with reduced immunogenicity.

[CUTTING EDGE] Cutting Edge: Multiple Sclerosis-Like Lesions Induced by Effector CD8 T Cells Recognizing a Sequestered Antigen on Oligodendrocytes

2008-07-18

CD8 T cells are emerging as important players in multiple sclerosis (MS) pathogenesis, although their direct contribution to tissue damage is still debated. To assess whether autoreactive CD8 T cells can contribute to the pronounced loss of oligodendrocytes observed in MS plaques, we generated mice in which the model Ag influenza hemagglutinin is selectively expressed in oligodendrocytes. Transfer of preactivated hemagglutinin-specific CD8 T cells led to inflammatory lesions in the optic nerve, spinal cord, and brain. These lesions, associating CD8 T cell infiltration with focal loss of oligodendrocytes, demyelination, and microglia activation, were very reminiscent of active MS lesions. Thus, our study demonstrates the potential of CD8 T cells to induce oligodendrocyte lysis in vivo as a likely consequence of direct Ag-recognition. These results provide new insights with regard to CNS tissue damage mediated by CD8 T cells and for understanding the role of CD8 T cells in MS.

[CUTTING EDGE] Cutting Edge: The Transmembrane E3 Ligase GRAIL Ubiquitinates the Costimulatory Molecule CD40 Ligand during the Induction of T Cell Anergy

2008-07-18

Activation of naive T lymphocytes is regulated through a series of discrete checkpoints that maintain unresponsiveness to self. During this multistep process, costimulatory interactions act as inducible signals that allow APCs to selectively mobilize T cells against foreign Ags. In this study, we provide evidence that the anergy-associated E3 ubiquitin ligase GRAIL (gene related to anergy in lymphocytes) regulates expression of the costimulatory molecule CD40L on CD4 T cells. Using its luminal protease-associated domain, GRAIL binds to the luminal/extracellular portion of CD40L and facilitates transfer of ubiquitin molecules from the intracellular GRAIL RING (really interesting new gene) finger to the small cytosolic portion of CD40L. Down-regulation of CD40L occurred following ectopic expression of GRAIL in naive T cells from CD40^–/– mice, and expression of GRAIL in bone marrow chimeric mice was associated with diminished lymphoid follicle formation. These data provide a model for intrinsic T cell regulation of costimulatory molecules and a molecular framework for the initiation of clonal T cell anergy.

[CUTTING EDGE] Cutting Edge: Priming of NK Cells by IL-18

2008-07-18

Recent evidence suggests that NK cells require priming to display full effector activity. In this study, we demonstrate that IL-18 contributed to this phenomenon. IL-18 signaling-deficient NK cells were found to be unable to secrete IFN- in response to ex vivo stimulation with IL-12. This was not due to a costimulatory role of IL-18, because blocking IL-18 signaling during the ex vivo stimulation with IL-12 did not alter IFN- production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN- upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN- transcription by NK cells was comparable in IL-18 signaling-deficient and -sufficient NK cells. This suggests that priming by IL-18 leads to an improved translation of IFN- mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Type I IFN-Induced, NKT Cell-Mediated Negative Control of CD8 T Cell Priming by Dendritic Cells

Bochtler, P., Kroger, A., Schirmbeck, R., Reimann, J. — 2008-07-18

We investigated the negative effect of type I IFN (IFN-I) on the priming of specific CD8 T cell immunity. Priming of murine CD8 T cells is down-modulated if Ag is codelivered with IFN-I-inducing polyinosinic:polycytidylic acid (pI/C) that induces (NK cell- and T/B cell-independent) acute changes in the composition and surface phenotype of dendritic cells (DC). In wild-type but not IFN-I receptor-deficient mice, pI/C reduces the plasmacytoid DC but expands the CD8⁺ conventional DC (cDC) population and up-regulates surface expression of activation-associated (CD69, BST2), MHC (class I/II), costimulator (CD40, CD80/CD86), and coinhibitor (PD-L1/L2) molecules by cDC. Naive T cells are efficiently primed in vitro by IFN-I-stimulated CD8 cDC (the key APC involved in CD8 T cell priming) although these DC produced less IL-12 p40 and IL-6. pI/C (IFN-I)-mediated down modulation of CD8 T cell priming in vivo was not observed in NKT cell-deficient CD1d^–/– mice. CD8 cDC from pI/C-treated mice inefficiently stimulated IFN-, IL-4, and IL-2 responses of NKT cells. In vitro, CD8 cDC that had activated NKT cells in the presence of IFN-I primed CD8 T cells that produced less IFN- but more IL-10. The described immunosuppressive effect of IFN-I thus involves an NKT cell-mediated change in the phenotype of CD8 cDC that favors priming of IL-10-producing CD8 T cells. In the presence of IFN-I, NKT cells hence impair the competence of CD8 cDC to prime proinflammatory CD8 T cell responses.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] The Transcription Factor Fli-1 Modulates Marginal Zone and Follicular B Cell Development in Mice

2008-07-18

Fli-1 belongs to the Ets transcription factor family and is expressed primarily in hematopoietic cells, including most cells active in immunity. To assess the role of Fli-1 in lymphocyte development in vivo, we generated mice that express a truncated Fli-1 protein, lacking the C-terminal transcriptional activation domain (Fli-1^CTA). Fli-1^CTA/Fli-1^CTA mice had significantly fewer splenic follicular B cells, and an increased number of transitional and marginal zone B cells, compared with wild-type controls. Bone marrow reconstitution studies demonstrated that this phenotype is the result of lymphocyte intrinsic effects. Expression of Ig and other genes implicated in B cell development, including Pax-5, E2A, and Egr-1, are reduced, while Id1 and Id2 are increased in Fli-1^CTA/Fli-1^CTA mice. Proliferation of B cells from Fli-1^CTA/Fli-1^CTA mice was diminished, although intracellular Ca²⁺ flux in B cells from Fli-1^CTA/Fli-1^CTA mice was similar to that of wild-type controls after anti-IgM stimulation. Immune responses and in vitro class switch recombination were also altered in Fli-1^CTA/Fli-1^CTA mice. Thus, Fli-1 modulates B cell development both centrally and peripherally, resulting in a significant impact on the in vivo immune response.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Allele-Selective Effect of PA28 in MHC Class I Antigen Processing

2008-07-18

PA28 is an IFN--inducible proteasome activator and its genetic ablation causes complete loss of processing of certain Ags, but not all of them. The reason why this occurs and how PA28 influences the formation of peptide repertoires for MHC class I molecules remains unknown. In this study, we show the allele-specific role of PA28 in Ag processing. Retrovirus-transduced overexpression of PA28 decreased expression of K^d (D^d) while it increased K^b and L^d on the cell surface. By contrast, overexpression of PA28C5, a mutant carrying a deletion of its five C-terminal residues and capable of attenuating the activity of endogenous PA28, produced the opposite effect on expression of those MHC class I molecules. Moreover, knockdown of both PA28 and β by small-interfering RNA profoundly augmented expression of K^d and D^d, but not of L^d, on the cell surface. Finally, we found that PA28-associated proteasome preferentially digested within epitopic sequences of K^d, although correct C-terminal flankings were removed, which in turn hampered production of K^d ligands. Our results indicate that whereas PA28 negatively influences processing of K^d (D^d) ligands, thereby, down-regulating Ag presentation by those MHC class I molecules, it also efficiently produces K^b (L^d) epitopes, leading to up-regulation of the MHC molecules.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] HIF-1{alpha} Is Up-Regulated in Activated Mast Cells by a Process That Involves Calcineurin and NFAT

Walczak-Drzewiecka, A., Ratajewski, M., Wagner, W., Dastych, J. — 2008-07-18

Mast cells play important roles in many pathological conditions where local hypoxia is observed, including asthma, rheumatic diseases, and certain types of cancer. Here, we investigated how expression of the hypoxia-inducible factor 1, subunit gene (HIF1A), is regulated in mast cells. The product of HIF1A is hypoxia-inducible factor 1 (HIF-1), is a major nuclear transcription factor modulating gene expression in response to hypoxic conditions. We observed that under hypoxic conditions, exposure of mast cells to ionomycin and substance P resulted in significant up-regulation of HIF1A expression as compared with resting mast cells incubated under identical conditions. The ionomycin-mediated increase in HIF-1 protein levels was sensitive to the transcription inhibitor actinomycin D and to inhibitors of calcineurin, cyclosporin A (CsA), and FK506. The increased HIF-1 protein level was paralleled by a severalfold increase in HIF-1 mRNA that could be also inhibited with actinomycin D and CsA. The HIF1A promoter activity was significantly increased in ionomycin-activated mast cells, and the promoter activity could be inhibited by CsA and FK506. Furthermore, in situ mutagenesis experiments showed that the ionomycin-mediated HIF1A promoter activity depends on a conservative NFAT-binding site. Thus, accumulation of HIF-1 in activated mast cells requires up-regulation of HIF1A gene transcription and depends on the calcineurin-NFAT signaling pathway.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] IFN Regulatory Factor-1 Negatively Regulates CD4+CD25+ Regulatory T Cell Differentiation by Repressing Foxp3 Expression

2008-07-18

Regulatory T (Treg) cells are critical in inducing and maintaining tolerance. Despite progress in understanding the basis of immune tolerance, mechanisms and molecules involved in the generation of Treg cells remain poorly understood. IFN regulatory factor (IRF)-1 is a pleiotropic transcription factor implicated in the regulation of various immune processes. In this study, we report that IRF-1 negatively regulates CD4⁺CD25⁺ Treg cell development and function by specifically repressing Foxp3 expression. IRF-1-deficient (IRF-1^–/–) mice showed a selective and marked increase of highly activated and differentiated CD4⁺CD25⁺Foxp3⁺ Treg cells in thymus and in all peripheral lymphoid organs. Furthermore, IRF-1^–/– CD4⁺CD25^– T cells showed extremely high bent to differentiate into CD4⁺CD25⁺Foxp3⁺ Treg cells, whereas restoring IRF-1 expression in IRF-1^–/– CD4⁺CD25^– T cells impaired their differentiation into CD25⁺Foxp3⁺ cells. Functionally, both isolated and TGF-β-induced CD4⁺CD25⁺ Treg cells from IRF-1^–/– mice exhibited more increased suppressive activity than wild-type Treg cells. Such phenotype and functional characteristics were explained at a mechanistic level by the finding that IRF-1 binds a highly conserved IRF consensus element sequence (IRF-E) in the foxp3 gene promoter in vivo and negatively regulates its transcriptional activity. We conclude that IRF-1 is a key negative regulator of CD4⁺CD25⁺ Treg cells through direct repression of Foxp3 expression.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Acquisition of Suppressive Function by Activated Human CD4+CD25- T Cells Is Associated with the Expression of CTLA-4 Not FoxP3

2008-07-18

The role of CTLA-4 in regulatory T cell (Treg) function is not well understood. We have examined the role of CTLA-4 and its relationship with the transcription factor FoxP3 using a model of Treg induction in human peripheral blood. Activation of human CD4⁺CD25^– T cells resulted in the appearance of a de novo population of FoxP3-expressing cells within 48 h. These cells expressed high levels of CTLA-4 and cell sorting on expression of CTLA-4 strongly enriched for FoxP3⁺-expressing cells with suppressive function. Culture in IL-2 alone also generated cells with suppressive capacity that also correlated with the appearance of CTLA-4. To directly test the role of CTLA-4, we transfected resting human T cells with CTLA-4 and found that this method conferred suppression, similar to that of natural Tregs, even though these cells did not express FoxP3. Furthermore, transfection of FoxP3 did not induce CTLA-4 and these cells were not suppressive. By separating the expression of CTLA-4 and FoxP3, our data show that FoxP3 expression alone is insufficient to up-regulate CTLA-4; however, activation of CD4⁺CD25^– T cells can induce both FoxP3 and CTLA-4 in a subpopulation of T cells that are capable of suppression. These data suggest that the acquisition of suppressive behavior by activated CD4⁺CD25^– T cells requires the expression of CTLA-4, a feature that appears to be facilitated by, but is not dependent on, expression of FoxP3.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Mechanisms Underlying Blockade of Allograft Acceptance by TLR Ligands

2008-07-18

Immune activation via TLRs is known to prevent transplantation tolerance in multiple animal models. To investigate the mechanisms underlying this barrier to tolerance induction, we used complementary murine models of skin and cardiac transplantation in which prolonged allograft acceptance is either spontaneous or pharmacologically induced with anti-CD154 mAb and rapamycin. In each model, we found that prolonged allograft survival requires the presence of natural CD4⁺Foxp3⁺ T regulatory cells (Tregs), and that the TLR9 ligand CpG prevents graft acceptance both by interfering with natural Treg function and by promoting the differentiation of Th1 effector T cells in vivo. We further demonstrate that although Th17 cells differentiate from naive alloreactive T cells, these cells do not arise from natural Tregs in either CpG-treated or untreated graft recipients. Finally, we show that CpG impairs natural Treg suppressor capability and prevents Treg-dependent allograft acceptance in an IL-6-independent fashion. Our data therefore suggest that TLR signals do not prevent prolonged graft acceptance by directing natural Tregs into the Th17 lineage or by using other IL-6-dependent mechanisms. Instead, graft destruction results from the ability of CpG to drive Th1 differentiation and interfere with immunoregulation established by alloreactive natural CD4⁺Foxp3⁺ Tregs.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] IFN-{gamma} Induces the Erosion of Preexisting CD8 T Cell Memory during Infection with a Heterologous Intracellular Bacterium

Dudani, R., Murali-Krishna, K., Krishnan, L., Sad, S. — 2008-07-18

Memory T cells are critical for the control of intracellular pathogens and require few signals for maintenance; however, erosion of established preexisting memory CD8⁺ T cells has been shown to occur during infection with heterologous viral infections. We evaluated whether this also occurs during infection with various intracellular bacteria and what mechanisms may be involved. We demonstrate that erosion of established memory is also induced during infection of mice with various intracellular bacteria, such as Listeria monocytogenes, Salmonella typhimurium, and Mycobacterium bovis (bacillus Calmette-Guérin). The extent of erosion of established CD8⁺ T cell memory was dependent on the virulence of the heterologous pathogen, not persistence. Furthermore, when antibiotics were used to comprehensively eliminate the heterologous pathogen, the numbers of memory CD8⁺ T cells were not restored, indicating that erosion of preexisting memory CD8⁺ T cells was irreversible. Irrespective of the initial numbers of memory CD8⁺ T cells, challenge with the heterologous pathogen resulted in a similar extent of erosion of memory CD8⁺ T cells, suggesting that cellular competition was not responsible for erosion. After challenge with the heterologous pathogen, effector memory CD8⁺ T cells were rapidly eliminated. More importantly, erosion of preexisting memory CD8⁺ T cells was abrogated in the absence of IFN-. These studies help reveal the paradoxical role of IFN-. Although IFN- promotes the control of intracellular bacterial replication during primary infection, this comes at the expense of erosion of preexisting memory CD8⁺ T cells in the wake of infection with heterologous pathogens.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] SCART Scavenger Receptors Identify a Novel Subset of Adult {gamma}{delta} T Cells

Kisielow, J., Kopf, M., Karjalainen, K. — 2008-07-18

Although there has been great progress in the characterization of β T cell differentiation, selection, and function, T cells have remained poorly understood. One of the main reasons for this is the lack of T cell-specific surface markers other than the TCR chains themselves. In this study we describe two novel surface receptors, SCART1 and SCART2. SCARTs are related to CD5, CD6, and CD163 scavenger receptors but, unlike them, are found primarily on developing and mature T cells. Characterization of SCART2 positive immature and peripheral T cells suggests that they undergo lineage specification in the thymus and belong to a new IL-17-producing subset with distinct homing capabilities.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Identification of Initiator B Cells, a Novel Subset of Activation-Induced Deaminase-Dependent B-1-Like Cells That Mediate Initiation of Contact Sensitivity

Kerfoot, S. M., Szczepanik, M., Tung, J. W., Askenase, P. W. — 2008-07-18

Contact sensitivity (CS) is related to delayed-type hypersensitivity and is a well-characterized prototype of T cell-mediated inflammation. However, the inflammatory response associated with CS is additionally dependent on Ag-specific IgM produced by a subpopulation of B cells in response to sensitization. Upon re-exposure to hapten, this IgM mediates rapid vascular activation and subsequent recruitment of proinflammatory T cells to the local site. Interference with this pathway prevents the full development of the classic delayed inflammatory response and is therefore termed the "CS initiation" pathway. In this study, we show that CS initiation is defective in mice deficient in activation-induced deaminase, an enzyme central to the process of somatic hypermutation. Using adoptive transfer experiments, we demonstrate that the defect is specific to a B-1-like population of B cells and that transfer of WT cells reconstitutes CS initiation mechanisms in deficient recipients. We went on to identify a novel subpopulation of Ag-binding B cells in the spleens of sensitized mice that possess initiation activity (CD19⁺CD5⁺Thy-1^intIgM^highIgD^high) that we name "initiator B cells." Analysis of BCR H chain genes isolated from these cells revealed evidence of activation-induced deaminase-mediated somatic hypermutation. The sensitivity of CS initiation to very low amounts of sensitizing hapten suggests that the responsible B cells have increased IgM receptor gene mutations enabling selection to generate Abs with sufficient affinity to mediate the response.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] An Endogenous Prostaglandin Enhances Environmental Phthalate-Induced Apoptosis in Bone Marrow B Cells: Activation of Distinct but Overlapping Pathways

Bissonnette, S. L., Teague, J. E., Sherr, D. H., Schlezinger, J. J. — 2008-07-18

Phthalate esters are ubiquitous environmental contaminants that are produced for a variety of common industrial and commercial purposes. We have shown that mono-(2-ethylhexyl) phthalate (MEHP), the toxic metabolite of di-(2-ethylhexyl) phthalate, induces bone marrow B cell apoptosis that is enhanced in the presence of the endogenous prostaglandin 15-deoxy-^{(12, 14)}-PGJ₂ (15d-PGJ₂). Here, studies were performed to determine whether 15d-PGJ₂-mediated enhancement of MEHP-induced apoptosis represents activation of an overlapping or complementary apoptosis pathway. MEHP and 15d-PGJ₂ induced significant apoptosis within 8 and 5 h, respectively, in a pro/pre-B cell line and acted cooperatively to induce apoptosis in primary pro-B cells. Apoptosis induced with each chemical was accompanied by activation of a combination of initiator caspases (caspases-2, -8, and -9) and executed by caspase-3. Apoptosis induced with MEHP and 15d-PGJ₂ was reduced in APAF1 null primary pro-B cells and accompanied by alteration of mitochondrial membranes, albeit with different kinetics, indicating an intrinsically activated apoptosis pathway. Significant Bax translocation to the mitochondria supports its role in initiating release of cytochrome c. Both chemicals induced Bid cleavage, a result consistent with a truncated Bid-mediated release of cytochrome c in an apoptosis amplification feedback loop; however, significantly more Bid was cleaved following 15d-PGJ₂ treatment, potentially differentiating the two pathways_. Indeed, Bid cleavage and cytochrome c release following 15d-PGJ₂ but not MEHP treatment was profoundly inhibited by Z-VAD-FMK, suggesting that 15d-PGJ₂ activates apoptosis via two pathways, Bax mobilization and protease-dependent Bid cleavage. Thus, endogenous 15d-PGJ₂-mediated enhancement of environmental chemical-induced apoptosis represents activation of an overlapping but distinct signaling pathway.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Low- versus High-Baseline Epinephrine Output Shapes Opposite Innate Cytokine Profiles: Presence of Lewis- and Fischer-Like Neurohormonal Immune Phenotypes in Humans?

2008-07-18

Immunogenetic mechanisms operating within the immune system are known to influence cytokine profiles and disease susceptibility. Yet the role of the individual’s neurohormonal background in these processes remains undefined. Hormonal imbalances are documented in immune-related diseases, but it is unclear whether this represents a secondary phenomenon or a primary "defect" related to specific neurohormonal immune phenotype(s). We report that in a large subpopulation of healthy humans the baseline epinephrine output (but not cortisol and sex steroid hormones) correlated inversely with proinflammatory and positively with anti-inflammatory cytokine production. Thus, low vs high epinephrine excretors had a 2- to 5-fold higher TNF- and IL-12 production but 2-fold lower IL-10 production induced by LPS ex vivo. In alternative settings, we found low baseline levels and profoundly blunted stress-induced epinephrine responses but high TNF- levels in Lewis vs Fischer inbred rats. Additionally, isoproterenol, a β adrenoreceptor agonist suppressed LPS-induced TNF- production, with more pronounced effect in Lewis than in Fischer rats. In human monocytes, epinephrine and the β₂ adrenoreceptor agonist fenoterol potently inhibited LPS-induced TNF- and IL-12, but stimulated IL-10 production. The order of potency for hormones able to inhibit IL-12 production ex vivo was: epinephrine > norepinephrine > = 1,25-(OH)₂ vitamin D₃ > hydrocortisone. This indicates that baseline epinephrine conditions cytokine responsiveness and through this mechanism intrinsic hypo- or hyperactive adrenal medullas in some individuals may shape opposite cytokine profiles. Since Lewis and Fischer rats have opposite susceptibility to experimental immunological diseases, this suggests that the parallel human phenotypes could be linked to differing responsiveness and susceptibility to infections and immune/inflammatory-related conditions.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] The Agonists of TLR4 and 9 Are Sufficient to Activate Memory B Cells to Differentiate into Plasma Cells In Vitro but Not In Vivo

Richard, K., Pierce, S. K., Song, W. — 2008-07-18

Memory B cells can persist for a lifetime and be reactivated to yield high affinity, isotype switched plasma cells. The generation of memory B cells by Ag immunization requires adjuvants that generally contain TLR agonists. However, requirements for memory B cell activation and the role of TLRs in this activation are not well understood. In this study, we analyzed the response of memory B cells from immunized mice to TLR9 and 4 agonists CpG oligodeoxynucleotides (ODN) and LPS. Mouse memory B cells express both TLR9 and 4, and respond to both CpG ODN and LPS in vitro by differentiating into high affinity IgG secreting plasma cells. In contrast, neither CpG ODN nor LPS alone is sufficient to activate memory B cells in vivo. Ag is required for the clonal expansion of Ag-specific memory B cells, the differentiation of memory B cells to high affinity IgG secreting plasma cells, and the recall of high affinity Ab responses. The Ag-specific B cells that have not yet undergone isotype switching showed a relatively higher expression of TLR4 than memory B cells, which was reflected in a heightened response to LPS, but in both cases yielded mostly low affinity IgM secreting plasma cells. Thus, although memory B cells are sensitive to TLR agonists in vitro, TLR agonists alone appear to have little affect on B cell memory in vivo.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Influence of a Non-NK Complex Region of Chromosome 6 on CD4+ Invariant NK T Cell Homeostasis

2008-07-18

The number and function of immunoregulatory invariant NKT (iNKT) cells are genetically controlled. A defect of iNKT cell ontogeny and function has been implicated as one causal factor of NOD mouse susceptibility to type 1 diabetes. Other factors of diabetes susceptibility, such as a decrease of regulatory T cell function or an increase in TLR1 expression, are corrected in diabetes-resistant Idd6 NOD.C3H 6.VIII congenic mice. Thus, we surmised that the iNKT cell defects found in NOD mice may also be rescued in congenic mice. Unexpectedly, we found, in both the thymus and the periphery, a 50% reduction in iNKT cell number in NOD.C3H 6.VIII mice as compared with NOD mice. This reduction only affected CD4⁺ iNKT cells, and left the double negative iNKT cells unchanged. In parallel, the production of IL-4 and IFN- following -GalCer stimulation was proportionally reduced. Using three subcongenic strains, we have narrowed down the region controlling iNKT development within Idd6 (5.8 Mb) to Idd6.2 region (2.5 Mb). Idd6 region had no effect on NK cell number and in vivo cytotoxic activity. These results indicate that the role of iNKT cells in diabetes development is equivocal and more complex than initially considered. In addition, they bring strong evidence that the regulation of CD4⁺ iNKT cell production is independent from that of DN iNKT cells, and involves genes of the Idd6 locus.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] TCR Antagonism by Peptide Requires High TCR Expression

Jones, D. S., Reichardt, P., Ford, M. L., Edwards, L. J., Evavold, B. D. — 2008-07-18

Current models of T cell activation focus on the kinetics of TCR-ligand interactions as the central parameter governing T cell responsiveness. However, these kinetic parameters do not adequately predict all T cell behavior, particularly the response to antagonist ligands. Recent studies have demonstrated that TCR number is a critical parameter influencing the responses of CD4⁺ T cells to weak agonist ligands, and receptor density represents an important means of regulating tissue responsiveness in other receptor ligand systems. To systematically address the impact of TCR expression on CD8⁺ T cell responses, mAbs to the TCR -chain and T cells expressing two TCR species were used as two different methods to manipulate the number of available TCRs on P14 and OT-I transgenic T cells. Both methods of TCR reduction demonstrated that the efficacy of antagonist peptides was significantly reduced on T cells bearing low numbers of available receptors. In addition, the ability of weak agonists to induce proliferation was critically dependent on the availability of high numbers of TCRs. Therefore, in this report we show that TCR density is a major determinant of CD8⁺ T cell reactivity to weak agonist and antagonist ligands but not agonist ligands.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] IL-21-Induced Isotype Switching to IgG and IgA by Human Naive B Cells Is Differentially Regulated by IL-4

Avery, D. T., Bryant, V. L., Ma, C. S., de Waal Malefyt, R., Tangye, S. G. — 2008-07-18

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-β. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1⁺ cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA⁺ cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1⁺ cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] The E3 Ubiquitin Ligase Ro52 Negatively Regulates IFN-{beta} Production Post-Pathogen Recognition by Polyubiquitin-Mediated Degradation of IRF3

2008-07-18

Induction of type I IFNs is a fundamental cellular response to both viral and bacterial infection. The role of the transcription factor IRF3 is well established in driving this process. However, equally as important are cellular mechanisms for turning off type I IFN production to limit this response. In this respect, IRF3 has previously been shown to be targeted for ubiquitin-mediated degradation postviral detection to turn off the IFN-β response. In this study, we provide evidence that the E3 ligase Ro52 (TRIM21) targets IRF3 for degradation post-pathogen recognition receptor activation. We demonstrate that Ro52 interacts with IRF3 via its C-terminal SPRY domain, resulting in the polyubiquitination and proteasomal degradation of the transcription factor. Ro52-mediated IRF3 degradation significantly inhibits IFN-β promoter activity, an effect that is reversed in the presence of the proteasomal inhibitor MG132. Specific targeting of Ro52 using short hairpin RNA rescues IRF3 degradation following polyI:C-stimulation of HEK293T cells, with a subsequent increase in IFN-β production. Additionally, shRNA targeting of murine Ro52 enhances the production of the IRF3-dependent chemokine RANTES following Sendai virus infection of murine fibroblasts. Collectively, this demonstrates a novel role for Ro52 in turning off and thus limiting IRF3-dependent type I IFN production by targeting the transcription factor for polyubiquitination and subsequent proteasomal degradation.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Macrophages Pulsed with Streptococcus pneumoniae Elicit a T Cell-Dependent Antibody Response upon Transfer into Naive Mice

Vasilevsky, S., Colino, J., Puliaev, R., Canaday, D. H., Snapper, C. M. — 2008-07-18

Macrophages are less effective than DC at priming naive CD4⁺ T cells, suggesting that DC are unique in initiating T cell-dependent Ab responses. We compared the ability of DC and macrophages, pulsed in vitro with Streptococcus pneumoniae, to elicit protein- and polysaccharide-specific Ig isotype production upon adoptive transfer into naive mice. S. pneumoniae-activated DC secreted more proinflammatory and anti-inflammatory cytokines, expressed higher levels of surface MHC class II and CD40, and presented S. pneumoniae or recombinant pneumococcal surface protein A (PspA) to a PspA-specific T hybridoma more efficiently than macrophages. However, upon adoptive transfer into naive mice, S. pneumoniae-pulsed macrophages elicited an IgM or IgG anti-PspA and anti-polysaccharide response comparable in serum titers and IgG isotype distribution to that induced by DC. The IgG anti-PspA response, in contrast to the IgG anti-polysaccharide, to S. pneumoniae-pulsed macrophages was T cell-dependent. S. pneumoniae-pulsed macrophages that were paraformaldehyde-fixed before transfer or lacking expression of MHC class II or CD40 were highly defective in eliciting an anti-PspA response, although the anti-polysaccharide response was largely unaffected. To our knowledge, these data are the first to indicate that macrophages can play an active role in the induction of a T cell-dependent humoral immune response in a naive host.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Polyclonal Adaptive Regulatory CD4 Cells That Can Reverse Type I Diabetes Become Oligoclonal Long-Term Protective Memory Cells

Godebu, E., Summers-Torres, D., Lin, M. M., Baaten, B. J. G., Bradley, L. M. — 2008-07-18

Type 1 diabetes is a CD4 cell-dependent disease that results from destruction of insulin-producing β cells in pancreatic islets. An ideal therapy would reverse diabetes shortly after onset when islet function in not yet fully ablated, and also prevent re-emergence of disease through the generation of memory cells that control the autoimmune response. In this study, we show that adaptive/induced polyclonal regulatory (TR) cells, which contain islet-reactive cells, fulfill these criteria in the NOD mouse model. CD4 cells induced to express FoxP3, IL-10, and TGF-β1 in response to TCR signaling and TGF-β1 can reverse diabetes with clinical restoration of prediabetic serum levels of IL-10. Unlike naturally occurring TR cells, these adaptive TR cells persist indefinitely (>1 year) as FoxP3⁺, CD25^– memory cells that self-renew. Establishment of memory is accompanied by narrowing of the T cell repertoire to usage of a single TCR β-chain, Vβ11, implying selection by Ag. With islet-specific adaptive TR cells, we show that memory is functionally stable and transferable. Therefore, adaptive TR cells, which can be readily generated from normal CD4 populations and become focused by Ag with induction of memory, may provide a treatment and a vaccine for the long-term cure of diabetes making them attractive as immunotherapeutic agents.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] CTLA4 Expression Is an Indicator and Regulator of Steady-State CD4+FoxP3+ T Cell Homeostasis

2008-07-18

The presence of FoxP3⁺ regulatory T cells (Tregs) is necessary for control of deleterious immune responses in the steady state; however, mechanisms for maintaining the frequency and quality of endogenous Tregs are not well defined. In this study, we used in vivo modulators of the CD28 and CTLA4 pathways administered to intact mice to reveal mechanisms controlling the homeostasis and phenotype of endogenous Tregs. We demonstrate that expression of the negative costimulatory regulator CTLA4 on FoxP3⁺ Tregs in vivo is a direct consequence of their rapid, perpetual homeostasis. Up-regulation of CTLA4 expression occurs only on FoxP3⁺ Tregs undergoing extensive proliferation and can be abrogated by inhibiting the CD28 pathway, coinciding with a reduction in FoxP3⁺ Treg proliferation and frequency. We further demonstrate that CTLA4 negatively regulates steady-state Treg homeostasis, given that inhibiting CTLA4 signaling with an anti-CTLA4 blocking Ab greatly enhances Treg proliferation and overall Treg frequency. Our findings provide new insight into the origin and role of CTLA4 expression on natural FoxP3⁺ Tregs and reveal opposing effects of costimulation modulators on the steady-state level and quality of Tregs, with implications regarding their effects on endogenous Tregs in patients receiving immunotherapy.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Activated CD8 T Cells Redistribute to Antigen-Free Lymph Nodes and Exhibit Effector and Memory Characteristics

Brinkman, C. C., Sheasley-O'Neill, S. L., Ferguson, A. R., Engelhard, V. H. — 2008-07-18

Exogenous dendritic cells display restricted trafficking when injected in vivo and stimulate CD8 T cell responses that are localized to a small number of lymphoid compartments. By examining these responses in the presence and absence of FTY720, a drug that causes sequestration of T cells in lymph nodes, we demonstrate that a significant fraction of divided CD8 T cells redistribute into Ag-free lymph nodes within 3 days of activation. Despite variation in the level of expression of CD62L, redistribution of these cells is CD62L-dependent. Redistributed CD8 T cells exhibit characteristics of differentiated effectors. However, when re-isolated from Ag-free lymph nodes 3 days after activation and transferred into naive mice, they persist for at least 3 wk and expand upon Ag challenge. Thus, CD8 T cells that redistribute to Ag-free lymph nodes 3 days after immunization contain memory precursors. We suggest that this redistribution process represents an important mechanism for establishment of lymph node resident central memory, and that redistribution to Ag-free nodes is an additional characteristic to be added to those that distinguish memory precursors from terminal effectors.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Pemphigus Vulgaris IgG Directly Inhibit Desmoglein 3-Mediated Transinteraction

Heupel, W.-M., Zillikens, D., Drenckhahn, D., Waschke, J. — 2008-07-18

The autoimmune blistering skin disease pemphigus is caused by autoantibodies against keratinocyte surface Ags. In pemphigus vulgaris (PV), autoantibodies are primarily directed against desmosomal cadherins desmoglein (Dsg) 3 and Dsg 1, whereas pemphigus foliaceus (PF) patients only have Abs against Dsg 1. At present, it is unclear whether Dsg autoantibodies contribute to pemphigus pathogenesis by direct inhibition of Dsg transinteraction. Using atomic force microscopy, we provide evidence that PV-IgG directly interfere with homophilic Dsg 3 but, similar to PF-IgG, not with homophilic Dsg 1 transinteraction, indicating that the molecular mechanisms in PV and PF pathogenesis substantially differ. PV-IgG (containing Dsg 3 or Dsg 1 and Dsg 3 autoantibodies) as well as PV-IgG Fab reduced binding activity of Dsg 3 by ~60%, comparable to Ca²⁺ depletion. Similarly, the mouse monoclonal PV Ab AK 23 targeting the N-terminal Dsg 3 domain and AK 23 Fab reduced Dsg 3 transinteraction. In contrast, neither PV-IgG nor PF-IgG blocked Dsg 1 transinteraction. In HaCaT monolayers, however, both PV- and PF-IgG caused keratinocyte dissociation as well as loss of Dsg 1 and Dsg 3 transinteraction as revealed by laser tweezer assay. These data demonstrate that PV-IgG and PF-IgG reduce Dsg transinteraction by cell-dependent mechanisms and suggest that in addition, Abs to Dsg 3 contribute to PV by direct inhibition of Dsg transinteraction.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Functional Regulatory T Cells Accumulate in Aged Hosts and Promote Chronic Infectious Disease Reactivation

2008-07-18

Declines in immune function are well described in the elderly and are considered to contribute significantly to the disease burden in this population. Regulatory T cells (T_regs), a CD4⁺ T cell subset usually characterized by high CD25 expression, control the intensity of immune responses both in rodents and humans. However, because CD25 expression does not define all T_regs, especially in aged hosts, we characterized T_regs by the expression of FOXP3, a transcription factor crucial for T_reg differentiation and function. The proportion of FOXP3⁺CD4⁺ T_regs increased in the blood of the elderly and the lymphoid tissues of aged mice. The expression of functional markers, such as CTLA-4 and GITR, was either preserved or increased on FOXP3⁺ T_regs from aged hosts, depending on the tissue analyzed. In vitro depletion of peripheral T_regs from elderly humans improves effector T cell responses in most subjects. Importantly, T_regs from old FoxP3-GFP knock-in mice were suppressive, exhibiting a higher level of suppression per cell than young T_regs. The increased proportion of T_regs in aged mice was associated with the spontaneous reactivation of chronic Leishmania major infection in old mice, likely because old T_regs efficiently suppressed the production of IFN- by effector T cells. Finally, in vivo depletion of T_regs in old mice attenuated disease severity. Accumulation of functional T_regs in aged hosts could therefore play an important role in the frequent reactivation of chronic infections that occurs in aging. Manipulation of T_reg numbers and/or activity may be envisioned to enhance the control of infectious diseases in this fragile population.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Dual Signaling of MyD88 and TRIF Is Critical for Maximal TLR4-Induced Dendritic Cell Maturation

Shen, H., Tesar, B. M., Walker, W. E., Goldstein, D. R. — 2008-07-18

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-β (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Identification of an Evolutionarily Conserved Transcriptional Signature of CD8 Memory Differentiation That Is Shared by T and B Cells

2008-07-18

After Ag encounter, naive lymphocytes differentiate into populations of memory cells that share a common set of functions including faster response to Ag re-exposure and the ability to self-renew. However, memory lymphocytes in different lymphocyte lineages are functionally and phenotypically diverse. It is not known whether discrete populations of T and B cells use similar transcriptional programs during differentiation into the memory state. We used cross-species genomic analysis to examine the pattern of genes up-regulated during the differentiation of naive lymphocytes into memory cells in multiple populations of human CD4, CD8, and B cell lymphocytes as well as two mouse models of memory development. We identified and validated a signature of genes that was up-regulated in memory cells compared with naive cells in both human and mouse CD8 memory differentiation, suggesting marked evolutionary conservation of this transcriptional program. Surprisingly, this conserved CD8 differentiation signature was also up-regulated during memory differentiation in CD4 and B cell lineages. To validate the biologic significance of this signature, we showed that alterations in this signature of genes could distinguish between functional and exhausted CD8 T cells from a mouse model of chronic viral infection. Finally, we generated genome-wide microarray data from tetramer-sorted human T cells and showed profound differences in this differentiation signature between T cells specific for HIV and those specific for influenza. Thus, our data suggest that in addition to lineage-specific differentiation programs, T and B lymphocytes use a common transcriptional program during memory development that is disrupted in chronic viral infection.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Endometrial NK Cells Are Special Immature Cells That Await Pregnancy

2008-07-18

NK cells populate the human endometrium before pregnancy. Unlike decidual NK cells that populate the decidua during pregnancy, the NK cells present in the human endometrium, before pregnancy, have not been fully characterized. In this study, we provide a detailed analysis of the origin, phenotype, and function of endometrial NK cells (eNK). We show that eNK cells have a unique receptor repertoire. In particular, they are negative for NKp30 and chemokine receptor expression, which distinguishes them from any other NK subset described so far. We further show that eNK cells lack NK-specific functional phenotype and activity such as cytokine secretion and cytotoxicity, before IL-15 stimulation. Following such stimulation, endometrial NK cells acquire phenotype and function that are similar to those of decidual NK cells. We therefore suggest that eNK cells are inactive cells (before IL-15 activation and in relation to the known NK activity) that are present in the endometrium before conception, waiting for pregnancy.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Expression of Macrophage Migration Inhibitory Factor by Neuroblastoma Leads to the Inhibition of Antitumor T Cell Reactivity In Vivo

Zhou, Q., Yan, X., Gershan, J., Orentas, R. J., Johnson, B. D. — 2008-07-18

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8⁺ and CD4⁺ T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8⁺ T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8⁺ T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Bilirubin Possesses Powerful Immunomodulatory Activity and Suppresses Experimental Autoimmune Encephalomyelitis

Liu, Y., Li, P., Lu, J., Xiong, W., Oger, J., Tetzlaff, W., Cynader, M. — 2008-07-18

Bilirubin, an abundant bile pigment in mammalian serum, was once considered a toxic waste product and has more recently been recognized as a potent antioxidant of physiological importance. However, its potential biological functions in other fields are not well understood. Herein we show that bilirubin is also a powerful immunomodulatory agent. Bilirubin significantly inhibited Ag-specific and polyclonal T cell responses, while other similar antioxidants completely lacked this effect. Bilirubin suppressed CD4⁺ T cell responses at multiple steps. High levels of bilirubin could induce apoptosis in reactive CD4⁺ T cells. Bilirubin at nonapoptotic concentrations suppressed CD4⁺ T cell reactivity through a wide range of actions, including inhibition of costimulator activities, suppression of immune transcription factor activation, and down-regulation of inducible MHC class II expression. Further studies suggest that bilirubin actions were direct, rather than via induction of immune deviation or regulatory T cells. In vivo, treatment with bilirubin effectively suppressed experimental autoimmune encephalomyelitis in SJL/J mice. In contrast, depletion of endogenous bilirubin dramatically exacerbated this disease. In summary, our results identify bilirubin as an important immunomodulator that may protect mammals against autoimmune diseases, thereby indicating its potential in the treatment of multiple sclerosis and other immune disorders.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Stromal Cells Confer Lymph Node-Specific Properties by Shaping a Unique Microenvironment Influencing Local Immune Responses

Ahrendt, M., Hammerschmidt, S. I., Pabst, O., Pabst, R., Bode, U. — 2008-07-18

Lymph nodes (LN) consist not only of highly motile immune cells coming from the draining area or from the systemic circulation, but also of resident stromal cells building the backbone of the LN. These two cell types form a unique microenvironment which is important for initiating an optimal immune response. The present study asked how the unique microenvironment of the mesenteric lymph node (mLN) is influenced by highly motile cells and/or by the stromal cells. A transplantation model in rats and mice was established. After resecting the mLN, fragments of peripheral lymph node (pLN) or mLN were inserted into the mesentery. The pLN and mLN have LN-specific properties, resulting in differences of, for example, the CD103⁺ dendritic cell subset, the adhesion molecule mucosal addressin cell adhesion molecule 1, the chemokine receptor CCR9, the cytokine IL-4, and the enzyme retinal dehydrogenase 2. This new model clearly showed that during regeneration stromal cells survived and immune cells were replaced. Surviving high endothelial venules retained their site-specific expression (mucosal addressin cell adhesion molecule 1). In addition, the low expression of retinal dehydrogenase 2 and CCR9 persisted in the transplanted pLN, suggesting that stromal cells influence the lymph node-specific properties. To examine the functional relevance of this different expression pattern in transplanted animals, an immune response against orally applied cholera toxin was initiated. The data showed that the IgA response against cholera toxin is significantly diminished in animals transplanted with pLN. This model documents that stromal cells of the LN are active players in shaping a unique microenvironment and influencing immune responses in the drained area.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Th1, Th2, and Th17 Effector T Cell-Induced Autoimmune Gastritis Differs in Pathological Pattern and in Susceptibility to Suppression by Regulatory T Cells

2008-07-18

Th cells can be subdivided into IFN--secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H⁺K⁺-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] IL-2 Receptor {beta}-Chain Signaling Controls Immunosuppressive CD4+ T Cells in the Draining Lymph Nodes and Lung during Allergic Airway Inflammation In Vivo

2008-07-18

IL-2 influences both survival and differentiation of CD4⁺ T effector and regulatory T cells. We studied the effect of i.n. administration of Abs against the - and the β-chains of the IL-2R in a murine model of allergic asthma. Blockade of the β- but not the -chain of the IL-2R after allergen challenge led to a significant reduction of airway hyperresponsiveness. Although both treatments led to reduction of lung inflammation, IL-2 signaling, STAT-5 phosphorylation, and Th2-type cytokine production (IL-4 and IL-5) by lung T cells, IL-13 production and CD4⁺ T cell survival were solely inhibited by the blockade of the IL-2R β-chain. Moreover, local blockade of the common IL-2R/IL-15R β-chain reduced NK cell number and IL-2 production by lung CD4⁺CD25⁺ and CD4⁺CD25^– T cells while inducing IL-10- and TGF-β-producing CD4⁺ T cells in the lung. This cytokine milieu was associated with reduced CD4⁺ T cell proliferation in the draining lymph nodes. Thus, local blockade of the β-chain of the IL-2R restored an immunosuppressive cytokine milieu in the lung that ameliorated both inflammation and airway hyperresponsiveness in experimental allergic asthma. These findings provide novel insights into the functional role of IL-2 signaling in experimental asthma and suggest that blockade of the IL-2R β-chain might be useful for therapy of allergic asthma in humans.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Modulation of T Cell Activation by Stomatin-Like Protein 2

2008-07-18

T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Lack of Plasma Protein Hemopexin Dampens Mercury-Induced Autoimmune Response in Mice

2008-07-18

Several factors affect the autoimmune response, including iron-dependent modulation of T cells. Hemopexin is the plasma protein with the highest binding affinity to heme. It mediates heme-iron recovery in the liver, thus controlling heme-iron availability in peripheral cells. The aim of the present study was to investigate the role of hemopexin in the progress of an autoimmune response. To this end, we chose a mouse model of mercury-induced autoimmunity and evaluated the susceptibility of hemopexin-null mice to mercury treatment compared with wild-type controls. In this study we show that lack of hemopexin dampens mercury-induced autoimmune responses in mice. Hemopexin-null mice produced fewer antinuclear autoantibodies and had reduced deposits of immune complexes in the kidney after mercuric chloride treatment compared with wild-type mice. These features were associated with a reduction in activated T cells and lower absolute B cell number in spleen and impaired IgG1 and IgG2a production. In contrast, in hemopexin-null mice the response to OVA/CFA immunization was maintained. In addition, hemopexin-null mice had reduced transferrin receptor 1 expression in T cells, possibly due to the increase in heme-derived iron. Interestingly, CD4⁺T cells isolated from mercury-treated hemopexin-null mice show reduced IFN--dependent STAT1 phosphorylation compared with that of wild-type mice. Our data suggest that hemopexin, by controlling heme-iron availability in lymphocytes, modulates responsiveness to IFN- and, hence, autoimmune responses.

[CELLULAR IMMUNOLOGY AND IMMUNE REGULATION] Pancreatitis-Associated Protein 2 Modulates Inflammatory Responses in Macrophages

2008-07-18

Pancreatitis-associated proteins (PAP) are stress-induced secretory proteins that are implicated in immunoregulation. Previous studies have demonstrated that PAP is up-regulated in acute pancreatitis and that gene knockdown of PAP correlated with worsening severity of pancreatitis, suggesting a protective effect for PAP. In the present study, we investigated the effect of PAP2 in the regulation of macrophage physiology. rPAP2 administration to clonal (NR8383) and primary macrophages were followed by an assessment of cell morphology, inflammatory cytokine expression, and studies of cell-signaling pathways. NR8383 macrophages which were cultured in the presence of PAP2 aggregated and exhibited increased expression of IL-1, IL-6, TNF-, and IL-10; no significant change was observed in IL-12, IL-15, and IL-18 when compared with controls. Chemical inhibition of the NFB pathway abolished cytokine production and PAP facilitated nuclear translocation of NF-B and phosphorylation of IB inhibitory protein suggesting that PAP2 signaling involves this pathway. Cytokine responses were dose dependent. Interestingly, similar findings were observed with primary macrophages derived from lung, peritoneum, and blood but not spleen. Furthermore, PAP2 activity was inhibited by the presence of serum, inhibition which was overcome with increased PAP2. Our results demonstrate a new function for PAP2: it stimulates macrophage activity and likely modulates the inflammatory environment of pancreatitis.

[MOLECULAR AND STRUCTURAL IMMUNOLOGY] Mutational Characterization of Pancreatitis-Associated Protein 2 Domains Involved in Mediating Cytokine Secretion in Macrophages and the NF-{kappa}B Pathway

Viterbo, D., Bluth, M. H., Mueller, C. M., Zenilman, M. E. — 2008-07-18

Pancreatitis-associated protein 2 (PAP2) is a member of the Reg3 gene family and is classified as a group 7 C-type lectin-like protein. In rats, each of the three PAP isoforms has independent immunologic functional effects on macrophages. We have previously shown that PAP2 up-regulates inflammatory cytokines in macrophages in a dose-dependent manner and acts through NF-B mechanisms. In this study, we aimed to determine protein domains that are essential for the immunologic function of PAP2 by mutational or chemical analysis. The protein activity for each mutant was determined by measuring TNF-, IL-6, or IL-1 production in macrophages. Truncation of the first 25 residues on the N terminus of PAP2 did not affect protein activity whereas truncation of the last 30 residues of the C terminus of PAP2 completely inactivated the function of PAP2. Additionally, reduction of three disulfide bonds proved to be important for the activity of this protein. Further investigation revealed two invariant disulfide bonds were important for activity of PAP2 while the disulfide bond that is observed in long-form C-type lectin proteins was not essential for activity. Coupling the ability of PAP2 to up-regulate inflammatory cytokines via NF-B with its associated expression in acute pancreatitis, a condition with aberrant concentrations of inflammatory cytokines, we investigated whether PAP2 mutants mechanistically activate the NF-B-signaling pathway and demonstrate that preincubation with select rPAP2 mutant proteins affect translocation of this transcription factor into the nucleus.

[MOLECULAR AND STRUCTURAL IMMUNOLOGY] ICOS Ligation Recruits the p50{alpha} PI3K Regulatory Subunit to the Immunological Synapse

2008-07-18

ICOS ligation in concert with TCR stimulation results in strong PI3K activation in T lymphocytes. The ICOS cytoplasmic tail contains an YMFM motif that binds the p85 subunit of class IA PI3K, similar to the YMNM motif of CD28, suggesting a redundant function of the two receptors in PI3K signaling. However, ICOS costimulation shows greater PI3K activity than CD28 in T cells. We show in this report that ICOS expression in activated T cells triggers the participation of p50, one of the regulatory subunits of class IA PI3Ks. Using different T-APC cell conjugate systems, we report that p50 accumulates at the immunological synapse in activated but not in resting T cells. Our results demonstrate that ICOS membrane expression is involved in this process and that p50 plasma membrane accumulation requires a functional YMFM Src homology 2 domain-binding motif in ICOS. We also show that ICOS triggering with its ligand, ICOSL, induces the recruitment of p50 at the synapse of T cell/APC conjugates. In association with the p110 catalytic subunit, p50 is known to carry a stronger lipid kinase activity compared with p85. Accordingly, we observed that ICOS engagement results in a stronger activation of PI3K. Together, these findings provide evidence that p50 is likely a determining factor in ICOS-mediated PI3K activity in T cells. These results also suggest that a differential recruitment and activity of class IA PI3K subunits represents a novel mechanism in the control of PI3K signaling by costimulatory molecules.

[MOLECULAR AND STRUCTURAL IMMUNOLOGY] Amyloid Precursor-Like Protein 2 Increases the Endocytosis, Instability, and Turnover of the H2-Kd MHC Class I Molecule

2008-07-18

The defense against the invasion of viruses and tumors relies on the presentation of viral and tumor-derived peptides to CTL by cell surface MHC class I molecules. Previously, we showed that the ubiquitously expressed protein amyloid precursor-like protein 2 (APLP2) associates with the folded form of the MHC class I molecule K^d. In the current study, APLP2 was found to associate with folded K^d molecules following their endocytosis and to increase the amount of endocytosed K^d. In addition, increased expression of APLP2 was shown to decrease K^d surface expression and thermostability. Correspondingly, K^d thermostability and surface expression were increased by down-regulation of APLP2 expression. Overall, these data suggest that APLP2 modulates the stability and endocytosis of K^d molecules.

[MOLECULAR AND STRUCTURAL IMMUNOLOGY] Identification of Residues in the C{micro}4 Domain of Polymeric IgM Essential for Interaction with Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1)

2008-07-18

The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cµ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cµ4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.

[IMMUNOGENETICS] Tetrameric and Homodimeric Camelid IgGs Originate from the Same IgH Locus

Achour, I., Cavelier, P., Tichit, M., Bouchier, C., Lafaye, P., Rougeon, F. — 2008-07-18

In addition to producing conventional tetrameric IgGs, camelids have the particularity of producing a functional homodimeric IgG type lacking L (light) chains and only made up of two H (heavy) chains. This nonconventional IgG type is characterized by variable and constant regions referred to as V_HH and C_HH, respectively, and which differ from conventional V_H and C_H counterparts. Although the structural properties of homodimeric IgGs have been well investigated, the genetic bases involved in their generation are still largely unknown. In this study, we characterized the organization of genes coding for the H chains of tetrameric and homodimeric IgGs by constructing an alpaca (Lama pacos) genomic cosmid library. We showed that a single IgH locus in alpaca chromosome 4 contains all of the genetic elements required for the generation of the two types of Igs. The alpaca IgH locus is composed of a V region that contains both V_HH and V_H genes followed by a unique D_H-J_H cluster and C region genes, which include both C_HH and C_H genes. Although this general gene organization greatly resembles that of other typical mammalian V_n-D_n-J_n-C_n translocon IgH loci, the intermixed gene organization within the alpaca V and C regions reveals a new type of translocon IgH locus. Furthermore, analyses of cDNA coding for the membrane forms of IgG and IgM present in alpaca peripheral blood B cells are most consistent with the notion that the development of a B cell bearing homodimeric IgG passes through an IgM⁺ stage, similar to the case for conventional IgG.

[IMMUNOGENETICS] Selection of the Alternative Exon 1 from the cd5 Gene Down-Regulates Membrane Level of the Protein in B Lymphocytes

2008-07-18

The human cd5 gene has two alternative exons 1: exon 1A (E1A) which encodes the full-length (FL) CD5 protein and exon 1B (E1B) which encodes a truncated (TR) isoform. The FL variant of CD5 protein is translocated to the plasma membrane, while its TR variant is retained in the cytoplasm. Because there is an inverse relationship between the levels of FL-CD5 and TR-CD5 in B cells, we have addressed the issue of how the selection of exon 1 is determined. In leukemic B cells, DNA methyltransferase (DNMT)1-induced methylation of E1B prevents its transcription. Furthermore, the level of mRNA for DNMT1 correlates inversely with that of mRNA for CD5-E1B. However, suppression of E1B transcription is incomplete, and some molecules of TR-CD5 continue to be synthesized. Bortezomid-induced inhibition of the proteasome establishes that these TR-CD5 molecules are cleared through the ubiquitin-proteasome pathway. Transfection of CD5 mutants into COS-1 cells locates the ubiquitin-binding site at the second destruction box of the extracellular region of CD5. Activation of the B cells by anti-IgM, Staphylococcus aureus Cowan I (SAC), or PMA up-regulates DNMT1, and thereby CD5-E1A mRNA at the expense of CD5-E1B mRNA. Aberrant synthesis of TR-CD5 is thus offset by balanced degradation of excessive protein. Dysregulation of these mechanisms reduces the expression level of membrane CD5, and thereby diminishes the threshold of the response by cells expressing CD5.

[IMMUNOGENETICS] Genetic Analysis of SH2D4A, a Novel Adapter Protein Related to T Cell-Specific Adapter and Adapter Protein in Lymphocytes of Unknown Function, Reveals a Redundant Function in T Cells

2008-07-18

T cell-specific adapter (TSAd) protein and adapter protein in lymphocytes of unknown function (ALX) are two related Src homology 2 (SH2) domain-containing signaling adapter molecules that have both been shown to regulate TCR signal transduction in T cells. TSAd is required for normal TCR-induced synthesis of IL-2 and other cytokines in T cells and acts at least in part by promoting activation of the LCK protein tyrosine kinase at the outset of the TCR signaling cascade. By contrast, ALX functions as a negative-regulator of TCR-induced IL-2 synthesis through as yet undetermined mechanisms. In this study, we report a novel T cell-expressed adapter protein named SH2D4A that contains an SH2 domain that is highly homologous to the TSAd protein and ALX SH2 domains and that shares other structural features with these adapters. To examine the function of SH2D4A in T cells we produced SH2D4A-deficient mice by homologous recombination in embryonic stem cells. T cell development, homeostasis, proliferation, and function were all found to be normal in these mice. Furthermore, knockdown of SH2D4A expression in human T cells did not impact upon their function. We conclude that in contrast to TSAd and ALX proteins, SH2D4A is dispensable for TCR signal transduction in T cells.

[HOST DEFENSE] Lipoproteins of Listeria monocytogenes Are Critical for Virulence and TLR2-Mediated Immune Activation

2008-07-18

Numerous cell surface components of Listeria influence and regulate innate immune recognition and virulence. Here, we demonstrate that lipidation of prelipoproteins in Listeria monocytogenes is required to promote NF-B activation via TLR2. In HeLa cells transiently expressing TLR2, L. monocytogenes and Listeria innocua mutants lacking the prolipoprotein diacylglyceryl transferase (lgt) gene are unable to induce TLR2-dependent activation of NF-B, a property intrinsic to their isogenic parental strains. TLR2-dependent immune recognition is directed to secreted, soluble lipoproteins as evidenced by the sensitivity of the response to lipoprotein lipase. Studies of bone marrow-derived macrophages of C57BL/6 wild-type and TLR2-deficient mice infected with wild-type and lgt mutant strains indicate that the absence of host TLR2 receptor signaling has consequences similar to those of the absence of the bacterial TLR2 ligand, i.e., a delay in cellular immune responses directed toward the bacterium. Infection studies with the wild-type and TLR2^–/– mice indicated attenuation of the lgt deletion mutant in both mouse strains, implying multiple roles of lipoproteins during infection. Further characterization of the lgt mutant indicated that it is impaired for both invasion and intracellular survival and exhibits increased susceptibility to cationic peptides. Our studies identify lipoproteins as the immunologically active ligand of TLR2 and assign a critical role for this receptor in the recognition of these bacteria during infection, but they also reveal the overall importance of the lipoproteins for the pathogenicity of Listeria.

[HOST DEFENSE] Deletion of Flagellin's Hypervariable Region Abrogates Antibody-Mediated Neutralization and Systemic Activation of TLR5-Dependent Immunity

Nempont, C., Cayet, D., Rumbo, M., Bompard, C., Villeret, V., Sirard, J.-C. — 2008-07-18

TLRs trigger immunity by detecting microbe-associated molecular patterns (MAMPs). Flagellin is a unique MAMP because it harbors 1) an antigenic hypervariable region and 2) a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. In this study, the contribution of the flagellin domains in TLR5 activation was investigated. We showed that TLR5 signaling can be neutralized in vivo by flagellin-specific Abs, which target the conserved domain. However, deletions of flagellin’s hypervariable region abrogated the protein’s intrinsic ability to trigger the production of neutralizing Abs. The fact that MAMP-specific Abs block TLR-mediated responses shows that this type of neutralization is a novel mechanism for down-regulating innate immunity. The stimulation of mucosal innate immunity and adjuvancy to foreign Ag was not altered by the hypervariable domain deletions. In contrast, this domain is essential to trigger systemic innate immunity, suggesting that there are distinct mechanisms for TLR5 activation in systemic and mucosal compartments. In summary, specific MAMP determinants control the production of neutralizing Abs and the compartmentalization of innate responses.