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Cross-subtype neutralizing single domain antibodies against influenza present new opportunities for immunoprophylaxis and pandemic preparedness. Their simple modular structure and single open reading frame format are highly amenable to gene therapy-mediated delivery. We have previously described R1a-B6, an alpaca-derived single domain antibody (nanobody), that is capable of potent cross-subtype neutralization in vitro of H1N1, H5N1, H2N2, and H9N2 influenza viruses, through binding to a highly conserved epitope in the influenza hemagglutinin stem region. To evaluate the potential of R1a-B6 for immunoprophylaxis, we have reformatted it as an Fc fusion for adeno-associated viral (AAV) vector delivery. Our findings demonstrate that a single intramuscular injection in mice of AAV encoding R1a-B6 fused to Fc fragments of different isotypes equipped either, with or without antibody dependent cellular cytotoxicity (ADCC) activity, was able to drive sustained high-level expression (0.5–1.1 mg/mL) in sera with no evidence of reduction for up to 6 months. R1a-B6-Fc fusions of both isotypes gave complete protection against lethal challenge with both pandemic A/California/07/2009 (H1N1)pdm09 and avian influenza A/Vietnam/1194/2004 (H5N1). This data suggests that R1a-B6 is capable of cross-subtype protection and ADCC was not essential for R1a-B6 efficacy. Our findings demonstrate AAV delivery of cross-subtype neutralizing nanobodies may be an effective strategy to prevent influenza infection and provide long-term protection independent of a host induced immune response.

Expression of the costimulatory receptor 4-1BB is induced by TCR recognition of Ag, whereas 4-1BB ligand (4-1BBL) is highly expressed on activated APC. 4-1BB signaling is particularly important for survival of activated and memory CD8+ T cells. We wished to test whether coexpression of Ag and 4-1BBL by dendritic cells (DC) would be an effective vaccine strategy. Therefore, we constructed lentiviral vectors (LV) coexpressing 4-1BBL and influenza nucleoprotein (NP). Following s.c. immunization of mice, which targets DC, we found superior CD8+ T cell responses against NP and protection from influenza when 4-1BBL was expressed. However, functionally superior CD8+ T cell responses were obtained when two LV were coinjected: one expressing 4-1BBL and the other expressing NP. This surprising result suggested that 4-1BBL is more effective when expressed in trans, acting on adjacent DC. Therefore, we investigated the effect of LV expression of 4-1BBL in mouse DC cultures and observed induced maturation of bystander, untransduced cells. Maturation was blocked by anti–4-1BBL Ab, required cell–cell contact, and did not require the cytoplasmic signaling domain of 4-1BBL. Greater maturation of untransduced cells could be explained by LV expression of 4-1BBL, causing downregulation of 4-1BB. These data suggest that coexpression of 4-1BBL and Ag by vaccine vectors that target DC may not be an optimal strategy. However, 4-1BBL LV immunization activates significant numbers of bystander DC in the draining lymph nodes. Therefore, transactivation by 4-1BBL/4-1BB interaction following DC–DC contact may play a role in the immune response to infection or vaccination.

TLR4 interactor with leucine-rich repeats (TRIL) is a brain-enriched accessory protein that is important in TLR3 and TLR4 signaling. In this study, we generated Tril−/− mice and examined TLR responses in vitro and in vivo. We found a role for TRIL in both TLR4 and TLR3 signaling in mixed glial cells, consistent with the high level of expression of TRIL in these cells. We also found that TRIL is a modulator of the innate immune response to LPS challenge and Escherichia coli infection in vivo. Tril−/− mice produce lower levels of multiple proinflammatory cytokines and chemokines specifically within the brain after E. coli and LPS challenge. Collectively, these data uncover TRIL as a mediator of innate immune responses within the brain, where it enhances neuronal cytokine responses to infection.

Lentiviral vectors can efficiently transduce a variety of nondividing cells, including APCs. We assessed the immunogenicity of a lentiviral vector encoding the melanoma Ag NY-ESO-1 in HLA-A2 transgenic mice. Direct i.v. injection of NY-ESO-1 lentivirus induced NY-ESO-1157–165-specific CD8+ cells, detected ex vivo with an A2/H-2Kb chimeric class I tetramer. These NY-ESO-1157–165-specific CD8+ cells could be expanded by boosting with an NY-ESO-1 vaccinia virus and could kill NY-ESO-1157–165 peptide-pulsed targets in vivo. Such direct lentiviral vector injection was similar in potency to the injection of in vitro-transduced dendritic cells (DC). In addition, human monocyte-derived DC transduced by the NY-ESO-1 lentivirus stimulated an NY-ESO-1157–165-specific specific CTL clone. These data suggest that direct lentiviral transduction of DC in vivo might provide a powerful immunotherapeutic strategy.

MRL/MpJ-Faslpr/lpr/J (MRLlpr) mice develop lupus-like disease manifestations in an IL-21–dependent manner. IL-21 is a pleiotropic cytokine that can influence the activation, differentiation, and expansion of B and T cell effector subsets. Notably, autoreactive CD4+ T and B cells spontaneously accumulate in MRLlpr mice and mediate disease pathogenesis. We sought to identify the particular lymphocyte effector subsets regulated by IL-21 in the context of systemic autoimmunity and, thus, generated MRLlpr mice deficient in IL-21R (MRLlpr.IL-21R−/−). Lymphadenopathy and splenomegaly, which are characteristic traits of the MRLlpr model were significantly reduced in the absence of IL-21R, suggesting that immune activation was likewise decreased. Indeed, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R–deficient MRLlpr mice. Correspondingly, we observed a significant reduction in autoantibody titers. Activated CD4+ CD44+ CD62Llo T cells also failed to accumulate, and CD4+ Th cell differentiation was impaired, as evidenced by a significant reduction in CD4+ T cells that produced the pronephritogenic cytokine IFN-γ. T extrafollicular helper cells are a recently described subset of activated CD4+ T cells that function as the primary inducers of autoantibody production in MRLlpr mice. Importantly, we demonstrated that T extrafollicular helper cells are dependent on IL-21R for their generation. Together, our data highlighted the novel observation that IL-21 is a critical regulator of multiple pathogenic B and T cell effector subsets in MRLlpr mice.

IL-22 is made by a unique set of innate and adaptive immune cells, including the recently identified noncytolytic NK, lymphoid tissue-inducer, Th17, and Th22 cells. The direct effects of IL-22 are restricted to nonhematopoietic cells, its receptor expressed on the surface of only epithelial cells and some fibroblasts in various organs, including parenchymal tissue of the gut, lung, skin, and liver. Despite this cellular restriction on IL-22 activity, we demonstrate that IL-22 induces effects on systemic biochemical, cellular, and physiological parameters. By utilizing adenoviral-mediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Furthermore, we observed that IL-22 induces thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a systemic inflammatory state. We observed that IL-22 induces biochemical changes in the liver including induction of fibrinogen, CXCL1, and serum amyloid A that likely contribute to the reported cellular and physiological effects of IL-22. Based on these findings, we propose that downstream of its expression and impact in local tissue inflammation, circulating IL-22 can further induce changes in systemic physiology that is indicative of an acute-phase response.

Objective All γ-chain cytokines signal through JAK-3 and JAK-1 acting in tandem. We undertook this study to determine whether the JAK-3 selective inhibitor WYE-151650 would be sufficient to disrupt cytokine signaling and to ameliorate autoimmune disease pathology without inhibiting other pathways mediated by JAK-1, JAK-2, and Tyk-2. Methods JAK-3 kinase selective compounds were characterized by kinase assay and JAK-3–dependent (interleukin-2 [IL-2]) and –independent (IL-6, granulocyte–macrophage colony-stimulating factor [GM-CSF]) cell-based assays measuring proliferation or STAT phosphorylation. In vivo, off-target signaling was measured by IL-22– and erythropoietin (EPO)–mediated models, while on-target signaling was measured by IL-2–mediated signaling. Efficacy of JAK-3 inhibitors was determined using delayed-type hypersensitivity (DTH) and collagen-induced arthritis (CIA) models in mice. Results In vitro, WYE-151650 potently suppressed IL-2–induced STAT-5 phosphorylation and cell proliferation, while exhibiting 10–29-fold less activity against JAK-3–independent IL-6– or GM-CSF–induced STAT phosphorylation. Ex vivo, WYE-151650 suppressed IL-2–induced STAT phosphorylation, but not IL-6–induced STAT phosphorylation, as measured in whole blood. In vivo, WYE-151650 inhibited JAK-3–mediated IL-2–induced interferon-γ production and decreased the natural killer cell population in mice, while not affecting IL-22–induced serum amyloid A production or EPO-induced reticulocytosis. WYE-151650 was efficacious in mouse DTH and CIA models. Conclusion In vitro, ex vivo, and in vivo assays demonstrate that WYE-151650 is efficacious in mouse CIA despite JAK-3 selectivity. These data question the need to broadly inhibit JAK-1–, JAK-2–, or Tyk-2–dependent cytokine pathways for efficacy.

Objective Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor α (TNFα) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNFα blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNFα blockade–induced exacerbation of skin inflammation in murine psoriasis-like skin disease. Methods Skin inflammation was induced in BALB/c scid/scid mice after they received CD4+CD45RBhighCD25− (naive CD4) T cells from donor mice. These mice were treated with either anti–interleukin-12 (anti–IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4+ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNFα. Cytokine gene expression from these differentiated cells was also determined. Results Neutralization of TNFα exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1β, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNFα also demonstrated a divergent role during priming and reactivation of naive T cells. Conclusion These results reveal a novel immunoregulatory role of TNFα on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments.

T-helper 17 (Th17) cells are a new lineage of CD4(+) T cells that are characterized by their production of interleukin-17A (IL-17A). Recent studies show that these cells can also express IL-17F, IL-22, and IL-21. IL-17A and IL-17F can form a heterodimeric cytokine, which mediates biological activities, at least in part, through shared receptors with IL-17A and IL-17F homodimers. The cytokines made by Th17 cells represent three distinct gene families, highlighting the unique biology of these cells. Accumulating data support a role for Th17 cells and these cytokines in inflammatory processes and in animal models of autoimmunity or inflammation. Emerging data in clinical trials support our understanding of the importance of Th17 cells in inflammatory disease. Future clinical studies will allow us to evaluate the role of each cytokine independently in contributing to human diseases with immune-mediated pathologies and to design optimal cytokine-targeted therapies for these diseases.

IL-17A and IL-17F are related homodimeric proteins of the IL-17 family produced by Th17 cells. In this study, we show that mouse Th17 cells also produce an IL-17F/A heterodimeric protein. Whereas naive CD4+ T cells differentiating toward the Th17 cell lineage expressed IL-17F/A in higher amounts than IL-17A/A homodimer and in lower amounts than IL-17F/F homodimer, differentiated Th17 cells expressed IL-17F/A in higher amounts than either homodimer. In vitro, IL-17F/A was more potent than IL-17F/F and less potent than IL-17A/A in regulating CXCL1 expression. Neutralization of IL-17F/A with an IL-17A-specific Ab, and not with an IL-17F-specific Ab, reduced the majority of IL-17F/A-induced CXCL1 expression. To study these cytokines in vivo, we established a Th17 cell adoptive transfer model characterized by increased neutrophilia in the airways. An IL-17A-specific Ab completely prevented Th17 cell-induced neutrophilia and CXCL5 expression, whereas Abs specific for IL-17F or IL-22, a cytokine also produced by Th17 cells, had no effects. Direct administration of mouse IL-17A/A or IL-17F/A, and not IL-17F/F or IL-22, into the airways significantly increased neutrophil and chemokine expression. Taken together, our data elucidate the regulation of IL-17F/A heterodimer expression by Th17 cells and demonstrate an in vivo function for this cytokine in airway neutrophilia.

Th17 cells are a distinct lineage of effector CD4+ T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-22, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th2 cells. Similar to IL-17A, IL-22 expression was initiated by transforming growth factor β signaling in the context of IL-6 and other proinflammatory cytokines. The subsequent expansion of IL-22–producing cells was dependent on IL-23. We further demonstrate that IL-22 was coexpressed in vitro and in vivo with both IL-17A and IL-17F. To study a functional relationship among these cytokines, we examined the expression of antimicrobial peptides by primary keratinocytes treated with combinations of IL-22, IL-17A, and IL-17F. IL-22 in conjunction with IL-17A or IL-17F synergistically induced the expression of β-defensin 2 and S100A9 and additively enhanced the expression of S100A7 and S100A8. Collectively, we have identified IL-22 as a new cytokine expressed by Th17 cells that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity.

Disseminated candidiasis is a frequent infection in neutropenic patients, in whom it causes 50% mortality, despite antifungal therapy. As the duration of neutropenia is the strongest predictor of survival in neutropenic patients with invasive fungal infections, neutrophil transfusions are a logical, therapeutic option. However, significant technical barriers have prevented the clinical use of neutrophil transfusions. To overcome these barriers, we identified a human phagocytic cell line that could be administered to candidemic hosts in lieu of freshly harvested neutrophils. HL-60 cells killed Candida albicans in vitro. Activation of HL-60 cells with dimethyl sulfoxide and retinoic acid abrogated the cells’ proliferation and augmented their killing of C. albicans. Administration of activated HL-60 cells to candidemic, neutropenic mice significantly improved survival (53% vs. 0%). Live HL-60 cells chemotaxed to sites of infection, phagocytized C. albicans, and reduced the fungal burden in key target organs. Although unactivated HL-60 cells also reduced tissue fungal burden in vivo, they did not improve survival as a result of their toxicity in infected mice. In contrast, no toxicity as a result of activated HL-60 cells was observed at up to 2 months of follow-up. To our knowledge, this is the first description of a cell line-based immunotherapy for an infectious disease. With further refinements, activated HL-60 cells have the potential to overcome the technical barriers to neutrophil transfusions.

Nonactivated CD4+CD25+ regulatory T cells constitutively express glucocorticoid-induced TNFR family-related receptor (GITR), a TNFR family member whose engagement was presumed to abrogate regulatory T cell-mediated suppression. Using GITR−/− mice, we report that GITR engagement on CD25−, not CD25+ T cells abrogates T cell-mediated suppression. Mouse APCs constitutively express GITR ligand (GITR-L), which is down-regulated following TLR signaling in vivo. Although GITR−/−CD25− T cells were capable of mounting proliferative responses, they were incapable of proliferation in the presence of physiological numbers of CD25+ T cells. Thus, GITR-L provides an important signal for CD25− T cells, rendering them resistant to CD25+-mediated regulation at the initiation of the immune response. The down-regulation of GITR-L by inflammatory stimuli may enhance the susceptibility of effector T cells to suppressor activity during the course of an infectious insult.

The B and T lymphocyte attenuator, BTLA, is a recently discovered Ig superfamily member. BTLA engagement results in downregulation of T-cell activation, and mice deficient in BTLA show increased incidence and severity of autoimmune disorders. A novel B7 family member, B7x (B7S1, B7-H4), has been proposed as the ligand for BTLA. A B7x–Ig fusion protein inhibits T-cell activation and interleukin-2 (IL-2) production. Thus, cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1) and BTLA comprise an expanding set of inhibitory receptors expressed on lymphocytes.

Highly polarized type 2 cytokine responses can be harmful and even lethal to the host if they are too vigorous or persist too long. Therefore, it is important to elucidate the mechanisms that down-regulate these reactions. Interleukin (IL)-13 has emerged as a central mediator of T helper cell (Th)2-dominant immune responses, exhibiting a diverse array of functional activities including regulation of airway hyperreactivity, resistance to nematode parasites, and tissue remodeling and fibrosis. Here, we show that IL-13 receptor (R)α2 is a critical down-regulatory factor of IL-13–mediated tissue fibrosis induced by the parasitic helminth Schistosoma mansoni. IL-13Rα2 expression was induced after the onset of the fibrotic response, IL-10, IL-13, and Stat6 dependent, and inhibited by the Th1-inducing adjuvant IL-12. Strikingly, schistosome-infected C57BL/6 and BALB/c IL-13Rα2–deficient mice showed a marked exacerbation in hepatic fibrosis, despite displaying no change in granuloma size, tissue eosinophilia, or mastocytosis. Fibrosis increased despite the fact that IL-13 levels decreased significantly in the liver and serum. Importantly, pathology was prevented when IL-13Rα2–deficient mice were treated with a soluble IL-13Rα2-Fc construct, formally demonstrating that their exacerbated fibrotic response was due to heightened IL-13 activity. Together, these studies illustrate the central role played by the IL-13Rα2 in the down-regulation of a chronic and pathogenic Th2-mediated immune response.

Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma, parasite immunity, and tumor recurrence. At least two distinct receptor components, IL-4 receptor (R)α and IL-13Rα1, mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13, IL-13Rα2, whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Rα2, mice deficient in IL-13Rα2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Rα2−/− mice despite the fact that serum IL-13 was absent and immune interferon γ production increased compared with wild-type mice. IL-13Rα2–deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Rα2 in regulating immune responses in wild-type mice.

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4+ T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Rα expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.

The cytokine potential of developing T helper (Th) cells is directly shaped both positively and negatively by the cytokines expressed by the effector Th cell subsets. Here we find that the recently identified cytokine, interleukin (IL)-21, is preferentially expressed by Th2 cells when compared with Th1 cells generated in vitro and in vivo. Exposure of naive Th precursors to IL-21 inhibits interferon (IFN)-γ production from developing Th1 cells. The repression of IFN-γ production is specific in that the expression of other Th1 and Th2 cytokines is unaffected. IL-21 decreases the IL-12 responsiveness of developing Th cells by specifically reducing both signal transducer and activator of transcription 4 protein and mRNA expression. These results suggest that Th2 cell-derived IL-21 regulates the development of IFN-γ–producing Th1 cells which could serve to amplify a Th2 response.

T cell activation is dependent upon signals delivered through the antigen-specific T cell receptor and accessory receptors on the T cell. A primary costimulatory signal is delivered through the CD28 receptor after engagement of its ligands, B7-1 (CD80) or B7-2 (CD86). Engagement of CTLA-4 (CD152) by the same B7-1 or B7-2 ligands results in attenuation of T cells responses. Recently, molecular homologs of CD28 and CTLA-4 receptors and their B7-like ligands have been identified. ICOS is a CD28-like costimulatory receptor with a unique B7-like ligand. PD-1 is an inhibitory receptor, with two B7-like ligands. Additional members of B7 and CD28 gene families have been proposed. Integration of signals through this family of costimulatory and inhibitory receptors and their ligands is critical for activation of immune responses and tolerance. Understanding these pathways will allow development of new strategies for therapeutic intervention in immune-mediated diseases.

IFNalpha/beta, IL-12, and IL-15 regulate NK cell activation and expansion, but signals triggering resolution of the NK response upon induction of adaptive immunity remain to be defined. We now report that IL-21, a product of activated T cells, may serve this function. Mice lacking IL-21R (IL-21R(-/-)) had normal NK cell development but no detectable responses to IL-21. IL-21 enhanced cytotoxic activity and IFNgamma production by activated murine NK cells but did not support their viability, thus limiting their duration of activation. Furthermore, IL-21 blocked IL-15-induced expansion of resting NK cells, thus preventing the initiation of further innate responses. In contrast, IL-21 enhanced the proliferation, IFNgamma production, and cytotoxic function of CD8(+) effector T cells in an allogeneic MLR. These observations suggest that IL-21 promotes the transition between innate and adaptive immunity.

CD4(+)CD25(+) immunoregulatory T cells represent a unique lineage of thymic-derived cells that potently suppress both in vitro and in vivo effector T cell function. We analyzed CD4(+)CD25(+) and CD4(+)CD25(-) T cells by DNA microarray, identifying 29 genes differentially expressed in the resting subpopulations, and 77 that were differentially expressed following activation. Most of these genes were elevated in the CD4(+)CD25(+) population, suggesting a previously activated phenotype. Among these were a number of genes that antagonize signaling, including members of the SOCS family, which may contribute to their anergic phenotype. Multiple cell surface receptors also had increased expression in CD4(+)CD25(+) cells, including GITR, a member of the TNF receptor superfamily. Importantly, antibodies to GITR abrogated suppression, demonstrating a functional role for this receptor in regulating the CD4(+)CD25(+) T cell subset.

The inducible costimulatory (ICOS) molecule is expressed by activated T cells and has homology to CD28 and CD152. ICOS binds B7h, a molecule expressed by APC with homology to CD80 and CD86. To investigate regulation of ICOS expression and its role in Th responses we developed anti-mouse ICOS mAbs and ICOS-Ig fusion protein. Little ICOS is expressed by freshly isolated mouse T cells, but ICOS is rapidly up-regulated on most CD4+ and CD8+ T cells following stimulation of the TCR. Strikingly, ICOS up-regulation is significantly reduced in the absence of CD80 and CD86 and can be restored by CD28 stimulation, suggesting that CD28-CD80/CD86 interactions may optimize ICOS expression. Interestingly, TCR-transgenic T cells differentiated into Th2 expressed significantly more ICOS than cells differentiated into Th1. We used two methods to investigate the role of ICOS in activation of CD4+ T cells. First, CD4+ cells were stimulated with beads coated with anti-CD3 and either B7h-Ig fusion protein or control Ig fusion protein. ICOS stimulation enhanced proliferation of CD4+ cells and production of IFN-γ, IL-4, and IL-10, but not IL-2. Second, TCR-transgenic CD4+ T cells were stimulated with peptide and APC in the presence of ICOS-Ig or control Ig. When the ICOS:B7h interaction was blocked by ICOS-Ig, CD4+ T cells produced more IFN-γ and less IL-4 and IL-10 than CD4+ cells differentiated with control Ig. These results demonstrate that ICOS stimulation is important in T cell activation and that ICOS may have a particularly important role in development of Th2 cells.

CTLA-4 (CD152) engagement can down-regulate T cell activation and promote the induction of immune tolerance. However, the strategy of attenuating T cell activation by engaging CTLA-4 has been limited by sharing of its natural ligands with the costimulatory protein CD28. In the present study, a CTLA-4-specific single-chain Ab (scFv) was developed and expressed on the cell surface to promote selective engagement of this regulatory molecule. Transfectants expressing anti-CTLA-4 scFv at their surface bound soluble CTLA-4 but not soluble CD28. Coexpression of anti-CTLA-4 scFv with anti-CD3ε and anti-CD28 scFvs on artificial APCs reduced the proliferation and IL-2 production by resting and preactivated bulk T cells as well as CD4+ and CD8+ T cell subsets. Importantly, expression of anti-CTLA-4 scFv on the same cell surface as the TCR ligand was essential for the inhibitory effects of CTLA-4-specific ligation. CTLA-4-mediated inhibition of tyrosine phosphorylation of components of the proximal TCR signaling apparatus was similarly dependent on coexpression of TCR and CTLA-4 ligands on the same surface. These findings support a predominant role for CTLA-4 function in the modification of the proximal TCR signal. Using T cells from DO11.10 and 2C TCR transgenic mice, negative regulatory effects of selective CTLA-4 ligation were also demonstrated during the stimulation of Ag-specific CD4+ and CD8+ T cells by MHC/peptide complexes. Together these studies demonstrate that selective ligation of CTLA-4 using a membrane-bound scFv results in attenuated T cell responses only when coengaged with the TCR during T cell/APC interaction and define an approach to harnessing the immunomodulatory potential of CTLA-4-specific ligation.

By the genetic selection of mouse cDNAs encoding secreted proteins, a B7-like cDNA clone termed mouse GL50 (mGL50) was isolated encoding a 322-aa polypeptide identical with B7h. Isolation of the human ortholog of this cDNA (hGL50) revealed a coding sequence of 309 aa residues with 42% sequence identity with mGL50. Northern analysis indicated GL50 to be present in many tissues including lymphoid, embryonic yolk sac, and fetal liver samples. Of the CD28, CTLA4, and ICOS fusion constructs tested, flow cytometric analysis demonstrated only mouse ICOS-IgG binding to mGL50 cell transfectants. Subsequent phenotyping demonstrated high levels of ICOS ligand staining on splenic CD19+ B cells and low levels on CD3+ T cells. These results indicate that GL50 is a specific ligand for the ICOS receptor and suggest that the GL50-ICOS interaction functions in lymphocyte costimulation.

Two components of a receptor complex for IL-13, the IL-4R and a low affinity IL-13-binding chain, IL-13Rα1, have been cloned in mice and humans. An additional high affinity binding chain for IL-13, IL-13Rα2, has been described in humans. We isolated a cDNA from the thymus that encodes the murine orthologue of the human IL-13Rα2. The predicted protein sequence of murine IL-13Rα2 (mIL-13Rα2) has 59% overall identity to human IL-13Rα2 and is closely related to the murine low affinity IL-13-binding subunit, IL-13Rα1. The genes for both mIL-13-binding chains map to the X chromosome. A specific interaction between mIL-13Rα2.Fc protein and IL-13 was demonstrated by surface plasmon resonance using a BIACORE instrument. Ba/F3 cells that were transfected with mIL-13Rα2 expressed 5000 molecules per cell and bound IL-13 with a single Kd of 0.5 to 1.2 nM. However, these cells did not proliferate in response to IL-13, and the IL-4 dose response was unaffected by high concentrations of IL-13. In contrast, the expression of mIL-13Rα1 by Ba/F3 cells resulted in a sensitive proliferative response to IL-13. Consistent with its lower affinity for IL-13, IL-13Rα1.Fc was 100-fold less effective than IL-13Rα2.Fc in neutralizing IL-13 in vitro. These results show that mIL-13Rα2 and mIL-13Rα1 are not functionally equivalent and predict distinct roles for each polypeptide in IL-13R complex formation and in the modulation of IL-13 signal transduction.

Although IL-4 induces expulsion of the gastrointestinal nematode parasite, Nippostrongylus brasiliensis, from immunodeficient mice, this parasite is expelled normally by IL-4-deficient mice. This apparent paradox is explained by observations that IL-4 receptor alpha chain (IL-4Ralpha)-deficient mice and Stat6-deficient mice fail to expel N. brasiliensis, and a specific antagonist for IL-13, another activator of Stat6 through IL-4Ralpha, prevents worm expulsion. Thus, N. brasiliensis expulsion requires signaling via IL-4Ralpha and Stat6, and IL-13 may be more important than IL-4 as an inducer of the Stat6 signaling that leads to worm expulsion. Additional observations made in the course of these experiments demonstrate that Stat6 signaling is not required for IL-4 enhancement of IgG1 production and actually inhibits IL-4-induction of mucosal mastocytosis.

The murine fibrosarcoma FS29 can be more efficiently killed by syngeneic lymphocytes when it has been engineered to secrete either interferon gamma (IFN-gamma), or interleukin 2 (IL-2). The mechanisms by which the two cytokines enhance target sensitivity differ. Supernatant from IFN-gamma-secreting cells can enhance the sensitivity of unmodified cells. The enhanced sensitivity correlates with MHC upregulation observed on both the IFN-gamma-secreting and supernatant-treated cells. In contrast, supernatant from IL-2-secreting cells does not affect the sensitivity of unmodified cells. IL-2 can be detected, by a bioassay, bound to the extracellular matrix of the secreting tumour cells.

Recent studies have demonstrated that rodent tumour cells engineered to secrete a variety of cytokines, or to express foreign antigens, MHC molecules or co-stimulatory molecules, are rejected by syngeneic animals. These observations have led to the initiation of a number of clinical trials using genetically modified tumour cells, to attempt to stimulate a patient anti-tumour immune response. In this study, a protocol has been developed to test in vitro the specific cytotoxic anti-tumour response generated from melanoma patient lymphocytes. The results showed that IL-12 in combination with IL-2 enhanced the autologous anti-melanoma CTL response, whereas B7.1 antigen expression on tumour cells did not increase anti-melanoma CTL generation. This method could be used to design more appropriate genetically modified tumour vaccines.

Vaccines that induce T cells, which recognize conserved viral proteins, could confer universal protection against seasonal and pandemic influenza strains. An effective vaccine should generate sufficient mucosal T cells to ensure rapid viral control before clinical disease. However, T cells may also cause lung injury in influenza, so this approach carries inherent risks. Here we describe intranasal immunization of mice with a lentiviral vector expressing influenza nucleoprotein (NP), together with an NFκB activator, which transduces over 75% of alveolar macrophages (AM). This strategy recalls and expands NP-specific CD8+ T cells in the lung and airway of mice that have been immunized subcutaneously, or previously exposed to influenza. Granzyme B-high, lung-resident T-cell populations persist for at least 4 months and can control a lethal influenza challenge without harmful cytokine responses, weight loss, or lung injury. These data demonstrate that AM can be harnessed as effective antigen-presenting cells for influenza vaccination.

The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.

Derivatives of the murine bone marrow-derived, IL-3-dependent cell line, BAF3, were isolated following recombinant retroviral infection which showed disregulated expression of human c-Myc oncoprotein. Such cells entered apoptosis and lost viability more rapidly than parental BAF3 cells when IL-3 was removed. c-Myc disregulation also resulted in an inability of BAF3 cells to survive in either sub-optimal concentrations of IL-3, or saturating concentrations of IL-4 and insulin-like growth factor-1. Furthermore, BAF3 cells with disregulated c-Myc expression were more sensitive to the induction of apoptosis by DNA-damaging agents. These effects of c-Myc disregulation could be inhibited by co-expression of the Bcl-2 oncoprotein. The implications of these data for the transformation of haematopoietic cells by disregulation of Myc expression are discussed.

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to PGs via the cyclooxygenase 1 and 2 pathways and to leukotrienes via the 5-lipoxygenase pathway. We used adoptive transfer and relapsing–remitting forms of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, in two different strains of mice (SJL or C57BL/6) to demonstrate that blockade of cPLA2α with a highly specific small-molecule inhibitor during the tissue-damage effector phase abrogates the clinical manifestation of disease. Using the adoptive transfer model in SJL mice, we demonstrated that the blockade of cPLA2α during the effector phase of disease was more efficacious in ameliorating the disease pathogenesis than the blockade of each of the downstream enzymes, cyclooxygenase-1/2 and 5-lipooxygenase. Similarly, blockade of cPLA2α was highly efficacious in ameliorating disease pathogenesis during the effector phase of EAE in the adoptive transfer model of EAE in C57BL/6 mice. Investigation of the mechanism of action indicates that cPLA2α inhibitors act on APCs to diminish their ability to induce Ag-specific effector T cell proliferation and proinflammatory cytokine production. Furthermore, cPLA2α inhibitors may prevent activation of CNS-resident microglia and may increase oligodendrocyte survival. Finally, in a relapsing–remitting model of EAE in SJL mice, therapeutic administration of a cPLA2α inhibitor, starting from the peak of disease or during remission, completely protected the mice from subsequent relapses.

Mutation of Asp20 in human interleukin-2 (IL-2) to Lys is known to result in an IL-2 molecule with unchanged binding to the p55 subunit of the IL-2 receptor, but with greatly decreased affinity for the p75 subunit (Collins, L., Tsien, W.-H., Seals, C. et al. Proc. Natl. Acad. Sci USA 1988. 85: 7709). Here we demonstrate that Lys20 IL-2 competed with a reduced (10-fold) affinity for high-affinity IL-2 receptors on two murine cell lines HT2 and CTLL. In parallel with this difference in receptor interaction, Lys20 IL-2 stimulated half-maximal HT2 cell proliferation at a 10-fold higher concentration than wild-type IL-2. However, half-maximal stimulation of CTLL cells required a 100-fold higher concentration of Lys20 IL-2. A similar 100-fold reduction in bioactivity of Lys20 IL-2 was observed for primary, activated, human or murine lymphocytes. Anti-p55 antibodies increased the concentration of Lys20 IL-2 required to stimulate HT2 cells to that required for CTLL cells. These data suggest that CTLL cells, while able to bind Lys20 IL-2 with high affinity, are lacking a p55-dependent function necessary for optimal stimulation. Therefore, p55 has a dual role, being important both for high-affinity IL-2 binding and for optimal cell triggering.

This report addresses the role of individual IL-2 binding proteins of the IL-2 receptor in the stimulation of cell proliferation by IL-2. Murine IL-3-dependent cell lines were established which expressed the human p75 IL-2-binding protein, in the absence or presence of the human p55 IL-2-binding protein. Whereas p75 expression was sufficient to confer response to an intermediate (half-maximal stimulation at 100 pM) concentration of IL-2, additional expression of p55 increased the sensitivity of the cells to half-maximal stimulation at 10 pM IL-2. A mutant IL-2 molecule, Lys-20 IL-2 which is known to be defective of p75 interaction, was unable to stimulate cells expressing only p75: p55 co-expression could restore its activity. Under conditions of low p75 expression, Lys-20 IL-2 could act as an antagonist of wild-type IL-2 action. These data support a role for p55 in the enhancement of responsiveness to IL-2.

Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R–deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.

Although cancer immunology has made vigorous progress over the last decade, its future remains uncertain. Tumors have clearly proved subject to immune surveillance, leading to antigenic editing, and means of activating both T and B arms of the immune system have been devised. Therapeutic vaccination and monoclonal antibody therapy have so far proved disappointing, because tumors prove adept at evasion from immune control. Dual targeting could well counteract evasion, provided that the two targets are independent and are attacked simultaneously. This stage has nearly but not quite been reached in several forms of immunotherapy, particularly of B-cell cancers, although such treatment also carries hazards.

Murine bone marrow-derived cells, dependent on interleukin 3 (IL-3) for their growth in culture, undergo programmed cell, or apoptosis, upon cytokine withdrawal. Here it is reported that a variety of DNA damaging agents cause a more rapid onset of apoptosis in a factor-dependent cell line, BAF3, deprived of IL-3. In contrast, when cultured in the presence of IL-3, or other growth promoting factors, BAF3 cells are highly resistant to X-irradiation and the cytotoxic drugs etoposide and cisplatin. Overexpression of the bcl2 gene product also protects BAF3 cells from DNA damage. The presence of IL-3 is not required during the initial events of DNA damage or its repair. In the absence of IL-3, cells still complete the repair of DNA breaks within 15 min, and continue to cycle for 5 h. At this time, IL-3 is necessary to prevent the accelerated onset of DNA cleavage from a G2 arrest point.

The murine haemopoietic cell line, BAF3, undergoes apoptosis when the growth factor IL-3 is withdrawn. Two flow cytometric methods for quantifying the apoptotic cells are described. Cell sorting followed by DNA gel electrophoresis, and both light and electron microscopy have been used to identify the apoptotic cells. In the first method the cells are fixed in ethanol, stained with propidium iodide and a DNA histogram recorded. The apoptotic cells give a ‘sub-G1’ peak. In the second method unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342. The apoptotic cells take up this dye more rapidly. In this latter method, the non-viable cells can also be enumerated by addition of propidium iodide. The value of the method has been demonstrated in a brief study of the effects of a panel of cytokines on growth and apoptosis.

To study the effects of regulatory proteins encoded by herpes viruses on the HIV long terminal repeat (LTR) in the presence or absence of HIV-encoded regulatory products, we prepared a proviral construct containing 5' and 3' HIV LTR, but lacking the coding sequences of any HIV proteins. This construct allowed the effects of herpesvirus regulatory proteins on the HIV LTR to be assessed in a construct similar to the HIV provirus whilst also allowing their interactions with HIV-encoded regulatory proteins to be studied. In this system, the herpes simplex virus (HSV) protein IE1 (ICPO) but not the IE3 (ICP4) protein can activate the HIV LTR, whereas both proteins are active on a single plasmid-borne HIV LTR. Although the activation of the LTR by IE1 is strongly stimulated by the HIV Tat protein, it can also be observed in the absence of Tat, indicating that HSV infection via IE1 has the potential to activate an entirely silent, latent HIV provirus.

Animal models of rheumatoid arthritis (RA) are widely used for testing potential new therapies for RA. However, the question of which animal model is most predictive of therapeutic efficacy in human RA commonly arises in data evaluation. A retrospective review of the animal models used to evaluate approved, pending RA therapies, and compounds that were discontinued during phase II or III clinical trials found that the three most commonly used models were adjuvant-induced arthritis (AIA) in rats and collagen-induced arthritis (CIA) in rats and mice. Limited data were found for more recently developed genetically modified animal models. Examination of the efficacy of various compounds in these animal models revealed that a compound's therapeutic efficacy, rather than prophylactic efficacy, in AIA and CIA models was more predictive of clinical efficacy in human RA than data from either model alone.

Although granulocyte transfusion is a logical, therapeutic option for neutropenic patients with refractory infections, significant technical barriers have prevented its widespread use. A novel phagocyte transfusion strategy has been developed based on activation of a human myeloid cell line HL-60. To further define the potential for HL-60 cells to recapitulate white cell transfusions, a shortened duration of activation was evaluated, facile quality control markers were defined, and the impact of low-dose irradiation on cell function was determined. Three days of activation resulted in increased cell viability and in vitro candidacidal capacity but with slightly higher cell replication compared with 7 days of activation. Cell viability and several flow cytometric measurements were accurate, quality control markers for HL-60 activation. In combination with activation, low-dose irradiation abrogated replication while sparing the candidacidal effects of the HL-60 cells. Infusion of irradiated, activated HL-60 cells improved survival of neutropenic, candidemic mice significantly. In summary, activated, irradiated HL-60 cells are microbicidal, have virtually no replicative capacity, and are safe and effective at protecting neutropenic mice against an otherwise 100% fatal candidal infection. With continued development, this strategy to recapitulate neutrophil functions has the potential to serve as an effective alternative to granulocyte transfusions.

Interactions between PD-1 and its two differentially expressed ligands, PD-L1 and PD-L2, attenuate T cell activation and effector function. To determine the role of these molecules in autoimmune disease of the CNS, PD-1-/-, PD-L1-/- and PD-L2-/- mice were generated and immunized to induce experimental autoimmune encephalomyelitis (EAE). PD-1-/- and PD-L1-/- mice developed more severe EAE than wild type and PD-L2-/- mice. Consistent with this, PD-1-/- and PD-L1-/- cells produced elevated levels of the pro-inflammatory cytokines IFN-gamma, TNF, IL-6 and IL-17. These results demonstrate that interactions between PD-1/PD-L1, but not PD-1/PDL-2, are crucial in attenuating T cell responses in EAE.

Therapeutic cancer vaccines need to stimulate a refractory immune system to make an effective anti-tumour response. We have explored the use of lentiviral vectors to deliver tumour antigen genes to dendritic cells (DC) as a possible mechanism of immune stimulation. Direct injection of a lentiviral vector encoding the melanoma antigen NY-ESO-1 in HLA-A2 transgenic mice primed NY-ESO-1-specific CD8+ cells that could be expanded by boosting with an NY-ESO-1 vaccinia virus. The expanded cells could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. In order to examine the priming step directly, we constructed another lentiviral vector expressing the melanoma antigen Melan-A (MART-1). Here we show that Melan-A protein is also efficiently expressed after transduction of human DC cultured from peripheral blood mononuclear cells. When these transduced DC are co-cultured with autologous naïve T cells, they cause the expansion of cells that recognise the HLA-A2 restricted Melan-A(27-35) epitope. The expanded cells are functional in that they release IFN-gamma upon antigen stimulation. Melan-A lentiviral vector transduced DC caused a similar level of naïve T-cell expansion to Melan-A(27-35) peptide-pulsed DC in four experiments using different HLA-A2 positive donors. These data suggest that a vaccine based either on DC transduced with a lentiviral vector ex vivo, or on direct lentiviral vector injection, should be assessed in a phase I clinical trial.

To date, very few Ag-based regimens have been defined that could expand T regulatory (Treg) cells to reverse autoimmunity. Additional understanding of Treg function with respect to specificity and broad suppression should help overcome these limitations. Ig-proteolipid protein (PLP)1, an Ig carrying a PLP1 peptide corresponding to amino acid residues 139-151 of PLP, displayed potent tolerogenic functions and proved effective against experimental allergic encephalomyelitis (EAE). In this study, we took advantage of the Ig-PLP1 system and the PLP1-specific TCR transgenic 5B6 mouse to define a regimen that could expand Ag-specific Treg cells in vivo and tested for effectiveness against autoimmunity involving diverse T cell specificities. The findings indicate that in vivo exposure to aggregated Ig-PLP1 drives PLP1-specific 5B6 TCR transgenic cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3 and producing IL-10. These Treg cells were able to suppress PLP1 peptide-induced EAE in both SJL/J and F1 (SJL/J × C57BL/6) mice. However, despite being effective against disease induced with a CNS homogenate, the Treg cells were unable to counter EAE induced by a myelin basic protein or a myelin oligodendrocyte glycoprotein peptide. Nevertheless, activation with Ag before transfer into the host mice supports suppression of both myelin oligodendrocyte glycoprotein- and myelin basic protein peptide-induced EAE. Thus, it is suggested that activation of Treg cells by the cognate autoantigen is necessary for operation of broad suppressive functions.

IL-13 is a Th2 cytokine that plays crucial roles in the pathophysiology of allergy, asthma and helminth infection. The high affinity receptor for IL-13, IL-13Ralpha2, may act as a decoy receptor for IL-13. The anti-tumor effect of IL-13 and its soluble receptor IL-13Ralpha2 have been examined in different tumor systems. Previous studies have shown that IL-13 enhances anti-tumor responses in some model systems, whereas IL-13Ralpha2Fc prevents IL-13 mediated suppression of tumor immuno-surveillance in a different model system. In this study, we have used a cytokine (receptor) gene therapy approach and studied the immune responses mediated by IL-13 and IL-13Ralpha2Fc in poorly immunogenic B16F1 melanoma and immunogenic MethA fibrosarcoma tumor models. We find that IL-13 reduces the tumorigenicity of B16F1 melanoma and MethA fibrosarcoma cells in vivo, most likely through the recruitment of neutrophils and macrophages. IL-13 mediated anti-tumor responses do not lead to the generation of tumor-specific T cells. Neither IL-13Ralpha2Fc gene transduction nor in vivo treatment with soluble IL-13Ralpha2Fc has a statistically significant effect of tumor growth. IL-13Ralpha2 deficient host background does not alter tumor growth, suggesting that endogenous levels of IL-13 do not contribute to an anti-tumor response in these models. We conclude that IL-13, but not soluble IL-13Ralpha2, has anti-tumor activity in the models described here, possibly by enhancing innate anti-tumor immunity.

IL-21 is a key factor in the transition between innate and adaptive immune responses. We have used the cytokine gene therapy approach to study the antitumor responses mediated by IL-21 in the B16F1 melanoma and MethA fibrosarcoma tumor models in mice. Retrovirally transduced tumor cells secreting biologically functional IL-21 have growth patterns in vitro similar to that of control green fluorescent protein-transduced cells, but are completely rejected in vivo. We show that IL-21 activates NK and CD8+ T cells in vivo, thus mediating complete rejection of poorly immunogenic tumors. Rejection of IL-21-secreting tumors requires the presence of cognate IL-21R and does not depend on CD4+ T cell help. Interestingly, perforin, but not IFN-γ or other major Th1 and Th2 cytokines (IL-12, IL-4, or IL-10), is required for the IL-21-mediated antitumor response. Moreover, IL-21 results in 50% protection and 70% cure of nonimmunogenic tumors when given before and after tumor challenge, respectively, in C57BL/6 mice. We conclude that IL-21 immunotherapy warrants clinical evaluation as a potential treatment for cancer.

Cytokines play an important role in regulating the development and homeostasis of B cells by controlling their viability. In this study, we show that the recently described T cell-derived cytokine IL-21 induces the apoptosis of resting primary murine B cells. In addition, the activation of primary B cells with IL-4, LPS, or anti-CD40 Ab does not prevent IL-21-mediated apoptosis. The induction of apoptosis by IL-21 correlates with a down-regulation in the expression of Bcl-2 and Bcl-xL, two antiapoptotic members of the Bcl-2 family. Furthermore, the reconstitution of Bcl-xL or Bcl-2 expression protects primary B cells from IL-21-induced apoptosis. In addition, a short-term preactivation of B cells with anti-CD40 Ab confers protection from IL-21-mediated apoptosis through the up-regulation of Bcl-xL. These studies reveal a novel pathway that mediates B cell apoptosis via the IL-21R and suggest that IL-21 may play a role in regulating B cell homeostasis.

The catalytic subunit of the serine/threonine phosphatase 2A (PP2A) can interact with the cytoplasmic tail of CTLA-4. However, the molecular basis and the biological significance of this interaction are unknown. In this study, we report that the regulatory subunit of PP2A (PP2AA) also interacts with the cytoplasmic tail of CTLA-4. Interestingly, TCR ligation induces tyrosine phosphorylation of PP2AA and its dissociation from CTLA-4 when coligated. The association between PP2AA and CTLA-4 involves a conserved three-lysine motif in the juxtamembrane portion of the cytoplasmic tail of CTLA-4. Mutations of these lysine residues prevent the binding of PP2AA and enhance the inhibition of IL-2 gene transcription by CTLA-4, indicating that PP2A represses CTLA-4 function. Our data imply that the lysine-rich motif in CTLA-4 may be used to identify small molecules that block its binding to PP2A and act as agonists for CTLA-4 function.

The process of immunological costimulation between APC and T cells is mediated by protein ligand:receptor interactions. To date, costimulatory receptors known to be expressed by T cells include the structurally related proteins CD28 and the inducible costimulator (ICOS). The ligands to human and mouse ICOS, human GL50 (hGL50), and mouse GL50 (mGL50) were recently cloned and demonstrated to have sequence similarity to the CD28 ligands B7-1 and B7-2. Examination of mGL50 cDNA transcripts by 3′RACE revealed an alternatively spliced form, mGL50-B, that encoded a protein product with a divergent 27-aa intracellular domain. Both mGL50- and mGL50-B-transfected cells exhibited binding to human and mouse ICOS-Ig fusion protein, indicating that the alternate cytoplasmic domain of mGL50-B does not interfere with extracellular interactions with ICOS receptor. Flow cytometric and RT-PCR analysis of BALB/c and RAG1−/− mice splenocytes demonstrate that freshly isolated B cells, T cells, macrophages, and dendritic cells express both splice variant forms of ICOS ligand. Comparative analyses with the human ICOS ligand splice variants hGL50 and B7-H2 indicate that differential splicing at the junction of cytoplasmic exon 6 and exon 7 may be a common method by which GL50-ICOS immunological costimulatory processes are regulated in vivo.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor–mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon γ, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.