Inhibitory receptors play a crucial role in regulating CD8 T-cell function during chronic viral infection. T-cell Ig-and mucin-domaincontaining molecule-3 (Tim-3) is well known to negatively regulate T-cell responses, but its role in CD8 T-cell exhaustion during chronic infection in vivo remains unclear. In this study, we document coregulation of CD8 T cell exhaustion by Tim-3 and PD-1 during chronic lymphocytic choriomeningitis virus infection. Whereas Tim-3 was only transiently expressed by CD8 T cells after acute infection, virus-specific CD8 T cells retained high Tim-3 expression throughout chronic infection. The majority (approximately 65% to 80%) of lymphocytic choriomeningitis virus-specific CD8 T cells in lymphoid and nonlymphoid organs coexpressed Tim-3 and PD-1. This coexpression of Tim-3 and PD-1 was associated with more severe CD8 T-cell exhaustion in terms of proliferation and secretion of effector cytokines such as IFN-γ, TNF-α, and IL-2. Interestingly, CD8 T cells expressing both inhibitory receptors also produced the suppressive cytokine IL-10. Most importantly, combined blockade of Tim-3 and PD-1 pathways in vivo synergistically improved CD8 T cell responses and viral control in chronically infected mice. Taken together, our study defines a parameter for determining the severity of CD8 T cell dysfunction and for identifying virus-specific CD8 T cells that produce IL-10, and shows that targeting both PD-1 and Tim-3 is an effective immune strategy for treating chronic viral infections.

The inhibitory receptor programmed cell death 1 (PD-1) plays a major role in functional exhaustion of T cells during chronic infections and cancer, and recent clinical data suggest that blockade of the PD-1 pathway is an effective immunotherapy in treating certain cancers. Thus, it is important to define combinatorial approaches that increase the efficacy of PD-1 blockade. To address this issue, we examined the effect of IL-2 and PD-1 ligand 1 (PD-L1) blockade in the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We found that low-dose IL-2 administration alone enhanced CD8 + T cell responses in chronically infected mice. IL-2 treatment also decreased inhibitory receptor levels on virus-specific CD8 + T cells and increased expression of CD127 and CD44, resulting in a phenotype resembling that of memory T cells. Surprisingly, IL-2 therapy had only a minimal effect on reducing viral load. However, combining IL-2 treatment with blockade of the PD-1 inhibitory pathway had striking synergistic effects in enhancing virus-specific CD8 + T cell responses and decreasing viral load. Interestingly, this reduction in viral load occurred despite increased numbers of Tregs. These results suggest that combined IL-2 therapy and PD-L1 blockade merits consideration as a regimen for treating human chronic infections and cancer.

Summary Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a-Stat5b double knock-in (DKI) N-domain mutant mice that form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined dimer versus tetramer consensus motifs. Whereas Stat5- deficient mice exhibited perinatal lethality, DKI mice were viable; thus, STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4+CD25+ T cells, NK cells, and CD8+ T cells, with impaired cytokine-induced and homeostatic proliferation of CD8+ T cells. DKI CD8+ T cell proliferation following viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is dispensable for survival but is critical for cytokine responses and normal immune function, establishing a critical role for tetramerization in vivo.

IL-23 is a heterodimeric cytokine composed of a p19 subunit and the p40 subunit of IL-12. IL-23 has proinflammatory activity, inducing IL-17 secretion from activated CD4+ T cells and stimulating the proliferation of memory CD4+ T cells. We investigated the pathogenic role of IL-23 in CD4+ T cells in mice lacking the IL-1R antagonist (IL-1Ra−/−), an animal model of spontaneous arthritis. IL-23 was strongly expressed in the inflamed joints of IL-1Ra−/− mice. Recombinant adenovirus expressing mouse IL-23 (rAd/mIL-23) significantly accelerated this joint inflammation and joint destruction. IL-1β further increased the production of IL-23, which induced IL-17 production and OX40 expression in splenic CD4+ T cells of IL-1Ra−/− mice. Blocking IL-23 with anti-p19 Ab abolished the IL-17 production induced by IL-1 in splenocyte cultures. The process of IL-23-induced IL-17 production in CD4+ T cells was mediated via the activation of Jak2, PI3K/Akt, STAT3, and NF-κB, whereas p38 MAPK and AP-1 did not participate in the process. Our data suggest that IL-23 is a link between IL-1 and IL-17. IL-23 seems to be a central proinflammatory cytokine in the pathogenesis of this IL-1Ra−/− model of spontaneous arthritis. Its intracellular signaling pathway could be useful therapeutic targets in the treatment of autoimmune arthritis.

BackgroundCurrent influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health.Methodology/Principal FindingsTo develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms.Conclusions/SignificanceThese results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.

؉ T cell responses were further enhanced when the duration between priming and boosting was extended from 30 to 60 days. Our results demonstrate that heterologous prime-boost vaccine regimens with alternative-serotype Ad vectors elicited more functional memory CD8 ؉ T cells than any of the regimens containing Ad5. In summary, these results suggest that alternative-serotype Ad vectors will prove useful as candidates for vaccine development against human immunodeficiency virus type 1 and other pathogens and also emphasize the importance of a longer rest period between prime and boost for generating optimal CD8 ؉ T cell immunity.

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Interleukin-7 (IL-7) is a heavily glycosylated cytokine with a molecular mass of 25 kDa, and it is a member of the common ␥-chain receptor (CD132) cytokine family. IL-7 is secreted mainly by nonhematopoietic cells, such as thymic and intestinal epithelial cells, bone marrow stromal cells, keratinocytes, and reticular cells (1, 2). The receptor for IL-7, comprised of IL-7 receptor alpha (IL-7R␣; CD127) and the common ␥ chain, is expressed on various immune cells, including immature B cells, early thymocyte progenitors, and most mature T lymphocytes. IL-7 plays a role in the development of T cells, B cells, certain subsets of NK cells, and dendritic cells (DCs), as well as in the homeostasis of naive and memory T cells. In addition, IL-7 is important for ␥␦ T cell proliferation and NKT cell maintenance. However, the role of IL-7 in the differentiation of T follicular helper (Tfh) cells and the induction of humoral immunity remains unclear.Tfh cells are the unique CD4 ϩ T helper (Th) cells that provide cognate help to B cells to induce high-affinity antibody production in germinal centers (GCs) (3). GCs are specialized structures that develop within B cell follicles of secondary lymphoid tissues. GCs support intense B cell clonal expansion, somatic hypermutation, selection of high-affinity B cells, and class switching of immunoglobulin genes. These B cells ultimately are differentiated into both memory B cells and long-lived plasma cells that secrete high-affinity antibodies (2, 4).The differentiation of Tfh cells depends on the expression of a master transcriptional repressor, Bcl-6, which inhibits non-Tfh lineage differentiation (Th1, Th2, and Th17) by repressing Blimp-1 (5). Tfh cells express CXCR5 and a number of costimulatory molecules, such as ICOS and PD-1 (6). In addition, IL-6 and IL-21 have been shown to be important soluble regulators for the differentiation of Tfh cells (7-9). Recently, IL-27 was shown to increase the Tfh cell differentiation by upregulating IL-21 production (10). However, there have been conflicting reports that the absence of either IL-6 or IL-21 exhibits significant, moderate, or minimal effects on Tfh cell differentiation. The absence of both cytokines resulted in a more significant decrease in Tfh cell generation than each cytokine alone in vivo (11), indicating that IL-6 and IL-21 have redundant and/or additive functions in Tfh differentiation. It remains to be determined whether exogenous IL-6 or IL-21 induces the expansion of Tfh cells, as previous findings mentioned above were based on the experiments using knockout mice and/or blockade antibodies. Moreover, the generation of Tfh cells is not completely impaired even in the absence of IL-6 and IL-21 (11). These results indicate that these