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The regulatory circuits dictating CD8+ T cell responsiveness versus exhaustion during anti-tumor immunity are incompletely understood. Here we report that tumor-infiltrating antigen-specific PD-1+ TCF-1− CD8+ T cells express the immunosuppressive cytokine Fgl2. Conditional deletion of Fgl2 specifically in mouse antigen-specific CD8+ T cells prolongs CD8+ T cell persistence, suppresses phenotypic and transcriptomic signatures of T cell exhaustion, and improves control of the tumor. In a mouse model of chronic viral infection, PD-1+ CD8+ T cell-derived Fgl2 also negatively regulates virus-specific T cell responses. In humans, CD8+ T cell-derived Fgl2 is associated with poorer survival in patients with melanoma. Mechanistically, the dampened responsiveness of WT Fgl2-expressing CD8+ T cells, when compared to Fgl2-deficient CD8+ T cells, is underpinned by the cell-intrinsic interaction of Fgl2 with CD8+ T cell-expressed FcγRIIB and concomitant caspase 3/7-mediated apoptosis. Our results thus illuminate a cell-autonomous regulatory axis by which PD-1+ CD8+ T cells both express the receptor and secrete its ligand in order to mediate suppression of anti-tumor and anti-viral immunity. Mechanisms regulating CD8 T cell responsiveness are not fully understood. Here the authors show, using mouse genetic tools and human patient samples, that CD8 T-derived Fgl2, an immunosuppressive cytokine, binds autologous FcγRIIB receptor to induce CD8 T cell apoptosis and dampen anti-tumor or antivirus immunity. [ABSTRACT FROM AUTHOR]

Author(s): Daewoong Jo [1]; Danya Liu [1]; Shan Yao [1]; Robert D Collins [2]; Jacek Hawiger (corresponding author) [1] Suppressor of cytokine signaling (SOCS) 3 attenuates proinflammatory signaling mediated by [...]

We recently showed that 2B4 expression on memory T cells in human renal transplant recipients was associated with reduced rates of rejection. To investigate whether 2B4 functionally underlies graft acceptance during transplantation, we established an experimental model in which 2B4 was retrogenically expressed on donor-reactive murine CD8+ T cells (2B4rg), which were then transferred into naive recipients prior to skin transplantation. We found that constitutive 2B4 expression resulted in significantly reduced accumulation of donor-reactive CD8+ T cells following transplantation and significantly prolonged graft survival follow- ing transplantation. This marked reduction in alloreactivity was due to reduced proliferation of CD8+ Thy1.1+ 2B4rg cells as compared with control cells, underpinned by extracellular flux analyses demonstrating that 2B4-deficient (2B4KO) CD8+ cells activated in vitro exhibited increased glycolytic capacity and upregulation of gene expression profiles consistent with enhanced glycolytic machinery as compared with wild type controls. Furthermore, 2B4KO CD8+ T cells primed in vivo exhibited significantly enhanced ex vivo uptake of a fluorescent glucose analogue. Finally, the proliferative advantage associated with 2B4 deficiency was only observed in the setting of glucose sufficiency; in glucose-poor conditions, 2B4KO CD8+ T cells lost their proliferative advantage. Together, these data indicate that 2B4 signals function to alter T cell glucose metabolism, thereby limiting the proliferation and accumulation of CD8+ T cells. Targeting 2B4 may therefore represent a novel therapeutic strategy to attenuate unwanted CD8+ T cell responses. [ABSTRACT FROM AUTHOR]

Current models of CD4+ T cell help suggest a major role for CD154 binding to CD40 expressed on dendritic cells, with a lesser role for direct T:T interactions via CD40 expressed on CD8+ T cells. However, the contribution of CD8+ T cell-derived CD40 signals during the donor-reactive T cell response to a transplant has never been studied. In this study, we examined the graft-rejection kinetics and CD4+ and CD8+ donor-reactive T cell responses under conditions in which CD40 was genetically ablated only on APC, as well as under conditions in which CD40 was genetically ablated only on donor-reactive CD8+ T cells. Our results revealed a significant role for CD8+ T cell-expressed CD40 in the augmentation of donor-reactive CD8+ T cell responses following transplantation and showed that CD40 expressed on CD8+ T cells must be inhibited to allow conversion of CD4+ T cells into induced regulatory T cells. Thus, this study identifies a major role for CD8+ T cell-derived CD40 signals as a critical switch factor that both promotes optimal differentiation of cytokine-producing CD8+ effector T cell responses and inhibits the differentiation of Ag-specific Foxp3+ induced regulatory T cells in vivo. [ABSTRACT FROM AUTHOR]

Genetic programming of B lymphocytes plays a key role in development of autoimmune diabetes in NOD mice. The genetic programs for autoimmunity, inflammation and apoptosis are regulated by NF-kappa B and other stress-responsive transcription factors (SRTFs). In response to inflammatory, immune, and oxidant stress, these SRTFs translocate to the nucleus via a set of nuclear import adapters known as karyopherins/importins. Karyopherin alpha 2 (Rch1, KPNA2) is the target for a cell-penetrating peptide (cSN50) that prevents the nuclear import of SRTFs. Here we report that cSN50 prevents overt diabetes in an accelerated model in which 10-11 week old female NOD mice received cyclophosphamide (Cy) (200mg/kg). Following Cy administration, NOD splenocytes displayed increased nuclear localization of NF-kappa B, and NOD B lymphocytes showed upregulation of CD80 and CD86. These preliminary findings indicate that Cy-induced acceleration is consistent with a model of antigen presenting cell/B cell-mediated autoimmunity. We treated Cy-challenged NOD mice with cSN50 for two days beginning 45 hours after Cy. Only 10% of cSN50 treated mice developed hyperglycemia during 250 days of followup compared to 90% of saline- or control peptide-treated animals (p=0.0002, log-rank). After 24 hours of cSN50 therapy, islets from mice receiving cSN50 demonstrated nearly complete resolution of insulitis by immunohistochemistry. Mice treated with cSN50 had reduced B lymphocyte numbers in pancreas, spleen and pancreatic lymph nodes. No changes were noted in the proportion of activated T ceils (CD69+), regulatory T cells (foxp3+), or T cell-derived cytokines. In contrast, cSN50-treated animals demonstrated increased serum levels of IL-6 (p<0.003, ANOVA) and MCP-1 (p<0.02) during therapy, factors that may be islet protective. These cytokines/chemokines were B cell-derived as restimulation of splenocytes from cSN50-treated mice with B cell agonists but not T cell agonists recapitulated this cytokine/chemokine profile. Cumulatively, these results provide initial evidence that re-programming of B lymphocytes by nuclear import modulation suppresses autoimmune diabetes after the establishment of insulitis. [ABSTRACT FROM AUTHOR]

The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II- or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell-dependent responses to diverse Ags during the life of an individual. [ABSTRACT FROM AUTHOR]

The affinity of a TCR binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low-affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic Ag and mediate graft rejection. We found that low-affinity-primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared with high-affinity-primed memory T cells. Low-affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low-affinity-primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low- but not high-affinity-primed animals. These data establish a functional connection between TNF signaling and TCR-priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. [ABSTRACT FROM AUTHOR]