Bcl-2 Is Necessary to Counteract Bim and Promote Survival of TCRαß+CD8αα+ Intraepithelial Lymphocyte Precursors in the Thymus.

The precursors of TCRαß+CD8αα+ intraepithelial lymphocytes (IEL) arise in the thymus through a complex process of agonist selection. We and others have shown that the proapoptotic protein, Bim, is critical to limit the number of thymic IEL precursors (IELp), as loss of Bim at the CD4+CD8+ double-positive stage of development drastically increases IELp. The factors determining this cell death versus survival decision remain largely unknown. In this study, we used CD4CreBcl2f/f mice to define the role of the antiapoptotic protein Bcl-2 and CD4CreBcl2f/fBimf/f mice to determine the role of Bcl-2 in opposing Bim to promote survival of IELp. First, in wild-type mice, we defined distinct subpopulations within PD-1+CD122+ IELp, based on their expression of Runx3 and α4ß7. Coexpression of α4ß7 and Runx3 marked IELp that were most dependent upon Bcl-2 for survival. Importantly, the additional loss of Bim restored Runx3+α4ß7+ IELp, showing that Bcl-2 antagonizes Bim to enable IELp survival. Further, the loss of thymic IELp in CD4CreBcl2f/f mice also led to a dramatic loss of IEL in the gut, and the additional loss of Bim restored gut IEL. The loss of gut IEL was due to both reduced seeding by IELp from the thymus as well as a requirement for Bcl-2 for peripheral IEL survival. Together, these findings highlight subset-specific and temporal roles for Bcl-2 in driving the survival of TCRαß+CD8αα+ IEL and thymic IELp.

IL-10-producing Tfh cells accumulate with age and link inflammation with age-related immune suppression.

Aging results in profound immune dysfunction, resulting in the decline of vaccine responsiveness previously attributed to irreversible defects in the immune system. In addition to increased interleukin-6 (IL-6), we found aged mice exhibit increased systemic IL-10 that requires forkhead box P3-negative (FoxP3-), but not FoxP3+, CD4+T cells. Most IL-10-producing cells manifested a T follicular helper (Tfh) phenotype and required the Tfh cytokines IL-6 and IL-21 for their accrual, so we refer to them as Tfh10 cells. IL-21 was also required to maintain normal serum levels of IL-6 and IL-10. Notably, antigen-specific Tfh10 cells arose after immunization of aged mice, and neutralization of IL-10 receptor signaling significantly restored Tfh-dependent antibody responses, whereas depletion of FoxP3+ regulatory and follicular regulatory cells did not. Thus, these data demonstrate that immune suppression with age is reversible and implicate Tfh10 cells as an intriguing link between "inflammaging" and impaired immune responses with age.

The Variable Genomic NK Cell Receptor Locus Is a Key Determinant of CD4+ T Cell Responses During Viral Infection.

Increasing evidence points to a key role for NK cells in controlling adaptive immune responses. In studies examining the role of CD1d on CD4+ T cell responses, we found that a line of CD1d-deficient mice on the C57BL/6J background had a homozygous 129 locus on chromosome 6 containing the entire NK cell gene cluster. Mice possessing this locus (C57BL/6.NKC129) displayed a >10-fold reduction in antigen-specific CD4+ T cell responses after intracranial infection with lymphocytic choriomeningitis virus (LCMV). Neither parental strain displayed defects in viral-specific CD4+ T cell responses. Interestingly, following infection, increased numbers of NK cells accumulated in the lymph nodes of C57BL/6.NKC129 mice and displayed enhanced in vivo functionality. Moreover, depletion of NK cells with anti-asialo-GM-1 antibody in C57BL/6.NKC129 mice resulted in a >20-fold increase in viral-specific CD4+ T cell responses. Mechanistically, we found that dendritic cell antigen presentation and early type I IFN production were significantly decreased in C57BL/6.NKC129 mice, but were restored in perforin-deficient C57BL/6.NKC129 mice or following NK depletion. Together, these data reveal that the variable genomic regions containing the activating/inhibitory NK cell receptors are key determinants of antigen-specific CD4+ T cell responses, controlling type I IFN production and the antigen-presenting capacity of dendritic cells.

mTOR Inhibitor Therapy Diminishes Circulating CD8+ CD28- Effector Memory T Cells and Improves Allograft Inflammation in Belatacept-refractory Renal Allograft Rejection.

BACKGROUND: Renal allograft rejection is more frequent under belatacept-based, compared with tacrolimus-based, immunosuppression. We studied kidney transplant recipients experiencing rejection under belatacept-based early corticosteroid withdrawal following T-cell-depleting induction in a recent randomized trial (Belatacept-based Early Steroid Withdrawal Trial, clinicaltrials.gov NCT01729494) to determine mechanisms of rejection and treatment. METHODS: Peripheral mononuclear cells, serum creatinine levels, and renal biopsies were collected from 8 patients undergoing belatacept-refractory rejection (BRR). We used flow cytometry, histology, and immunofluorescence to characterize CD8 effector memory T cell (TEM) populations in the periphery and graft before and after mammalian target of rapamycin (mTOR) inhibition. RESULTS: Here, we found that patients with BRR did not respond to standard antirejection therapy and had a substantial increase in alloreactive CD8 T cells with a CD28/DR/CD38/CD45RO TEM. These cells had increased activation of the mTOR pathway, as assessed by phosphorylated ribosomal protein S6 expression. Notably, everolimus (an mTOR inhibitor) treatment of patients with BRR halted the in vivo proliferation of TEM cells and their ex vivo alloreactivity and resulted in their significant reduction in the peripheral blood. The frequency of circulating FoxP3 regulatory T cells was not altered. Importantly, everolimus led to rapid resolution of rejection as confirmed by histology. CONCLUSIONS: Thus, while prior work has shown that concomitant belatacept + mTOR inhibitor therapy is effective for maintenance immunosuppression, our preliminary data suggest that everolimus may provide an available means for effecting "rescue" therapy for rejections occurring under belatacept that are refractory to traditional antirejection therapy with corticosteroids and polyclonal antilymphocyte globulin.

Paracrine IL-2 Is Required for Optimal Type 2 Effector Cytokine Production.

IL-2 is a pleiotropic cytokine that promotes the differentiation of Th cell subsets, including Th1, Th2, and Th9 cells, but it impairs the development of Th17 and T follicular helper cells. Although IL-2 is produced by all polarized Th subsets to some level, how it impacts cytokine production when effector T cells are restimulated is unknown. We show in this article that Golgi transport inhibitors (GTIs) blocked IL-9 production. Mechanistically, GTIs blocked secretion of IL-2 that normally feeds back in a paracrine manner to promote STAT5 activation and IL-9 production. IL-2 feedback had no effect on Th1- or Th17-signature cytokine production, but it promoted Th2- and Th9-associated cytokine expression. These data suggest that the use of GTIs results in an underestimation of the presence of type 2 cytokine-secreting cells and highlight IL-2 as a critical component in optimal cytokine production by Th2 and Th9 cells in vitro and in vivo.