In FY2015 we have made progress in the following areas: 1) Following infection with many viral pathogens, the host must mount an adaptive T cell response. T cells typically recognize peptide/MHC complexes derived from the virus in the context of co-stimulatory as well as innate inflammatory signals. In many cases, these innate inflammatory signals are required for a fully functional and adequate anti-viral Teff response. Perhaps the best characterized of the innate inflammatory signals produced following viral infection are Type I Interferons (IFNs). Type I IFNs, consisting of IFN-alpha and beta are produced in large quantities following viral infection and they potentiate the anti-viral Teff responses. Signaling through the IFN-alpha/beta receptor (IFNAR) expressed on CD8+ and CD4+ T cells is intrinsically required for Teff expansion following LCMV infection, and Type I IFNs exert their effect on CD8 T cells by extending their survival. The specific antigens driving Treg expansion following viral infection and the requirements for innate inflammatory signals in most models are not well characterized. We have addressed whether the innate inflammatory signals required for peptide antigen-specific Teff CD4+ and CD8+ T cell activation are also required for Treg generation/function/homeostasis. Given the immunomodulatory properties of Treg, we have considered a possible role for IFNAR signaling in Treg development/function. To specifically address the effects of type I IFNs on Treg, we studied mixed bone morrow (BM) chimeras between wild-type (WT) and IFNAR knockout (KO) mice, and heterozygous female mice expressing a Treg-specific deletion of the IFNAR. In these two models, IFNAR signaling promotes the development of the Treg lineage in the thymus and their survival in the periphery. IFNAR KO Treg had a higher expression of the pro-apoptotic gene Bim and higher frequency of active caspase positive cells. IFNAR KO Treg from chimeric mice displayed a more nave phenotype, accompanied by lower levels of CD25 and phosphorylated STAT5. Therefore, in Treg IFNAR signaling may directly or indirectly affect phosphorylation of STAT5. In mixed chimeras with Scurfy fetal liver, Treg derived from IFNAR KO BM were unable to control T effector cell activation and tissue inflammation. Under stress conditions or in a competitive environment, IFNAR signaling may be required to maintain Treg homeostasis and function. 2.The development and maturation of iNKT cells within the thymus is a tightly regulated process involving a unique profile of transcription factor expression and cytokine production. The transcription factor PLZF represents an early factor involved in the regulation of this process that leads to the eventual polarization of multiple iNKT cell sublineages, characterized by the expression of GATA3, RORgammat, and T-bet. It is known that genetic factors influence this development pathway within the thymus, as inbred mouse strains from different backgrounds demonstrate prominent variations in the composition of each iNKT cell sublineage. Such changes among the iNKT cells can further influence the development of conventional or innate-like CD8 SP cells within the thymus. However, the mechanisms by which genetic factors impact the different iNKT cell sublineages remain unknown. Innate-like or memory-like CD8+ T cells have been characterized based on their increased expression of the phenotypic surface makers of memory cells (CD44 and CD122), increased expression of the T-box transcription factor Eomesodermin (Eomes), and increased production of IFN-gamma upon stimulation. This phenotype results from the overproduction of IL-4 by PLZF+ thymic NKT cells. Innate CD8+ T cells were present in BALB/c, but not in C57BL/6 mice. We have analyzed iNKT development in the thymus of several different recombinant inbred strains of mice (BALB/c X C57BL/6 and DBA/2 XC57BL/6). We have identified several candidate genes that appear to control iNKT development and are in the process of further characterization of these genes. 3.Regulatory T cells (Treg) represent a unique population of CD4+ T cells expressing the lineage-specific transcription factor Foxp3 and are both thymically derived and induced within the periphery, particularly at mucosal sites. Treg are a heterogeous population composed of effector/memory/activated (Foxp3+CD44high) and nave/quiescent (Foxp3+CD44low) subpopulations. Only the CD44 high subpopulation can exert suppressive function. Recently, the GPI-linked surface protein, Ly-6C, has been identified as a potentially more specific marker to characterize peripheral Tregs. Ly-6C+ Tregs resemble the CD44low subset, while the Ly-6C- Treg subset expresses an activated phenotype with increased expression of CD44 and higher levels of the cell cycle-associated antigen Ki-67. Understanding the factors that control Treg activation, proliferation, and survival in the steady state is critical for furthering our ability to enhance or suppress Treg function therapeutically. CD28 and CTLA-4 have been shown to play roles in Treg immune homeostasis and suppressor function, respectively, by interacting with their ligands CD80/CD86 on antigen presenting cells. We have examined the differential effects of CD28-CTLA-4/CD80/CD86 interactions and MHC class II-TCR interactions on Treg subset homeostasis and compared the results to the effects of manipulation of these pathways on the homeostasis of Tconv naive and effector/memory CD4+ and CD8+ T cell subpopulations. We describe a complex pathway in which CTLA-4 plays a unique cell intrinsic role in sensing signals from both the CD80/CD86 and MHC class II-TCR pathways resulting in modulation of the homeostasis of both Treg memory and Tconv memory subpopulations. It should be emphasized that the homeostasis of memory phenotype Treg, CD4+ and CD8+ T cells is regulated differently from their nave cell counterparts. All the changes we have observed by blocking MHC class II, CD80/CD86, and CTLA-4 signaling were for the most part only observed in the memory cell compartments. One explanation for the increased expansion observed among the memory T cell populations, following blockade of MHC Class II, is likely an impairment of the cell intrinsic effects of CTLA-4 function leading to decreased Treg suppression. The effects of MHC blockade were mimicked by anti-CTLA-4 treatment suggesting that both TCR signals and stimulatory signals delivered by CD80/CD86 are required for optimal CTLA-4 function. Our results have important implications for the analysis of the effects of certain checkpoint inhibitors on tumor immunity. Treg-specific CTLA-4 deficiency results in enhanced tumor immunity. While anti-CTLA-4 treatment resulted in a marked enhancement of the numbers of activated Treg, it also resulted in the proliferation and and increase in the absolute number of memory CD4+ T cells and an increase in the proliferation of memory CD8+ T cells. Whether these changes in the homeostasis of CD4+ and CD8+ memory T cells translate into a more effective anti-tumor response remains to be investigated. Although global blockade of MHC class II is usually regarded as an immunosuppressive therapy, in our studies, it resulted in marked expansion of memory CD4+ and CD8+ T cells suggesting that in the primed host inhibition of MHC class II TCR interactions might blunt Treg cell suppression and promote CD4+ and CD8+ memory T cell effector function in the tumor bearing host.

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Metidji, Amina; Rieder, Sadiye Amcaoglu; Glass, Deborah Dacek et al. (2015) IFN-?/? receptor signaling promotes regulatory T cell development and function under stress conditions. J Immunol 194:4265-76
Roychoudhuri, Rahul; Hirahara, Kiyoshi; Mousavi, Kambiz et al. (2013) BACH2 represses effector programs to stabilize T(reg)-mediated immune homeostasis. Nature 498:506-10
Holt, Michael P; Shevach, Ethan M; Punkosdy, George A (2013) Endogenous mouse mammary tumor viruses (mtv): new roles for an old virus in cancer, infection, and immunity. Front Oncol 3:287
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