1) Further understanding of the role of Treg during chronic infection has been limited by our lack of knowledge regarding the stimuli that drive Treg activation and expansion in these situations. We have identified a subpopulation of Treg that rapidly and specifically expand early during chronic but not acute infection. The expanded Treg are restricted to cells expressing particular TCR Vbeta segments (Vbeta5 in C57BL/6 mice and Vbeta5 and 12 in BALB/c mice). Interestingly, the Vbeta-specific T cell expansion was restricted only to Treg and not Foxp3- Teff cells expressing the same V-beta segments, suggesting that the process is Treg-specific. By using congenic mice that lack all endogenous Mtv proviruses (Mtv/null), we have shown that the expansion of Vbeta-specific Treg during chronic LCMV infection is secondary to Mtv-superantigen (Sag)-encoded in the mouse genome. The mechanism by which chronic LCMV infection results in Mtv-Sag-dependent Treg activation seems to depend and require presentation of the endogenous SAg by DC. Taken together, these studies define a unique mechanism of Treg activation and expansion following chronic viral infection. 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 similarly required for Sag-mediated expansion of Treg following chronic LCMV infection. We have demonstrated that Sag-mediated Treg expansion requires intrinsic IFNAR signaling similar to Teff. While Teff cells required cell intrinsic signaling via the adapter protein, MyD88, in response to IL-33, Sag-specific Treg expansion does not require intrinsic MyD88 signaling. However, MyD88-dependent CD8+ T cell activation itself is required. The Treg cell intrinsic requirement for IFNAR was clearly demonstrated by studies using mixed bone marrow chimeras between WT and IFNAR deficient mice. To conclusively demonstrate that CD8+ Teff responses were also needed for Treg expansion, we made used LCMV congenital carrier mice that lack CD8+ T cells that recognize LCMV. No Treg expansion was detectable in these mice, but significant expansion of Treg could be induced by transfer of LCMV-specific CD8+ T cells from LCMV-specific TCR transgenic mice. Collectively, these findings shed further light on the similarities and differences between the specific requirements for Treg versus Teff activation. The mechanisms by which anti-viral specific CD8+ T cells mediate Treg expansion in this model also remain unknown. There appears to be a reciprocal relationship between Treg and anti-viral CD8+ T cells. CD8+ Teff cells may expand Treg to inhibit immunopathology, but overexpansion of Treg may directly or indirectly promote CD8+ T cell exhaustion. (2) Given the immunomodulatory properties of Treg, we have considered a possible role for IFNAR signaling in Treg development/function. Treg from WT and IFNAR deficient (KO) mice were equal in number and in vitro/in vivo suppressive activity. Therefore, we set up a model to test IFNAR KO Treg fitness in a competitive environment using bone marrow chimeras reconstituted with a 1:1 mixture of WT and IFNAR KO cells. While the WT to IFNAR KO ratio of all T cells was significantly skewed toward WT in these mice, skewing was most pronounced in the Treg compartment. The disadvantage of the IFNAR KO Treg in the competitive environment was likely due to a defect in survival, as the ratio of IFNAR KO Treg was not markedly decreased in the thymus, and peripheral IFNAR KO Treg had a significantly higher frequency of active caspase positive cells consistent with enhanced cell death. The fitness of IFNAR KO Treg was further examined in a disease model by testing their capacity to rescue the scurfy disease phenotype. IFNAR KO Treg in chimeric mice reconstituted with scurfy and IFNAR KO bone marrow were unable to control Teff cell activation and tissue inflammation. Collectively, these studies demonstrate that type I IFNs play a critical role in the homeostasis and function of Foxp3+ Treg cell particularly under conditions of stress including immune reconstitution following lethal irradiation. 3). We have also begun to explore the potential role of Mtv infection on other aspects of immune responses and have focused our studies on NK T cells and innate CD8+ T cells.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 via 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 promyelocytic leukemia zinc-finger (PLZF)+ thymic NKT cells. Innate CD8+ T cells were present in BALB/c, but not in C57BL/6 mice. Innate CD8+ T cells were not present in the thymus of Mtv-null mice. Thymic iNKT cells from BALB/c mice had increased expression of PLZF in iNKT cells compared to BALB/Mtv-null mice. Thus, the presence of genes encoded by endogenous retroviruses appears to have a profound influence on the development of iNKT cells.
