In FY2014 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. 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. A similar competitive disadvantage was noted in heterozygous female mice in which 50% of the Treg failed to express the IFNAR. 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. Similar results were observed when the Tregs were derived from mice with a Treg-specific deletion of the IFNAR. 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. We have also examined the effects of interferon-alpha in a model of autoimmune disease, experimental autoimmune encephalomyelitis (EAE). Previous studies have demonstrated that Type I IFNs primarily act on myeloid cells during the course of EAE. We have re-examined the role of Type I IFNs in EAE by generating mixed bone marrow chimeras between wild type (WT) mice and IFNAR-/- mice. IFN plays a role in the T lymphocyte homeostasis as the ratio of WT to IFNAR-/- CD4+ T cells is skewed toward WT and the skewing is more marked in the Treg compartment. Surprisingly, after induction of EAE, the ratio of WT to IFNAR-/- T cells is reversed in the periphery and the mixed chimeras develop more severe disease than the WT/WT chimeras. Almost all of the CD4+Foxp3+ T cells in the CNS of chimeric mice are derived from the IFNAR-/- donor, but the percentage of Foxp3+ T cells was similar in WT/WT and WT/IFNAR-/- chimeras. CD4+Foxp3+ T cells from the IFNAR-/- donor expressed higher levels of the alpha4 integrin, which may account for their increased numbers in the CNS during EAE. Mice with a conditional deletion of the IFNAR in Treg rapidly developed a very severe form of EAE. Taken together, these results demonstrate that signaling via the IFNAR is required for Treg suppressor function in EAE. 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. Tregs appear to be a critical population controlling the priming of naive T cells during acute infection and also the activation of T memory cells during during chronic infection. It is therefore critical to understand the factors that control Treg homeostasis. As a population, Foxp3+ Treg cells undergo rapid homeostatic proliferation that is comparable to the memory T conventional cell pool. However, it remains to be determined whether this proliferation constitutes a property of all or a subset of Treg cells. We have identified Ly6C as a cell surface marker that can distinguish two distinct populations of Treg cells. The Ly6C+ population (30%) is quiescent, while the Ly-6C- population (60%) contains a substantial number (50%) of rapidly dividing cells as measured by Ki-67 positivity or incorporation of BrdU. The two populations of Treg appear to be stable upon transfer to immunocompetent recipients. The expression of Ly6C on developing Treg cells in the thymus is minimal, but Foxp3+Ly-6+ Tregs appear in the periphery as early as 1.5 weeks of age. The factors that drive Treg homeostatic proliferation have thus far been elusive. While treatment of mice with anti-MHC class II or absence of microbial antigens have no effect, blockade of IL-2 moderately reduces, but blockade of CD80/CD86 derived signals profoundly reduces Treg homeostatic proliferation. Further analysis of these two Treg populations should offer important insights to the basic biology of Treg homeostasis/function and may have important implications for the manipulation of Tregs for cellular therapy.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
Zip Code
Gangaplara, Arunakumar; Martens, Craig; Dahlstrom, Eric et al. (2018) Type I interferon signaling attenuates regulatory T cell function in viral infection and in the tumor microenvironment. PLoS Pathog 14:e1006985
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
Zhu, Jinfang; Jankovic, Dragana; Oler, Andrew J et al. (2012) The transcription factor T-bet is induced by multiple pathways and prevents an endogenous Th2 cell program during Th1 cell responses. Immunity 37:660-73
Punkosdy, George A; Blain, Melissa; Glass, Deborah D et al. (2011) Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens. Proc Natl Acad Sci U S A 108:3677-82
Pasquali, Paolo; Thornton, Angela M; Vendetti, Silvia et al. (2010) CD4+CD25+ T regulatory cells limit effector T cells and favor the progression of brucellosis in BALB/c mice. Microbes Infect 12:3-10