Studies of the Function of Naturally Occurring and Adaptive T Regulatory Cells

National Institute Of Allergy And Infectious Diseases

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Abstract

Four distinct areas were studied in this project in FY2017: 1. A functional immune system is dependent on the maintenance of gene expression and transcription factors play critical roles in regulating gene expression at specific stages of development. The Ikaros transcription factor family is one such family whose expression is indispensable for immune system development and function. We have been studying the role of one member of this family, Helios, in the function of Treg cells. We had previously demonstrated that Helios was selectively expressed by 70-80% of mouse and human Foxp3+ peripheral Tregs and suggested that expression of Helios allowed the differentiation of thymic-derived Tregs (tTregs) from Tregs induced in peripheral sites (pTregs). Mice with a Treg-specific deletion of Helios showed signs of a systemic autoimmune disease beginning at 2 months of age. At 6 months of age, the mice exhibited generalized splenomegaly, lymphadenopathy, expansion of Th1 Teff cells, hypergammaglobulinemia, and increased size/numbers of lymphoid follicles and germinal centers. The most prominent abnormality observed in mice with a Treg-specific deletion of Helios was the failure of the deficient Treg to control T follicular helper (Tfh) function. In contrast, mice with a T cell-specific deletion of Helios were phenotypically normal which raised the possibility that Helios may also function in T conventional (Tconv) cells. We have demonstrated that Helios is transiently upregulated in a subset of activated CD4+Foxp3- Tconv cells. We therefore examined the capacity of Helios-deficient, naive Tconv cells to respond to their cognate antigen in vivo and found that Helios was not required for proliferation, differentiation into helper subsets, or the production of effector cytokines. Unexpectedly, we observed that Helios deficiency increased the susceptibility of naive T cells to differentiate into Tregs under inflammatory conditions. Additionally, we found that Helios is necessary for the reactivation of memory T cells, as Helios-deficient T cells failed to proliferate upon secondary challenge with antigen. Subsequently, we demonstrated that the defective memory T cell reactivation was secondary to the presence of antigen-specific pTregs. Taken together, these findings suggest that the increased pTreg differentiation in the periphery in mice with a deletion of Helios in all T cells may prevent the development of autoimmunity normally associated with Helios-deficient Tregs. 2. In addition to Helios, a second member of the Ikaros gene family, Eos, is preferentially expressed in Treg cells. However, the exact role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated Eosfl/fl X Foxp3-YFP-cre (cKO) mice. We found that a selective deficiency of Eos in Tregs does not result in a decrease of their suppressive capacity in vitro. However, absence of Eos resulted in an increased percentage of activated CD4+ Tconv cells in aged mice (3-month-old) accompannied by an increase in the percentage of Th1 phenotype cells with an increased capacity to secrete IFN-gamma. cKO mice developed severe EAE following immunization with MOG. The serum of cKO mice showed elevated levels of IgE, IgA, IgG and IgM as well as the presence of anti-nuclear antibodies. Both the kidneys and small intestine of cKO mice developed large lymphoid infiltrates. The absence of the development of disease in mice with a global deletion of Eos also suggests that Eos plays a function in Tconv cells. 3. Interaction of TFH cells with B cells not only helps TFH to fully commit to this lineage, but also provides B cells with survival and differentiation cues. A GC response is critical for the development of an immune response against pathogens, but a dysregulated response could lead to autoantibody production. TFR cells, a subset of Foxp3+ Tregs, localize into follicles to regulate GC responses. As our studies on both Helios and Eos deficient Tregs demonstrated a defect in the suppressive function of TFR cells in vivo, we are attempting to elucidate the mechanism (s) and cellular target (s) utilized by TFR cells to suppress GC responses. We first developed assays to quantitate TFR function in vitro. In the absence of TFR cells, TFH from primed mice induce both naive B cell differentiation into GC B cells and class switching in the presence of anti-CD3 alone or anti-IgM/anti-CD3 in a contact dependent manner. Addition of TFR cells from primed mice efficiently suppressed GC B cell proliferation, differentiation and class switching in the anti-CD3 alone cultures, but only moderately suppressed BCR-stimulated B cells. Under anti-CD3 conditions, IL-4-deficient TFH cells did not promote B cell differentiation and class switching. In contrast, IL-21R-deficient B cells differentiated into GC B cells with a reduced number of IgG1+ cells. When IL-4 deficient TFH cells were co-cultured with IL-21R-deficient B cells, both GC differentiation and class switching were reduced. Under anti-IgM/anti-CD3 conditions, IL-4-deficient TFH cells promoted B cell differentiation, but class switching was reduced, while IL-21R-deficient B cells differentiated normally into GC B cells and class switching was reduced. These studies suggest that at least in vitro, TFR cells regulate the GC responses by acting directly on TFH cells most likely by inhibiting cytokine production. 4. Tregs are critical to the maintenance of immune homeostasis and tolerance. Treg cells, CD4+Tconv cells, and CD8+ T cells represent heterogeneous populations composed of memory phenotype (MP, CD44hi) and naive phenotype (NP, CD44low) subpopulations. One of the most prominent characteristics of MP Tregs and MP CD4+ and CD8+ T cells is their high degree of cell cycling in vivo (10% proliferate/day). Very little is known about the contribution of TCR signals alone or the interrelationships between TCR and co-stimulatory signals on Treg homeostasis. The major goal of our studies was to examine the effects of cytokines (IL-2, TNF, IL-7 IL-33), the effects of co-stimulatory receptors (CD28, ICOS) and their receptors, the effects of co-inhibitory (CTLA-4, PD-1, TIGIT) receptors, as well the role of MHC class II interactions on Treg subset homeostasis. None of the cytokines tested appeared to play a role in MP Treg proliferation in vivo. In contrast, CTLA-4-Ig markedly reduced the cycling and numbers of MP Treg and MP CD4+ T cells. Complete inhibition of MP Treg proliferation was observed when mice were treated with a unique blocking anti-CD28 domain reagent. Blockade of MHC class II-TCR interaction led to selective expansion of MP Treg cells and MP CD4+ and CD8+ T cells that was reversed upon co-treatment with CTLA-4-Ig or the blocking anti- CD28. Treatment with anti-CTLA-4 antibody altered MP Treg cell and MP CD4+ and CD8+ T cell homeostasis in a manner similar to anti-MHC class II. Inhibition of PD-L1/PD-1 interactions also enhanced MP Treg proliferation, but to a much lesser extent than anti-CTLA-4. Treg cell homeostasis is thus controlled by a complex pathway in which CD28 is the primary driver of proliferation, while TCR, CTLA-4 and PD-1 function as the brakes.