Four distinct areas were studied in the this project in FY2015: 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. A subpopulation (70-80%) of Foxp3+ T regulatory (Treg) cells in both mouse and man express the transcription factor, Helios, but the role of Helios in Treg function is still unknown. Selective deletion of Helios in Tregs (Helios-/-) leads to systemic immune activation, hypergammaglobulinemia, and enhanced germinal center formation in the absence of immune pathology. Helios-/- Treg suppressor function was normal in vitro and in inflammatory bowel disease in vivo. Helios-/- Treg failed to control the expansion of pathogenic T cells derived from scurfy mice and failed to mediate T follicular regulatory cell function. In competitive settings, Helios-/- Treg were at a disadvantage indicating that Helios regulates Treg fitness. Thus, Helios controls certain aspects of Treg survival, differentiation, and suppressive function. 2. We have also studied a second member of the Ikaros gene family, Eos, which was purported to be essential for Treg cell function and the maintenance of Treg stability. We have utilized mice with a global deficiency of Eos to re-examine the role of Eos expression in both Treg and T conventional (Tconv) cells. Treg from Eos deficient (Eos-/-) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos-/- Treg were as effective as Treg from wild type (WT) mice in suppression of inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos-/- mice was as effective as BM from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of Scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconv cells and was required for IL-2 production, CD25 expression and proliferation in vitro by CD4+ Tconv cells. Eos-/- mice developed more severe Experimental Autoimmune Encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies demonstrated that Eos expression is not required for the development and function of Treg, but that Eos plays an important role in the activation and differentiation of Tconv cells. 3. A critical step for the formation of a germinal center (GC) is the availability of T cell help. T follicular helper (TFH) cells are effector T cells that provide help and by selecting high-affinity B cells in germinal centers (GC). This interaction not only helps TFH to fully commit to the this lineage, but also provides B-cells with survival, differentiation and class-switching cues. Although is clear that a GC response is critical for the development of an immune response against pathogens, a dysregulated response could lead to fatal responses such as the ones seen in autoimmune disorders. T follicular regulatory (TFR) cells are a subset of Foxp3+ Tregs which BCL6 and CXCR5. CXCR5 expression on TFR allows them to localize into the follicles where they control TFH and GC responses. In order to elucidate the mechanism (s) and cellular target (s) used by TFR to suppress the GC responses, we developed both in vivo and in vitro assays for the quantitation of TFR function. In the absence of TFR in vitro, TFH and GC-TFH are able to induce B cell proliferation and differentiation into GL7+ Fas+ (GC B) cells as well as class switch induction (IgG1); in the presence of TFR cells, B cell differentiation and switching is dramatically reduced. Similarly, when nave T cells are transferred to TCRalpha/beta-/- mice they differentiate into TFH cells upon immunization. However, the GC response is reduced by co-transferring Tregs that acquire CXCR5+ expression and localize to the follicles. 4. The mechanism(s) utlilized by Tregs to suppress immune responses, particularly in vivo, remain poorly defined. We have used antigen-specific inducible Tregs (iTregs) to inhibit T effector (Teff) cell expansion in vivo and to treat autoimmune diseases. As an initial approach to further define the mechanism of action of iTregs in vivo, we have developed flow cytometry and confocal microscopy assays to directly observe and quantitate the interactions between Treg, Teff cells, and DC. Magnetically separated DC were loaded with OVA or PCC peptide and injected via the footpad the day before i.v. transfer of naive antigen-specific T cells and antigen-specific iTregs. Co-transfer of iTregs inhibited antigen induced proliferation of the Teff cells and resulted in inhibition of CD44 upregulation. Co-transfer of iTreg with in vitro induced antigen-specific Th1 cells also resulted in inhibition of Teff cell proliferation, but failed to inhibit Teff cell cytokine production. Confocal microscopy of the popliteal lymph node revealed that the homing potential of iTregs was similar to that of conventional T cells. Surprisingly, co-transfer of a large excess of iTregs did not inhibit Teff-DC cluster formation. These findings suggest that adoptively transferred antigen specific iTregs can inhibit local immune responses in a way that cannot be simply explained by competition for space on the DC surface or by competition for antigen.
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