Four distinct areas were studied in the this project in FY2014: 1. We have analyzed the role of the transcription factor Helios (Ikzf2) in Treg function. Helios is a transcription factor belonging to the zinc finger containing Ikaros family comprised of five transcription factors-- Ikaros, Helios, Aiolos, Eos and Pegasus. We initially demonstrated that Helios was selectively expressed in Foxp3+ Treg cells. Helios was also shown to be a potential Foxp3 target gene. The expression and function of other Ikaros family members in Treg cells has not been extensively studied. We have developed a novel monoclonal antibody (mAb) to N-terminal region of Helios that reacts with both mouse and human Helios. We demonstrated that Helios expression is limited to only 70-80% of Foxp3+ Treg in either species. Our previous studies strongly suggested that Helios was a marker for tTreg. The function of Helios and the role it may play in tTreg function is still unknown. Foxp3 conditional Helios deficient mice were generated to address these major questions.Selective deletion of Helios in Tregs results in the gradual development of a systemic autoimmune disease characterized by T effector cell (Teff) activation, differentiation to a Th1 phenotype, and enhanced germinal center formation. Although Treg development and suppressor function in vitro are normal,Helios deficient Tregs expressed an activated phenotype and also upregulated genes characteristic of a Th1 phenotype leading to increased IFN-gamma expression. Helios deficient mice also lack T follicular regulatory (TFR) cells. Histological findings show increased germinal center formation in salivary gland,spleen and lymph nodes and an increase in the number of T follicular helper (TFH) cells. Mixed bone marrow chimeras between wild type and Helios deficient mice rapidly develop a autoimmune syndrome with a marked increase in Th1 Teff cells. These findings suggest that Helios plays a critical role in the stability and activity of tTregs and may specifically control the differentiation of TFR cells and indirectly antibody production. 2. A critical step for the formation of a germinal center (GC) is the availability of T cell help. TFH cells are known to provide help to B cells. This interaction not only helps in the commitment of this T-cell to fully commit to the TFH cell lineage, but it also provides B-cells with survival, differentiation and class-switching cues. B cells then proceed to expand into a GC, allowing for the production of antibodies. 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 diseases. Recently, a new Treg subset was characterized by several groups and identified as CXCR5+BCl6+FoxP3+ and named T follicular regulatory (TFR) cells. CXCR5 expression on TFRs 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 TFRs 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 are able to induce B cell proliferation and differentiation into GL7+ plasma cells, whereas in the presence of TFRs, B cell differentiation is reduced. Similarly, when nave T cells are transferred to TCRalpha-/- mice they differentiate into TFH cells upon immunization. However, the GC response is inhibited by co-transfer of Tregs that develop CXCR5+ expression and localize in the follicles. 3. 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. 4. In a major collaboration with the laboratory of Dr. E. Medof, we have begun to dissect the potential role of a component of the innate immune system in the induction of iTregs. Antigen-specific T cell activation in the presence of DC results in the local synthesis of C3a, C5a and their G-protein coupled receptors (GPCRs, C3aR/C5aR). Activation of C3aR and C5aR signaling results in the delivery of strong costimulatory and survival signals to the effector T cells. When C3aR/C5a signaling is inhibited or blocked CD4+ cells become Foxp3+ T regulatory cells (iTregs). Thus, signaling in CD4+ T cells themselves via the C3aR and C5aR on the one hand potentiates immune responses, while the absence of this signaling diverts naive T cells to an iTreg response. These studies have been extended to analyze the mechanisms involved in the induction of mucosal tolerance. Antigen-specific T conventional cells that do not express the C3aR/C5aR, but not wild type T cells, can readily be induced to become pTreg following the oral administration of antigen. These pTreg are stable upon adoptive transfer and with time undergo epigenetic changes in the Foxp3 promoter that enhance their stabiliity. These studies highlight that complement, widely associated with innate immunity, plays an integral role in modulating Treg function/induction and dominant immunologic tolerance.
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