Three distinct areas were studied in the this project: 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. Because the expression of Helios clearly divided the peripheral pool of Foxp3+ T cells into two defined subpopulations, we attempted to determine whether these two populations were derived from distinct progenitors or whether they manifested distinct functions. The Foxp3+Helios- population appears to be exclusively generated in the periphery because thymic Foxp3+ cells from young mice (3-7d old) are exclusively Foxp3+Helios+. Foxp3+Helios- cells are not observed in the spleen until d12 of life. We initially demonstrated that Helios was not expressed in human or mouse T cells induced to express Foxp3 by TCR stimulation in the absence of APC in vitro. Antigen-specific Foxp3+ T cells generated in vivo from peripheral Foxp3- cells by oral administration of antigen were uniformly Helios- or when Foxp3- cells converted to Foxp3+ cells following transfer to lymphopenic recipients. Other groups have demonstrated that Foxp3+ T cells induced from Tconv cells in germ-free mice following reconstitution of the mice with bacteria are also Helios-. 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. We have found that the selective deletion of Helios in Foxp3+ T cells alters the function of Tregs. Although both Treg development and suppressor function in vitro are normal, the percentage of CD44hi T effector (Teff cells) is increased beginning at 2 months of age. A more marked increase in CD44h Teff cells is seen at 6 months of age and this is accompanied by increased production of IFN-gamma and expression of CXCR3, suggesting a Th1 autoimmune phenotype. Histological findings show increased germinal center formation in salivary gland,spleen and lymph nodes. Furthermore, it appears that the number of T follicular helper cells is increased, while the number of T follicular Treg (TFR) is decreased. These results suggest that Helios may specifically control the differentiation of TFR cells and indirectly antibody production. 2. The contributions of tTregs and pTregs to maintaining immune homeostasis remain to be determined. 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. The Medof lab has previously demonstrated that that antigen-specific T cell activation in the presence of dendritic cells (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. We have examined the reverse situation in which C3aR/C5a signaling is inhibited or blocked. When C3aR and C5aR signals are not transduced into CD4+ cells, PI-3Kgamma-AKT-mTOR signaling ceases, PKA activation increases, transforming growth factor-beta (TGF-beta1) signaling initiates, and CD4+ cells become Foxp3+ T regulatory cells (iTregs). Endogenous TGF-beta1 suppresses C3aR and C5aR signaling by preventing C3a and C5a production and upregulating C5L2, an alternate C5a receptor. 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. Absent C3aR and C5aR signaling decreases costimulatory molecule and interleukin-6 production and augments interleukin-10 production. The resulting iTregs exert robust suppression, possess enhanced stability, and suppress ongoing autoimmune disease. These studies highlight that complement, widely associated with innate immunity, plays an integral role in modulating Treg function/induction and dominant immunologic tolerance. 3. We have taken advantage of our ability to generate large numbers of antigen-specific iTreg in vitro to explore their capacity to modulate DC function. A number of studies have suggested that DCs are one of the major targets of Treg-mediated suppression. Some studies have suggested that Treg-mediated suppression of DC function is mediated by the interaction of CTLA-4 on Treg with CD80/CD86 on the DC resulting in down- regulation of CD80/CD86 expression and a decrease in co-stimulation. We have re-examined the effects of Treg on mouse DC function in a model in which antigen-specific, induced Treg (iTreg) are co-cultured with DC in the absence of Teff cells. iTreg-treated DC are markedly defective in their capacity to present antigen to nave T cells. iTreg from CTLA-4 deficient mice failed to induce downregulation of CD80/CD86, but DCs treated with CTLA-4 deficient iTreg still exhibited impaired capacity to activate nave T cells. The iTreg-induced defect in DC function could be completely reversed by anti-IL-10 and IL-10 deficient iTregs failed to down-regulate DC function. iTreg-treated DCs expressed high levels of MARCH1, an E3 ubiquitin ligase, which degrades CD86 and MHC-II on the DC and expressed lower levels of CD83, a molecule involved in neutralizing the function of MARCH1. Both the enhanced expression of MARCH1 and the decreased expression of CD83 were mediated by IL-10 produced by the iTreg. Taken together, these studies demonstrate that a major suppressive mechanism of DC function by iTreg is secondary to the effects of IL-10 on MARCH1 and CD83 expression. These studies suggest that IL-10 production by Tregs is a prominent Treg suppressor mechanism in a wide variety of conditions in addition to its critical role in Treg function at mucosal surfaces. The present study supports the concept that the major target of Treg-produced IL-10 is the antigen presenting DC with resultant inhibition of Teff cell priming.
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