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. Ikaros is expressed in all immune cells. The Helios gene (Ikzf2) encodes protein isoforms with zinc-finger domains exhibiting considerable homology to Ikaros family proteins. Human and mouse Helios share 97% identity in their amino acid sequences. We initially demonstrated that Helios was selectively expressed in Foxp3+ Treg cells using differential display and similar results were obtained in a number of microarray comparisons of Foxp3+ Treg cells and conventional T (Tconv) 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. However, one report has claimed that Eos interacts with Foxp3 to mediate gene silencing through chromatin modifications. We have developed a novel monoclonal antibody (mAb) to a 55 amino acid fragment from the non-conserved N-terminal region of Helios that reacts with both mouse and human Helios. This mAb was selected by its ability to identify Helios intracellularly. We demonstrated that its expression is limited to only 70-80% of Foxp3+ Treg in either species. In addition, Helios is expressed at the DN2 stage of thymic development in the mouse, prior to Foxp3 expression. 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 and do not reach the percentage observed in adult animals until after weaning. Foxp3+Helios+ T cells generated in the thymus and the Foxp3+Helios- generated in the periphery differ phenotypically as neonatal thymic and splenic Foxp3+Helios+ T cells express high levels of CD44 and CD25 while Foxp3+Helios- T cells in the neonate are predominantly CD25- with variable levels of expression of CD44. 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. Studies by 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-. Collectively, these studies support our thesis that Helios expression is a marker of tTreg. We have also not been able to define a function for Helios in Foxp3+ Treg cells in vitro or in vivo. Thus far, our studies with Foxp3+Helios- Treg cells from either CD4-Cre, Vav-Cre mice or with human T cells with siRNA-mediated knock down of Helios have failed to define an abnormality in Treg function. It remains possible that Helios is so critical to the development and function of Foxp3+ Treg cells that redundant pathways are in place so that other members of the Ikaros family can substitute for Helios in its absence. Although the contributions of nTregs and iTregs to maintaining immune homeostasis remain to be determined, knowledge of how these cells are induced physiologically is key to understanding how T cell immunity is regulated as well as highly relevant for managing diverse human disorders. 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 the alternative pathway (AP) complement components C3, factor B (fB), factor D (fD) in conjunction with C5 and the G-protein coupled receptors (GPCRs) C3a/C5a receptors (C3aR/C5aR). At the same time, both T cells and DC downregulate their expression of the cell surface C3/C5 convertase inhibitor decay accelerating factor (DAF or CD55). In the absence of the inhibitory effect of DAF, C3 and C5 convertases stably assemble from the locally produced C3/fB/fD on the surfaces of both the DC and T cell. These enzymes act on the nascent C3 and C5 to generate C3a and C5a. The C3a and C5a anaphylatoxins engage the upregulated C3aR and C5aR on the DCs and CD4+ T cells. Activation of C3aR and C5aR signaling results in the delivery of strong costimulatory and survival signals to the effector T cells. In contrast, we have demonstrated that T cell activation in the absence of C3aR/C5aR signaling results in the induction of a high percentage of iTregs. C3aR/C5aR signaling was disabled by using mice doubly deficient in C3aR and C5aR, in the presence of C3aR/C5aR pharmaceutical antagonists (C3aR-A/C5aR-A) or by adding anti-C3a/C5a mAbs specific for neo-epitopes in the C3a/C5a ligands. However, induction of Foxp3+ T cells was dependent on the endogenous production of TGF-beta. Collectively, our studies demonstrate that important forward and backward feedback loops are involved in the induction of iTregs. First, the lack of C3aR/C5aR signaling in DCs results in enhanced TGF-beta production by the DC and the consequent suppression of complement synthesis and C3a/C5a production by the DC. The lack of C3a/C5a production by the DC deprives the responder T cells of their major source of C3a/C5a that can transmit C3aR/C5aR signals into them. The DC production of TGF-beta prevents C3a/C5a production by the DC or the responder CD4+ T cell, resulting in endogenous TGF-beta production and auto-inductive TGF-beta signaling by the CD4+ T cell and its conversion to an iTreg. The endogenous production of TGF-beta by the iTreg reciprocally feeds back to maintain the DC in an immature state. Absent C3aR/C5aR signaling by both partners thereby contributes to a local immunosuppressive milieu.
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