Helios, a member of the Ikaros transcription factor family, is preferentially expressed at the mRNA level by regulatory T cells (Treg cells). We evaluated Helios protein expression using a newly generated mAb and demonstrated that it is expressed in all thymocytes at the double negative 2 stage of thymic development. Although Helios was expressed by 100% of CD4+CD8−Foxp3+ thymocytes, its expression in peripheral lymphoid tissues was restricted to a subpopulation (~70%) of Foxp3+ T cells in mice and humans. Neither mouse nor human naive T cells induced to express Foxp3 in vitro by TCR stimulation in the presence of TGF-β expressed Helios. Ag-specific Foxp3+ T cells induced in vivo by Ag feeding also failed to express Helios. Collectively, these results demonstrate that Helios is potentially a specific marker of thymic-derived Treg cells and raises the possibility that a significant percentage of Foxp3+ Treg cells are generated extrathymically.

The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), while Helios− Treg were induced from Foxp3− T conventional (Tconv) cells in the periphery (pTreg). To compare the two Treg subpopulations, we generated Helios‐GFP reporter mice and crossed them to Foxp3‐RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype, had a slightly higher suppressive capacity in vitro and expressed a more highly demethylated TSDR but were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of activated, autoreactive splenocytes from scurfy mice. When Helios+ and Helios− Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios− Treg when transferred to lymphopenic mice. Gene expression profiling demonstrated a large number of differentially expressed genes and showed that Helios− Treg expressed certain genes normally expressed in CD4+Foxp3− T cells. TCR repertoire analysis indicated very little overlap between Helios+ and Helios− Treg. Thus, Helios+ and Helios− Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.

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To determine the relationship between the structure and function of proteins coded for by the Ly-6 gene complex, we have transfected a cDNA for a Ly-6.2 specificity into COS-7 cells. A number of monoclonal antibodies which have been shown to be capable of inducing T cell activation and which have been previously considered to recognize distinct proteins all reacted with the same transfected gene product. The approach used in these studies should be useful to further elucidate the complexities of the Ly-6 alloantigen system.

In studies with alloantisera and monoclonal antibodies (mAb) a number of antigenic determinants have been defined that are the products of the Ly-6 locus on murine chromosome 2 and that are expressed primarily on B and T lymphoid cells. It remains controversial whether these antigenic determinants are encoded by a single gene or a multigene complex. We have characterized a new rat mAb, D7, which recognizes a cell surface antigen whose expression on nonactivated peripheral lymphocytes varies from strain to strain. The phenotype of the staining profile, i.e., high or low percentage of D7-positive cells, mapped to the Ly-6 locus as assayed by strain distribution studies, RI lines, and Ly-6 congenic strains. The binding of D7 to Ly-6.1-positive strains could be inhibited by mAb directed to the Ly-6E.1 specificity, whereas D7 could inhibit the binding of mAb specific for Ly-6A.2 to cells from Ly-6.2-positive strains. Coprecipitation studies followed by Western blot analysis confirmed that D7 reacts with both Ly-6E.1- and Ly-6A.2-bearing molecules. The most likely explanation for these findings is that Ly-6A.2 and Ly-6E.1 represent allelic specificities. Further dissection of the complexity of the Ly-6 antigen system and determination of its possible functional importance in lymphocyte activation should be greatly facilitated by the availability of xenogeneic mAb that recognize framework determinants on multiple Ly-6 products.

The CD59 Ag is a 20-kDa protein that is widely expressed on most leukocytes and RBC, is coupled to the membrane by a phosphatidylinositol-glycan anchoring structure, plays a role in cell interaction between monocytes and T cells, and also functions as an inhibitor of cytolysis by the terminal C components C5b-9. Because this molecule is structurally related to the murine Ly-6 family of Ag, we have investigated whether anti-CD59 mAb might be capable of activating human T lymphocytes in a manner similar to that described for antibodies to the murine Ly-6 Ag. In the presence of the appropriate co-stimulators, mAb to one of the two epitopes on CD59 were capable of inducing both a rise in intracytoplasmic free Ca2+, inositol phosphate production, IL-2 production, and T cell proliferation. Anti-CD59-induced inositol phosphate turnover and IL-2 production were dependent on co-expression of the CD3/TCR complex. CD59-loss mutants of the Jurkat cell line were completely responsive to stimulation by anti-CD3 thereby demonstrating that CD59 does not play a role as a signal transducer downstream from the TCR. Taken together, these results demonstrate that the CD59 Ag can play multiple distinct roles in the regulation of the immune response.

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Regulatory T (Treg) cells suppress immune activation in a dominant manner and play a critical role in the maintenance of self-tolerance. A subpopulation (60–75%) of Foxp3+T regulatory (Treg) cells express the transcription factor Helios. To examine the function of Helios in Treg cells, we have generated Treg-specific Helios deficient mice (cKO, Heliosflxflx Foxp3cre). Although both Treg development in the cKO mice and their in vitro suppressor function are normal, the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation with a Th1 phenotype, hypergammaglobulinemia, and enhanced germinal center formation. Initially, we observed significant lymphocytic infiltrates only in the salivary gland and not in any other organs typically affected by Treg dysregulation. Strikingly, the mice developed lipodystrophy, hepatic steotosis and insulin resistance. Further analysis revealed a significant lymphocytic infiltrate in both the inguinal and perigonadal adipose tissue, indicating autoimmune mediated destruction of the white adipose tissue (WAT). We have further shown that the lymphocytic infiltrate specific to WAT can be transferred from mice with lipodystrophy to immunodeficient mice, confirming the autoimmune nature of the lipodystrophy. Thus, Helios deficiency in Treg disrupts immune homeostasis in the adipose tissue, leading to the destruction of WAT and redirection of lipids to the liver, and ultimately causes metabolic dysfunction. Supported by the Intramural Research Program of the NIAID, NIH.