Studies of the Function of Naturally Occurring and Adaptive T Regulatory Cells

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

Three distinct areas were studied in FY23: 1. The transcription factor Helios is expressed in the majority of Foxp3+ Treg cells, where it is necessary for the maintenance of self-tolerance. Mice with a Foxp3-specific deficiency in Helios develop systemic immune activation with increased T follicular helper (Tfh) and T helper 1 (Th1) effector responses. The selective deletion of Helios in Tregs leads to a progressive systemic immune activation with a Th1 phenotype, hypergammaglobulinemia, and enhanced germinal center formation. These mice did not exhibit the common types of organ-specific autoimmunity that would be expected from mice with defective Treg cells. Strikingly, these mice developed an acquired lipodystrophy, hepatic steatosis, and insulin resistance. Further analysis of the lipodystrophy revealed a significant lymphocytic infiltrate in both the inguinal and perigonadal adipose tissue, indicating autoimmune mediated destruction of the white adipose tissue. Flow cytometry analysis of the infiltrates demonstrates that the destruction of the adipose tissue is mediated by CD8+ T cells through both cytotoxic granule and Fas-L-dependent mechanisms. To directly confirm the critical role of CD8+ T cells in disease pathogenesis, we crossed Heliosfl/fl x Foxp3-Cre mice to B2m deficient mice. The offspring maintain normal adipose tissue volume. Efforts are also underway to elucidate the autoantigen(s) that drives the expansion of the CD8+ T cells. Thus, Helios deficiency in Treg disrupts immune homeostasis in the adipose tissue, leading to the expansion and activation of CD8+ T cells, tissue destruction and development of a metabolic syndrome. While Helios is primarily expressed in Treg cells, it is also expressed in a small subset of conventional CD4+ T cells. However, when Helios is deleted in all CD4 cells, both conventional and Treg, by crossing the mice to CD4-Cre, the disease phenotype observed in mice with a selective deletion of Helios in Treg is abrogated, indicating that Helios must also play a significant role in the function of conventional CD4+ T cells. The Helios+ subset of conventional T cells was composed of activated/memory cells that expressed high levels of the CD44 antigen. To examine specific subsets of CD4+ memory cells, we performed single cell transcriptome analysis of CD44hi memory cells from WT mice and mice with the CD4 specific deletion of Helios. Characterization of the CD44hi, Helios+, population showed that Helios expression changed dramatically with age, so cells from young (5-6 weeks) and old (5-6 months) mice were included in the analysis. Both longitudinal (young vs old) and cross-sectional (WT vs KO) comparisons were made, with emphasis placed on cross-sectional comparisons at old age. The sequencing results indicated significant transcriptomic and numeric shifts in multiple subpopulations of the analyzed splenocytes, including the almost complete loss of one cluster in both the young and old Helios deficient mice. Taken together, the observed transcriptomic and clustering changes may explain the role Helios plays in the abrogation of autoimmunity in our model and may provide a starting point for the analysis of Helios function in CD4+ T conventional cells. 2. We have identified an anti-mouse pan MHC-I mAb (M1/42), which blocks the interaction of NK cell inhibitory receptors (Ly-49 antigens) with MHC-I but does not inhibit antigen presentation by MHC-I. Administration of M1/42 in vivo markedly activated IFN-gamma producing NK cells. NK cell-derived IFN-gamma stimulated APC to produce IL-12/-15/-18 cytokine cascades that further drove the proliferation of NK cells and memory phenotype (MP) T cells. M1/42 treatment profoundly augmented innate and adaptive immunity against PD-1-sensitive and -resistant transplanted tumors. We evaluated the effects of M1/42 therapy in several metastatic cancer models. M1/42 treatments successfully constrained B16F10 lung metastasis by enhancing CD8+ T cell infiltration and induced melanoma-specific MP CD8+ T cell expansion and activation. In contrast, treatment with conventional checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4 and anti-NKG2A) had no therapeutic effect. M1/42 also enhanced Th1-type innate and adaptive immune responses, and greatly restricted liver and lung metastasis produced by a pancreatic adenocarcinoma cell line injected intrasplenically or intravenously. Thus, the global inhibition of Ly-49/MHC-I interactions by M1/42 results in activation of a potent innate and adaptive immune response that restrains the growth of both transplanted tumors and tumor metastasis. Human killer cell inhibitory receptors (KIR) bind to HLA at a site overlapping the TCR binding site and blocking of KIR binding to MHC-I would inhibit TCR recognition. However, two anti-pan-human MHC-I abs (DX17 and W6/32) markedly induced NK cell proliferation and IFN-gamma production in cultures of human PBMC. DX17 potently blocked the interaction of leukocyte Ig-like inhibitory receptors LILRB1, B2, B3 and B5 with human MHC-I but had no effect on TCR-MHC-I interactions. The crystal structure of a DX17 Fab/MHC-I complex revealed that the footprint of DX17 overlaps the LILRB1 binding site on MHC-I. Administration of W6/32 Fab to humanized mice induced human NK and MP T cell proliferation. Marked reduction of tumor size was seen when tumor bearing NOG-hIL-15Tg mice were reconstituted with CD3-negative cells and then treated with W6/32 Fab. DX17 Fc silenced mAb (DX17 LALAPG) activated human tumor infiltrating lymphocytes and monocytes and decreased the size of transplanted tumors. These results indicate that inhibition of LILRB-MHC-I interactions by anti-MHC-I in humans may result in marked augmentation of anti-tumor immunity. 3. Eos is a member of the Ikaros transcription factor family and is highly expressed in Tregs. Mice with a selective deficiency of Eos in Treg develop systemic or organ-specific autoimmune disease at a young age. We have generated a specific anti-Eos monoclonal antibody. The percentage of Tregs expressing Eos rapidly increases with aging from almost 0% at birth to 30-50% of thymic Tregs and 35-60% of splenic Tregs at 3 weeks of life and then remains stable. Eos is expressed at very low levels on induced Tregs, conventional CD4+ T cells and CD8+ T cells in vivo or in vitro. In the thymus, Eos is only expressed on CD73+CCR6+CXCR4+ Tregs, which suggests that Eos+ thymic Tregs represent peripheral Tregs that have recirculated to the thymus. In thymus, Eos expression on Tregs is also highly correlated with CXCR4 and CCR6. Using these markers, we sorted Eos+ and Eos- Tregs and in vitro expanded them using anti-CD3/CD28 beads and IL-2; Eos expression remained stable. Using immunofluorescence microscopy, we have found that Eos is highly co-localized with Helios, another Ikaros family member and HDAC1, but not other Ikaros family members including Ikaros or Aiolos, suggesting that Eos and Helios may form heterodimers and together with HDAC1 collaborate in Treg function. Nevertheless, expression of Eos is independent of Helios and vice versa, as Treg from Helios deficient mice express normal or elevated levels of Eos, and Treg from Eos deficient mice express normal levels of Helios. Taken together, these strongly suggest that Eos plays a related, but distinct role, from Helios in Treg function.