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

National Institute Of Allergy And Infectious Diseases

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Abstract

Three distinct areas were studied in FY2022: 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. While both Treg development in mice with a conditional deletion of Helios in Treg (cKO) 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. Surprisingly, these mice did not appear to exhibit organ-specific autoimmunity that would be expected from mice with defective Treg cells. However, further observations in cKO mice have revealed that aged mice lack adipose tissue, develop steatosis, become insulin resistant, and redirect lipids to the liver, ultimately causing metabolic dysfunction. The lymphocytic infiltrate specific to white adipose tissue (WAT) can be transferred from mice with lipodystrophy to immunodeficient mice, confirming the autoimmune nature of the lipodystrophy. This model represents the first description of an autoimmune lipodystrophy. Characterization of the adipose tissue infiltrate by flow cytometry shows that there is a considerable expansion of CD4+ and CD8+ T cells. The lymphocytic infiltrate also contains a higher percentage of Treg cells, thus the failure of the cKO Treg to control the expansion and activation of CD8+ T cells is not due to defective Treg trafficking. Transcriptome analysis of splenic Tregs from the cKO mice reveals that Tregs from the cKO mice fail to upregulate specific effector molecules. Using this data, we have taken a targeted approach to analyzing the cKO Tregs from the adipose tissue. Flow cytometry analysis has confirmed that activation/effector molecules such as CD69, TIGIT, and KLRG1 are decreased in adipose tissue cKO Tregs. Further analysis showed that the tissue destruction in the cKO mice was mediated by CD8+ T cells through the delivery of cytotoxic granules and Fas-L mediated killing. Additional experiments are underway in which the cKO mice have been crossed to CD8 deficient mice. Initial analysis of these mice demonstrates that the adipose tissue still contains an infiltrate of expanded, activated CD4+ T cells. However, at 4 months of age, the mice do not appear to have any loss of adipose tissue, demonstrating the crucial role of CD8+ cells in the loss of tissue in the cKO mice. These mice will be aged longer to confirm the lack of adipose tissue destruction and the mice will be analyzed for metabolic changes. We are also taking an unbiased approach to analyze adipose tissue Tregs. Treg cells from WT, cKO and chimeric animals that contain WT and cKO Tregs in the same animal, have been purified and submitted for RNASeq analysis. We believe this approach will elucidate the molecular basis underlying the Treg defects that lead to the loss of regulatory function and ultimately, adipose tissue loss. Efforts are also underway to elucidate the autoantigen(s) that drives the expansion of T cells. Further experiments to identify autoantibody targets using a phage display library are underway, with the hopes that the autoreactive T cells respond to similar antigens. 2. Natural Killer (NK) cells are innate lymphocytes involved in the first line of immune defense and T cell adaptive immunity against viral infection and cancer, including metastasis. While on patrol, NK cells contact other cells and recognize MHC-I Ags via stochastically expressed MHC-I specific inhibitory receptors (Ly49s in mice and KIRs in humans) that prevent NK cell activation via cytoplasmic ITIM. The binding site on MHC-Class-I for Ly49 inhibitory receptors is distinct from that for TCRs. The loss of MHC-I expression on tumor cells (missing self) abrogates inhibitory signals, resulting in NK activation. Global inhibition of the NK inhibitory receptor interactions in vivo by a pan-anti-MHC-I monoclonal antibody markedly activated IFN-gamma producing NK cells, independent of Fc receptors. Administration of pan-anti-MHC-I to unmanipulated mice profoundly augmented innate and adaptive immunity against viral infection, PD1-resistant transplanted tumors, successfully restrained lung and liver metastasis, and protected the animals from tumor burden. We have also identified several anti-pan-human MHC-I mAb (W6/32, Dx17), but these failed to inhibit KIR-MHC-I interactions. Surprisingly, these mAb potently blocked the interaction of Leukocyte Ig-like inhibitory receptors LILRB1, B2, and B5 with human MHC-I, but had no effect on TCR-MHC-I interactions. Addition of Fab fragment of the mAbs to cultures of human PBMC markedly induced NK cell proliferation and IFN-gamma production. Furthermore, administration of the mAbs to humanized mice (PBMC reconstituted NSG or NK cell reconstituted NOG-IL-15) induced human NK and memory phenotype (MP) T cell proliferation and activation as well as enhanced tumor immunity. These results strongly suggest that similar to the effects of blocking Ly49-MHC-I interactions in the mouse, inhibition of LILRB-MHC-I interactions in man may result in marked augmentation of anti-tumor and anti-viral immunity in man. 3. Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in Treg cells. However, the role of Eos in Treg function is controversial. Deletion of Eos in Treg resulted in activation of CD4+Foxp3- and CD8+ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyelitis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-gamma/TNF-alpha production by CD8+ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7- 8 months, respectively). Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an unknown role in the function of CD4+Foxp3- and CD8+ T cells and potentially in non-T cells. To facilitate the analysis of Eos function, we have generated a specific anti-Eos mAb that reacts with wild type Treg, but not Treg from Eos deficient mice. We found that very few thymic Treg or peripheral Treg express Eos in young mice but that the percentage of Tregs expressing Eos rapidly increases with aging peaking at 30-50% of thymic Treg and 35-60% of splenic Tregs. All of the Eos expressing thymic Tregs co-express both CD73 and CCR6 indicating that Eos+ thymic Treg represent peripheral Treg that have recirculated to the thymus. Eos was expressed at very low levels on induced Tregs, conventional CD4+ T cells and CD8+ T cells in vivo or in vitro, regardless of their activation status. Using immunofluorescence microscopy, we found that Eos is highly co-localized with another Ikaros family member, Helios, which is expressed by 70-80% of Treg suggesting that Eos and Helios may form heterodimers and collaborate in Treg function. Nevertheless, expression of Eos was independent of Helios and vice versa, as Treg from Helios deficient mice expressed normal or elevated levels of Eos, and Treg from Eos deficient mice expressed normal levels of Helios.