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

National Institute Of Allergy And Infectious Diseases

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Abstract

Two areas were studied in FY25: 1. The transcription factor, Helios, is a marker of thymically derived Treg cells. C57BL/6 mice with a Treg specific deletion of Helios develop systemic immune activation and autoimmune lipodystrophy. There is now ample evidence that Treg cells play an important role in metabolic homeostasis and we are currently investigating the role that Helios may play in Treg mediated control of adipose tissue homeostasis. For this current project, we have now characterized adipose tissue infiltrate by flow cytometry and demonstrated that there is a considerable expansion of CD4+ and CD8+ T cells in the adipose tissue of Helios deficient mice and that the tissue destruction is mediated by CD8+ T cells through the delivery of cytotoxic granules and Fas-L mediated killing. We have also characterized the Treg transcriptome and using this data, we have taken a targeted approach to analyzing the Helios Treg conditional knock out (cKO) Tregs from the adipose tissue. Flow cytometry analysis has confirmed that activation/effector molecules such as CD69 and KLRG1 are decreased in adipose tissue cKO Tregs. Thus, in a manner like the periphery, Tregs fail to achieve or maintain an activated phenotype. However, in the adipose tissue, the activation is tissue specific and the Tregs fail to maintain IRF4 and TCF1, transcription factors important for Treg mediated control of adipose tissue homeostasis. Finally, we have performed ChIPSeq analysis on Treg cells and are now correlating this data with our transcriptome data to show that Helios directly controls transcription factors important for the maintenance of activation. The cKO that has been characterized above was generated on a C57BL/6 background. It is known that mice on different MHC backgrounds can develop different autoimmune disease. Thus, we backcrossed the cKO mice onto a BALB/c background for 10 generations. Our current data shows that Treg specific Helios deficiency on a BALB/c background is lethal by 10 weeks of age. By flow cytometry, we show that cKO mice have a significant expansion of TFH cells. Furthermore, these mice exhibit a dramatic loss of platelets at the time of death. Preliminary data shows that these mice have autoantibodies, likely to thrombopoietin receptor, a major protein expressed by platelets. Experiments are currently underway to show directly that cKO possess anti-thrombopoietin receptor antibodies that are responsible for platelet loss and death. Finally, the transcription factor Helios is also expressed in a subset of T conventional (Tconv) memory cells. While mice with Foxp3-Cre deletion of Helios develop systemic autoimmunity characterized by hyperproliferation of T cells and autoimmune lipodystrophy, mice with CD4-Cre deletion of Helios do not exhibit overt signs of autoimmunity, indicating an important role for Helios in Tconv. Previously, single-cell transcriptome analysis on CD4+CD44hiFoxp3- splenocytes from wildtype (WT) and CD4-Cre knockout mice (KO) revealed upregulation of genes associated with T follicular helper (Tfh) differentiation and T cell exhaustion in KO memory cells. Current data shows that, at steady state, KO splenocytes show an increased frequency of memory CD4+ cells due to an expansion of the Tfh compartment. However, KO memory CD4+ cells demonstrate increased markers associated with Tfh programming and exhaustion. Studies are currently underway using LCMV infection to assess how Helios influences Tfh differentiation and exhaustion in response to antigen exposure. 2. Eos, a member of the Ikaros family of transcription factors, is expressed by Tregs and has been postulated to play a role in Treg suppression and maintenance of Treg stability. We have demonstrated that Eos is expressed by ~50% of Treg in peripheral lymphoid tissues. Expression of Eos was limited to a subpopulation of thymic-derived, activated Treg and could not be detected in resting or activated T conventional cells. Eos associates with Helios, Foxp3 and Hdac1 and binds directly to the CD25 locus at a site identical to the Foxp3 binding site resulting in enhancement of CD25 expression. Studies in heterozygous female mice demonstrate that Eos is critical for Treg survival in the periphery and transition to an activated phenotype. Eos+ Treg also represent the major population of recirculating thymic-Treg and function by limiting Treg generation in the thymus by competing for IL-2 and depleting MHC II from thymic dendritic cells.