Grant Abstract: Epigenetic control of Foxp3 expression in induced T regulatory cells

Grant Number: 5R01AI128589-02
PI Name: Rao
Project Title: Epigenetic control of Foxp3 expression in induced T regulatory cells

Abstract: The goal of the parent project, AI 128589 (Epigenetic control of Foxp3 expression in induced T regulatory cells), was to investigate the molecular role of TET methylcytosine oxidases in the differentiation of naïve mouse CD4+ T cells into “induced” regulatory T cells (“iTregs”). TET proteins are Fe(II) and alpha-ketoglutarate-dependent dioxygenases that oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and beyond, thereby facilitating DNA demethylation. We and others have shown that the activity of TET proteins and certain other
Fe(II)/ alpha-ketoglutarate-dependent dioxygenases is dramatically potentiated by Vitamin C. iTreg differentiation and FOXP3 expression can be induced in both mouse and human CD4+ T cells by activation in the presence of TGFb and retinoic acid (RA), and we showed that addition of the TET activator Vitamin C to the culture media for iTreg generation resulted in a dramatic increase in iTreg stability and function. We also traced the mechanism of enhanced iTreg function to TET-mediated demethylation of two intronic enhancers in the Foxp3 locus, CNS1
and CNS2. In previous studies, we showed that TET proteins are involved in cell activation and differentiation in numerous cell types in the mouse, including in early embryogenesis and embryonic stem cells, thymic differentiation, Treg and iTreg differentiation, pro-B to pre-B cell differentiation, and class switch recombination in mature B cells. However, we have not examined any human cell types so far. Remarkably, as many as 10-20% of humans are
reported to be Vitamin C-deficient, and the role of Vitamin C in immune function has not been systematically explored at a molecular level. We address this important question here.
In this supplement request, we propose to study the effect of Vitamin C on other directions of mouse and human T cell differentiation. We will activate naïve CD4+ T cells in culture media containing appropriate cytokines, to drive their differentiation towards four different T helper (Th) lineages: Th1, Th2, Th17 and iTreg. Similarly, we will activate naive CD8+ T cells in IL-2-containing culture media so that they differentiate to become cytolytic effector T cells. In each case we will determine the effect of adding or omitting Vitamin C from the differentiation
cultures. As readouts, we will measure cytokine production, degranulation and cytolytic function, expression of surface receptors, transcriptional profiles and chromatin accessibility. We will also restimulate the cells and monitor the stability of expression of key lineage-determining transcription factors by RNA-sequencing. The proposed studies will enhance our understanding of a very fundamental question: how the TET activator Vitamin C alters the differentiation and function of mouse and human CD4+ and CD8+ T cells, and to what extent these
effects go through TET proteins and TET enzymatic activity. Our genome-wide data should help identify novel candidates for therapeutic intervention in immune-related disorders, including cancer immunotherapy, transplant rejection and autoimmune disease.

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