Genetic Analysis of T-cell Differentiation

Division Of Basic Sciences - Nci

Investigators

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Abstract

We study T cell development and function. T cells are essential for immune responses. Most express an antigen receptor (TCR) made of an alpha and a beta chain (ab T cells) and recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules, and express either of two surface glycoproteins that contribute to antigen recognition: CD4, which binds MHC-II, or CD8, which binds MHC-I. Consistent with such binding properties, MHC I-specific T cells generally are CD4-CD8 (CD8 T cells), whereas MHC II-specific T cells generally are CD4+CD8- (CD4 T cells). CD4 T cells are essential for life: CD4 T cell deficiency, whether innate or acquired, leads to recurrent or chronic infections and death. Studies of ab T cell development in the thymus have remained at the forefront of the laboratory activity in the past few years. We implemented single cell (sc) analyses of gene expression: single-cell RNA sequencing (scRNAseq, for gene expression itself), and single cell ATAC sequencing (scATACseq) to map chromatin accessibility. In addition to T cell development studies summarized below, we used these approaches to study T cell responses to viral infection and tumor antigens (Ciucci et al., 2019; Vacchio et al., 2019; Magen et al., 2019; Ciucci et al., 2022). A recent report from the laboratory (Chopp et al., 2020) combined scRNAseq and scATACseq to identify developmental trajectories during the intrathymic differentiation of ab T cell precursors. In parallel studies, we showed that the zinc finger transcription factor LRF (encoded by Zbtb7a) is necessary for the proper development of thymic precursors of "CD8alpha-alpha" intestinal intraepithelial T cells (CD8aa IEL), a major subset of gut T cells whose functions remain enigmatic (Nie et al, 2022). Using scRNAseq and a variety of adoptive transfer assays, we showed that LRF is necessary for the expression of the intestine-homing integrin beta7, for the migration of IEL precursors to the small intestine, and for their contribution to intestinal homeostasis and prevention of inflammation in an experimental model of colitis. Building on these recent findings, ongoing studies focus on two directions. First, we are exploring the mechanisms that promote the development of thymocytes carrying antigen receptors with high affinity for self-ligands (called "agonist-selected"), including CD8aa IEL precursors. We are notably investigating how in situ transcriptomics can shed light on cell interactions along intrathymic developmental trajectories of agonist signaled thymocytes. Second, we are leveraging our recent gene regulatory network inference studies (Chopp et al., 2020) to identify factors needed for CD4 T cell development and to study their actual functions, using genetic approaches. The long-term objective of these studies is to build in vitro strategies for the differentiation of CD4 T cells. Among the factors needed for CD4 T cell development in the thymus is the zinc finger transcription factor Thpok, encoded by Zbtb7b. Understanding Thpok functions has been a key objective of the laboratory research over the past 5-10 years. We reported the genome-wide distribution of Thpok binding sites in CD4 T cells and differentiating CD4-lineage thymocytes (Ciucci et al., 2019; Chopp et al., 2020). More recently, we showed that Thpok binds the nucleosome remodeling and deacetylase (NuRD) complex, and we identified the domain of Thpok involved in this binding (Gao et al., 2022). We also found that such binding mediates critical functions of Thpok in intrathymic CD4 T cell differentiation. Specifically, recruitment of NuRD is essential for Thpok repression of CD8-lineage genes, including the transcription factor Runx3 that controls CD8 T cell differentiation; in contrast, NuRD recruitment is dispensable for Thpok to promote Cd4 expression. We are currently examining whether NuRD recruitment is involved in the repression of cytotoxic genes by Thpok in post-thymic CD4 T cells, and whether such mechanisms could control acquisition of cytotoxic gene expression in recently described cytotoxic CD4 T cell populations.