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 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. CD4 T cell responses have remained an essential area of focus for the laboratory. After infection or immunization (e.g. vaccine), antigen-specific CD4 T cells proliferate and, depending on the local inflammatory context, differentiate into effector subtypes endowed with specific functions. Infections by intra-cellular pathogens (e.g. viruses) result in the generation of Th1 CD4 T cells producing interferon (IFN)-gamma and of follicular helper CD4 T cells (Tfh), which provide help to B cells for antibody maturation. Additionally, long-lived memory CD4 T cells, persisting after pathogen clearance, contribute to durable immunity, together with the B cell response. Accordingly, efficient differentiation of Tfh and memory CD4 T cells are key objectives of vaccination strategies. To gain insight into T cell development and function, we implemented single cell (sc) analyses of gene expression by RNA sequencing (scRNAseq). We used this approach to study T cell responses to viral infection and tumor antigens (Ciucci et al., 2019; Vacchio et al., 2019; Magen et al., 2019; Jaiswal et al., 2021). We recently extended these studies to chromatin accessibility (ATAC sequencing, scATACseq), which provides a broader view of the cell functional potential than analyses of actual gene expression. In the current report year, we combined scRNAseq and scATACseq to demonstrate that virus-specific memory and Tfh CD4 T cells develop through distinct transcriptional mechanisms, suggesting that these two fates represent distinct lineages that separate in the early phases of the immune response to infection (Ciucci et al., 2022). We also continued our studies or T cell development in the thymus, focusing on the main T cell subset, defined by expression of an antigen receptor (TCR) made of an alpha and a beta chain (ab T cells). A recent study from the laboratory (Chopp et al., 2020) combined scRNAseq and scATACseq to identify developmental trajectories during the intrathymic differentiation of ab T cell precursors, notably those carrying antigen receptors with high affinity for self ligand (called "agonist-selected"), including regulatory T cells. In the current year, we reported analyses of thymic precursors of an intriguing ab T cell subset homing to the intestinal epithelium and expressing the CD8alpha subunits of the CD8 molecule (Nie et al, 2022). Using scRNAseq, we identified immature and mature thymic precursors and showed that differentiation of the mature component depends on the zinc finger transcription factor LRF (encoded by Zbtb7a). Furthermore, we showed that LRF is necessary for the migration of these precursors to the small intestine, and for their contribution to intestinal homeostasis and prevention of inflammation in an experimental model of colitis. Using genetic, biochemical (chromatin immunoprecipitation) and computational approaches, we found that LRF is necessary for expression of the intestine-homing integrin beta7, and that LRF molecules bind the gene encoding this protein. We are currently expanding these studies, leveraging single cell analyses of transcriptome and chromatin status, aiming to (i) identify relevant transcriptomic patterns and cis-regulatory elements (from scRNAseq and scATACseq, respectively) involved in thymocyte differentiation, (ii) infer gene regulatory networks controlling these processes, and (iii) leverage this information to identify, using genetic approaches, factors needed for CD4 T cell development. 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 or the laboratory research over the past 5-10 years. We recently 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). In the current report year, we completed studies investigating how Thpok affects the expression of its target genes (including those encoding CD4, CD8, and molecules critical for CD4 T cell functions). Combining mass spectrometry, biochemical and genetic analyses, we identified the nucleosome remodeling and deacetylase (NuRD) complex as a novel Thpok cofactor. We showed that three amino-acid residues located within the amino-terminal region of Thpok (the BTB domain) are required for both NuRD binding and Thpok functions. We further showed that, conversely, NuRD recruitment recapitulates the functions of the Thpok BTB domain. We found that NuRD binding mediates Thpok repression of CD8+-lineage genes, including the transcription factor Runx3 that controls CD8 T cell differentiation, but is dispensable for Cd4 expression. Last, we showed that these functions cannot be performed by the BTB domain of the Thpok-related factor Bcl6, which fails to bind NuRD. This indicates that cofactor binding critically contributes to the functional specificity of BTB-zinc finger factors, which control the differentiation of most hematopoietic subsets.