Genetic Analysis of T-cell Differentiation

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

We study the transcriptional control of 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 and CD8 T cells differentiate in the thymus from precursors that express both CD4 and CD8 ('double-positive', DP). Because of their pivotal role in immune responses, CD4 T cells have remained the main focus of the laboratory research. Upon antigen encounter during infection, and depending on the local inflammatory context, short-lived effector CD4 T cells proliferate and differentiate into multiple subtypes, characterized by the cytokines they produce, including IFN-gamma (IFN-g) (Th1 cells, responding to intra-cellular pathogens), IL-4 (Th2 cells, notably in response to worm infections), and IL-17 (Th17, involved in controlling extra-cellular micro-organisms). Additionally, follicular helper CD4 T cells (Tfh) provide help to B cells for the generation of efficient antibodies; this is a critical function of CD4 T cells, disrupted in various inherited human immunodeficiency syndromes. Last, memory antigen-specific T cells persist after infection; together with the B cell response, they provide protection against re-infection by the same pathogens. Efficient differentiation of Tfh and memory CD4 T cells are key objectives of vaccination strategies. Our recent research has focused on two specific questions regarding CD4 T cell functions: (i) which mechanisms direct T cells into short-term effector vs. Tfh vs. memory cells in vivo and (ii) how do these paradigms, generated from studies of responses to infections, apply to CD4 T cell responses to cancer, in which chronic antigen exposure results in dysfunctional (exhausted) T cell responses. To address the first question, we used single-cell RNA sequencing (scRNAseq) of CD4 T cells responding to a prototypical mouse pathogen, Lymphocytic ChorioMeningitis Virus (LCMV). By evaluating the transcriptome of thousands of cells, scRNAseq offers an unprecedented opportunity to measure the diversity of T cell responses; thus, implementing these analyses has been a major laboratory investment in the past few years. We found CD4 T cells with transcriptomic patterns corresponding to Th1 and Tfh programs and identified corresponding gene expression signatures. We also identified a novel signature, called T central memory precursor (Tcmp), that included genes expressed in memory CD4 T cells but excluded typical Tfh and Th1 markers. Our previous observations that the transcription factor Thpok promotes the differentiation of CD4 T cells in the thymus and their 'pre-programming' for helper functions prompted us to examine whether it affects the functional differentiation of LCMV-responding CD4 T cell. Using genetic strategies to inactivate Thpok in mature CD4 T cells, we found that the Tfh and Tcmp but not the Th1 transcriptomic patterns were Thpok-dependent. This was in line with ongoing experiments analyzing the impact of Thpok on Tfh cell differentiation. Additionally, it prompted us to examine the impact of Thpok on CD4 T cell memory, with the following results. First, Thpok is needed to establish CD4 memory after LCMV infection: Thpok disruption both reduces the number of CD4 memory T cells and impairs their gene expression pattern, including reduced expression of the pro-survival proteins Bcl2 or IL-7Ra, and increased expression of IFN-g. Additionally, Thpok is needed for the production of the cytokine IL-2, an attribute of functional memory CD4 T cells, and restrains the expression of markers characteristic of T cell dysfunction, notably those expressed by exhausted T cells responding to chronic infection and by intra-tumoral T cells (e.g. Lag3, Tim3 and 2B4). Last, in addition to its cell-intrinsic effect on CD4 memory, Thpok expression in CD4 T cells is important to generate functional memory CD8 T cells. Altogether, these findings establish Thpok as a key controller of memory vs. short-term T cell responses. We analyzed the mechanistic bases for these functions of Thpok using both biochemical and genetic approaches. Using chromatin immunoprecipitation (ChIP) followed by deep-sequencing (ChIP-seq), we generated a genome-wide map of Thpok DNA binding sites in CD4 T cells. We detected Thpok binding to most genes it controls in LCMV-specific CD4 T cells, including those encoding Runx3 and Blimp1, two transcription factors characteristic of Th1 cells and repressed by Thpok. Using combined gene inactivation, we found that both Runx3 and Blimp1 repression mediated the function of Thpok in promoting the differentiation and functional fitness of memory CD4 T cells. Ongoing research examines the role of this circuitry in situations of antigen persistence. We are currently using these approaches to study the response of CD4 T cells to cancer. The last few years have emphasized the role of MHC II-presented antigens and CD4 T cells in tumor control. However, harnessing CD4 T cells for cancer immunotherapy implies deciphering the functional complexity of CD4 T cell responses (e.g. the anti-tumor impact of IFN-g-producing vs. the pro-tumor effects of regulatory CD4 T cells), their impact on diverse targets (CD8 T cells, B cell, innate immune and stromal cells, vasculature), and the multiplicity of controlling input signals. To address these challenges, current research aims at generating tractable models of CD4 T cell responses to cancer, with analyses combining high-throughput single-cell approaches, including scRNAseq, and Crispr-Ca9 mutagenesis.