The ontogeny of effector regulatory T cells

Abstract

Regulatory T (Treg) cells are critical for the maintenance of immune homeostasis and peripheral tolerance. Different subsets of Treg cells have recently been described with many studies showing the importance of context-specific differentiation of Treg cells, in particular within non-lymphoid organs. These non-lymphoid organ Treg cells have a fully suppressive Treg cell phenotype with an effector function and are termed effector (e)Treg cells. However, the ontogeny of eTreg cells have not yet been fully described. Additionally, molecular determinants of the eTreg cell program remain incompletely understood. My thesis examines the transcriptional events that regulate the generation of eTreg cells during their thymic development, their homeostasis and response to infection. Using different gene targeted mouse models at steady state and in viral infection models, I studied the intrinsic molecular mechanisms that contribute to eTreg cell differentiation. In particular, I focused on follicular Treg (TFR) cells, which constitute the eTreg cell subset of the germinal centre.

The molecular control of eTreg cell fate and function converges on the transcription factors IRF4 and Blimp-1. IRF4 is induced by antigen receptor signals and cooperates with AP-1 factors, BATF and JUN, to regulate transcriptional networks involved in lymphocyte differentiation, function and metabolism. For example, these factors regulate genes important for antibody class switch recombination in B cells and functional differentiation of distinct CD4 T helper (Th) subsets, including Th2, Th9, Th17 and T follicular helper cells. IRF4 expression in Treg cells is critical for effector differentiation, yet the precise mechanisms of how IRF4 regulates the transcriptional program of TFR cells remains unknown. Using a novel transgenic IRF4 reporter mouse we found that IRF4 is highly expressed in TFR cells. Using IRF4 knockout mouse models, we demonstrate that IRF4 is necessary for TFR cell generation in a Treg cell-intrinsic manner. IRF4 controls important aspects of the transcriptional program that drives TFR cell differentiation, including genes essential for Treg cell migration. Furthermore, I identified the transcription factor c-Maf to be essential for TFR cell generation and demonstrated its central role in maintaining a follicular program in Treg cells.

Subsequently, using ribonucleic acid (RNA)-sequencing, we generated a “follicular signature” of gene expression from the combined analysis of TFH and TFR cells. Integrated transcriptional analyses showed that in the absence of either IRF4 or c-Maf, the majority of the follicular signature genes were downregulated, indicating that these two transcriptional regulators, aside from Bcl6 are indispensable for follicular TFR cell development. Finally, analyses of IRF4 and c-Maf DNA binding sites, identified by chromatin induced precipitation (ChIP)-sequencing, in combination with open chromatin regions in follicular T cell specific loci, we showed that the precise orchestration of distinct sets of genes is required to promote conserved aspects of the follicular T cell fate. In conclusion, my thesis describes how a key transcriptional network orchestrates fundamental steps in TFR cell differentiation and function, which contributes to the understanding of eTreg cell biology.