Differential Requirement of Cd8 Enhancers E8I and E8VI in Cytotoxic Lineage T Cells and in Intestinal Intraepithelial Lymphocytes.

Alexandra Franziska Gülich,Patricia Hamminger,Maria Jonah Orola,Teresa Preglej,Caroline Tizian,Ichiro Taniuchi,Shinya Sakaguchi,Sawako Muroi,Wilfried Ellmeier,Marlis Alteneder

doi:10.3389/fimmu.2019.00409

Alexandra Franziska Gülich, Patricia Hamminger + Show 8 more

Open Access

https://doi.org/10.3389/fimmu.2019.00409

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Abstract

CD8 expression in T lymphocytes is tightly regulated by the activity of at least six Cd8 enhancers (E8I-E8VI), however their complex developmental stage-, subset-, and lineage-specific interplays are incompletely understood. Here we analyzed ATAC-seq data on the Immunological Genome Project database and identified a similar developmental regulation of chromatin accessibility of a subregion of E8I, designated E8I-core, and of E8VI. Loss of E8I-core led to a similar reduction in CD8 expression in naïve CD8+ T cells and in IELs as observed in E8I−/− mice, demonstrating that we identified the core enhancer region of E8I. While E8VI−/− mice displayed a mild reduction in CD8 expression levels on CD8SP thymocytes and peripheral CD8+ T cells, CD8 levels were further reduced upon combined deletion of E8I-core and E8VI. Moreover, activated E8I-core−/−E8VI−/− CD8+ T cells lost CD8 expression to a greater degree than E8I-core−/− and E8VI−/− CD8+ T cells, suggesting that the combined activity of both enhancers is required for establishment and maintenance of CD8 expression before and after TCR activation. Finally, we observed a severe reduction of CD4 CTLs among the TCRβ+CD4+ IEL population in E8I-core−/− but not E8VI−/− mice. Such a reduction was not observed in Cd8a−/− mice, indicating that E8I-core controls the generation of CD4 CTLs independently of its role in Cd8a gene regulation. Further, the combined deletion of E8I-core and E8VI restored CD4 CTL subsets, suggesting an antagonistic function of E8VI in the generation of CD4 CTLs. Together, our study demonstrates a complex utilization and interplay of E8I-core and E8VI in regulating CD8 expression in cytotoxic lineage T cells and in IELs. Moreover, we revealed a novel E8I-mediated regulatory mechanism controlling the generation of intestinal CD4 CTLs.

Highlights

CD8 plays an important role in the activation of cytotoxic T cells by serving as a coreceptor for MHC class I-restricted T cell receptors via its binding to the invariant α3 domain of MHC class I [1]
Five ATAC-seq peaks mapped to the evolutionary conserved regions (ECR)−3, −4, −7, −8, and −10, respectively (Figure 1B), which we have identified in a previous study [23] using the MULAN algorithm [37]
ECR-8 and ECR-4 mapped within E8I and E8VI, respectively, both of which display enhancer activity in mature CD8SP thymocytes and in naive CD8+ T cells [11, 12, 23]

Summary

Introduction

CD8 plays an important role in the activation of cytotoxic T cells by serving as a coreceptor for MHC class I-restricted T cell receptors via its binding to the invariant α3 domain of MHC class I [1]. A fraction of activated cytotoxic T cells upregulates Cd8a gene expression, leading to the formation of CD8αα in addition to CD8αβ heterodimers [7]. A series of transgenic reporter gene expression assays as well as the analyses of mice harboring single and combinatorial deletion of Cd8 enhancers revealed developmental stage-, lineage-, and subset-specific activities of these enhancers. Together, these studies revealed a highly complex and partially synergistic network of cis-regulatory elements driving CD8 expression [8,9,10]

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