Differential Requirement of Enhancers E8 and E8 in Cytotoxic Lineage T Cells and in Intestinal Intraepithelial Lymphocytes.

CD8 expression in T lymphocytes is tightly regulated by the activity of at least six enhancers (E8-E8), 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 E8, designated E8-core, and of E8. Loss of E8-core led to a similar reduction in CD8 expression in naïve CD8 T cells and in IELs as observed in mice, demonstrating that we identified the core enhancer region of E8. While 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 E8-core and E8. Moreover, activated core CD8 T cells lost CD8 expression to a greater degree than core and 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 TCRβCD4 IEL population in core but not mice. Such a reduction was not observed in mice, indicating that E8-core controls the generation of CD4 CTLs independently of its role in gene regulation. Further, the combined deletion of E8-core and E8 restored CD4 CTL subsets, suggesting an antagonistic function of E8 in the generation of CD4 CTLs. Together, our study demonstrates a complex utilization and interplay of E8-core and E8 in regulating CD8 expression in cytotoxic lineage T cells and in IELs. Moreover, we revealed a novel E8-mediated regulatory mechanism controlling the generation of intestinal CD4 CTLs.