Differential Requirement of Cd8 Enhancers E8(I) and E8(VI) 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 Cd8 enhancers (E8(I)-E8(VI)), 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(I), designated E8(I)-core, and of E8(VI). Loss of E8(I)-core led to a similar reduction in CD8 expression in naïve CD8(+) T cells and in IELs as observed in E8(I)(−/−) mice, demonstrating that we identified the core enhancer region of E8(I). While E8(VI)(−/−) 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(I)-core and E8(VI). Moreover, activated E8(I)-core(−/−)E8(VI)(−/−) CD8(+) T cells lost CD8 expression to a greater degree than E8(I)-core(−/−) and E8(VI)(−/−) 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 E8(I)-core(−/−) but not E8(VI)(−/−) mice. Such a reduction was not observed in Cd8a(−/−) mice, indicating that E8(I)-core controls the generation of CD4 CTLs independently of its role in Cd8a gene regulation. Further, the combined deletion of E8(I)-core and E8(VI) restored CD4 CTL subsets, suggesting an antagonistic function of E8(VI) in the generation of CD4 CTLs. Together, our study demonstrates a complex utilization and interplay of E8(I)-core and E8(VI) in regulating CD8 expression in cytotoxic lineage T cells and in IELs. Moreover, we revealed a novel E8(I)-mediated regulatory mechanism controlling the generation of intestinal CD4 CTLs.