Downregulation of chemokine receptor 9 facilitates CD4⁺CD8aa⁺ intraepithelial lymphocyte development

Intestinal intraepithelial lymphocytes (IELs) reside in the gut epithelial layer, where they help in maintaining intestinal homeostasis. Peripheral CD4(+) T cells can develop into CD4(+)CD8aa(+) IELs upon arrival at the gut epithelium via the lamina propria (LP). Although this specific differentiation of T cells is well established, the mechanisms preventing it from occurring in the LP remain unclear. Here, we show that chemokine receptor 9 (CCR9) expression is low in epithelial CD4(+)CD8aa(+) IELs, but CCR9 deficiency results in CD4(+)CD8aa(+) over-differentiation in both the epithelium and the LP. Single-cell RNA sequencing shows an enriched precursor cell cluster for CD4(+)CD8aa(+) IELs in Ccr9(-/-) mice. CD4(+) T cells isolated from the epithelium of Ccr9(-/-) mice also display increased expression of Cbf beta 2, and the genomic occupancy modification of Cbf beta 2 expression reveals its important function in CD4(+)CD8aa(+) differentiation. These results implicate a link between CCR9 downregulation and Cbfb2 splicing upregulation to enhance CD4(+)CD8aa(+) IEL differentiation.