Stepwise chromatin and transcriptional acquisition of an intraepithelial lymphocyte program.

Mesenteric lymph node (mLN) T cells undergo tissue adaptation upon migrating to intestinal lamina propria and epithelium, ensuring appropriate balance between tolerance and resistance. By combining mouse genetics with single-cell and chromatin analyses, we uncovered the molecular imprinting of gut epithelium on T cells. Transcriptionally, conventional and regulatory (T) CD4 T cells from mLN, lamina propria and intestinal epithelium segregate based on the gut layer they occupy; trajectory analysis suggests a stepwise loss of CD4 programming and acquisition of an intraepithelial profile. T cell fate mapping coupled with RNA sequencing and assay for transposase-accessible chromatin followed by sequencing revealed that the T cell program shuts down before an intraepithelial program becomes fully accessible at the epithelium. Ablation of CD4-lineage-defining transcription factor ThPOK results in premature acquisition of an intraepithelial lymphocyte profile by mLN T cells, partially recapitulating epithelium imprinting. Thus, coordinated replacement of the circulating lymphocyte program with site-specific transcriptional and chromatin changes is necessary for tissue imprinting.