CD49a expression and induction of cytotoxicity on human tissue-resident CD8+ T cells is controlled by RUNX2 and RUNX3 transcription factor activity [RNA-seq]

CD49a marks highly cytotoxic epidermal tissue-resident memory (TRM)-cells, but their molecular circuitry and relationships to circulating populations are poorly defined. We demonstrate enrichment of RUNX family transcription factor binding motifs in human epidermal CD8+CD103+CD49a+ TRM-cells, paralleled by high RUNX2 and RUNX3 protein expression. Clonal overlap between epidermal CD8+CD103+CD49a+ TRM-cells and circulating memory CD8+CD45RA–CD62L+ T-cells identified a reservoir of circulating cells with potential to seed cytotoxic TRM-cells in new sites. Upon IL-15 and TGF-β stimulation, subsets of circulating CD8+CD45RA–CD62L+ T-cells acquired CD49a expression and cytotoxic transcriptional profiles in a RUNX2 and RUNX3 dependent manner. In contrast, knock-out of RUNX3, but not RUNX2, prevented CD103 expression. In melanoma, high RUNX2, but not RUNX3, transcription correlated with a cytotoxic CD8+CD103+CD49a+ TRM cell signature and overall patient survival. Together, our results indicate that combined RUNX2 and RUNX3 activity promotes the differentiation of cytotoxic CD8+CD103+CD49a+ TRM-cells, providing immunosurveillance of infected and malignant cells. Bulk RNA-seq upon in vitro differentiation. Memory CD45RA- CD62L+ CD27+ CD8 T cells have been FACS sorted from 3 healthy donors and used for bulk RNA-seq (day 1) or differentiated in vitro for 14 days in Trm-like polarizing conditions including IL-15, TGF-b and CD3/28 stimulation in ECM-coated wells. Upon differentiation, CD8 T cells have been FACS sorted on the basis of CD69, CD103 and CD49a expression for bulk RNA-seq. Bulk RNA-seq upon RUNT transcription factors gene deletion. CRISPR/Cas9 gene deletion was performed for RUNX2, RUNX3 and a control gene on FACS sorted memory CD45RA- CD62L+ CD27+ CD8 T cells from 5 healthy donors. Subsequently, edited cells have been cultured in in vitro for 14 days in Trm-like polarizing conditions including IL-15, TGF-b and CD3/28 stimulation in ECM-coated wells. Upon differentiation, bulk CD8 T cells have been FACS sorted for bulk RNA-seq. Two different targeting guides were used for both RUNX2 (AB and AC) and RUNX3 (AD and AE). Bulk RNA-seq on skin Trm cells. CD103+ CD49a- and CD103+ CD49a+ CD8 Trm cells from the skin epidermis of 6 healthy donors were FACS sorted in RNA lyses buffer and used for bulk RNAseq. This dataset includes a re-analysis of 12 Samples in GSE83637 (GSM2211621-GSM2211624, GSM2211627-GSM2211634). The processed data for this re-analysis is included in the supplementary file GSE232236_GSE83637_RNAseq_raw_gene_count_matrix.txt. This file include raw read counts for each re-analysis Sample. **Submitter declares that raw data were not submitted due to patient privacy concerns**