T cell egress via lymphatic vessels limits the intratumoral T cell repertoire in melanoma

Antigen-specific CD8+ T cell accumulation in tumors is a prerequisite for effective immunotherapy, and yet, the mechanisms of lymphocyte transit remain poorly defined. We find that tumor-associated lymphatic vessels control T cell exit from tumors via the chemokine CXCL12, and intratumoral antigen encounter tunes CXCR4 expression on effector CD8+ T cells. Only high affinity antigen downregulates CXCR4 and upregulates the CXCL12 decoy receptor, ACKR3, thereby reducing CXCL12 sensitivity and promoting T cell retention. A diverse repertoire of functional tumor-specific CD8+ T cells exit the tumor, thereby limiting tumor control. CXCR4 inhibition and loss of lymphatic-specific CXCL12 boosts T cell retention and enhances tumor control. Strategies that limit T cell egress, therefore, provide a new tool to boost the response to immunotherapy. Overall design: YUMMER1.7 murine melanoma cells were intradermally implanted into 3 C57Bl/6 mice. At day 21, tumors were harvested and digested into cell suspensions. Cell suspensions were immunostained with CD45-APC antibodies and then enriched for Miltenyi APC microbeads and the QuadroMACs Magnetic Seperator and LS Columns. Enriched CD45+ cell suspensions were stained with DAPI and then sorted on SY3200 HAPS sorter to improve purity of live CD45+ cells. 2.5e5 cells were sorted from each tumor and then pooled. The data was output to fastq format, converted to 10X format, then into a Seurat object. The Seurat object was dimensionally reduced and clustered, with CD3d/CD3E+ cells identified, then dimensionally reduced and clustered again, with CD8+ cells identified.