Terminally Exhausted CD8 T Cells Resistant to PD-1 Blockade Promote Generation and Maintenance of Aggressive Cancer Stem Cells

Heterogeneity within the tumor-infiltrating lymphocytes (TIL) population limits immunotherapeutic efficacy against cancer. Between two subpopulations of exhausted CD8 +/- TILs (progeni-tor-exhausted; TPEX, terminally exhausted; TTEX), TTEX cells remain unresponsive to anti-programmed cell death protein 1(PD-1) therapy. Deciphering whether and how PD-1-resistant TTEX cells engage in tumor promotion could improve the response to immu-notherapy. Here, we report that TTEX cells actively participate in tumor progression by modulating cancer stem cells (CSC). TTEX cells strongly correlated with elevated CSC frequency in poorly immune-infiltrated (CD8 +/- TIL low) advanced human breast and ovarian carcinomas. TTEX directly upregulated CSC frequency in vitro, which was not affected by anti-PD-1 treatment. The TTEX-influenced CSCs were highly clonogenic and exhibited a multidrug-resistant phenotype, overexpressing drug efflux pumps like ABCC1 and ABCB1. These CSCs were highly invasive, dis-playing increased invadopodia development and elevated cofilin, CXCR4, and matrix metalloproteinase 7 (MMP7) expression. The invasive properties along with epithelial-mesenchymal plasticity of TTEX-educated CSCs increased metastasis in vivo. TTEX increased cell surface levels and activation of VEGFR2 in CSCs, and silencing or inhibition of VEGFR2 reversed the CSC-stimulatory effects of TTEX. LAMP3 and NRP1 on the surface of TTEX stimulated VEGFR2 in CSCs to promote aggressiveness. Cumulatively, these findings suggest that screening patients with carcinoma for both CD8 +/- TILs and TTEX frequency prior to anti-PD-1 therapy could improve patient outcomes. In addition, targeting the LAMP3/ NRP1-VEGFR2 axis could be a therapeutic strategy in advanced patients with carcinoma with limited CD8 +/- T-cell infiltration and high TTEX frequency.