Heterogeneity within the tumor infiltrating lymphocyte (TIL) population limits immunotherapeutic efficacy against cancer. Between two subpopulations of exhausted CD8+ TILs (progenitor-exhausted, TPEX; terminally-exhausted, TTEX), TTEX cells remain unresponsive to anti-PD1 therapy. Deciphering whether and how PD1-resistant TTEX cells engage in tumor promotion could improve the response to immunotherapy. 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-PD1 treatment. The TTEX-influenced CSCs were highly clonogenic and exhibited a multi-drug resistant phenotype, overexpressing drug efflux pumps like ABCC1 and ABCB1. These CSCs were highly invasive, displaying increased invadopodia development and elevated cofilin, CXCR4, and 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 carcinoma patients for both CD8+ TIL and TTEX frequency prior to anti-PD1-therapy could improve patient outcomes. Additionally, targeting the LAMP3/NRP1-VEGFR2 axis could be a therapeutic strategy in advanced carcinoma patients with limited CD8+ T cell infiltration and high TTEX frequency.

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<p>Contains supplementary table ST1, ST2 and ST3.</p>

<div>Abstract<p>Heterogeneity within the tumor-infiltrating lymphocytes (TIL) population limits immunotherapeutic efficacy against cancer. Between two subpopulations of exhausted CD8⁺ TILs (progenitor-exhausted; T_PEX, terminally exhausted; T_TEX), T_TEX cells remain unresponsive to anti–programmed cell death protein 1(PD-1) therapy. Deciphering whether and how PD-1–resistant T_TEX cells engage in tumor promotion could improve the response to immunotherapy. Here, we report that T_TEX cells actively participate in tumor progression by modulating cancer stem cells (CSC). T_TEX cells strongly correlated with elevated CSC frequency in poorly immune-infiltrated (CD8⁺ TIL low) advanced human breast and ovarian carcinomas. T_TEX directly upregulated CSC frequency <i>in vitro</i>, which was not affected by anti–PD-1 treatment. The T_TEX-influenced CSCs were highly clonogenic and exhibited a multidrug-resistant phenotype, overexpressing drug efflux pumps like ABCC1 and ABCB1. These CSCs were highly invasive, displaying increased invadopodia development and elevated cofilin, CXCR4, and matrix metalloproteinase 7 (MMP7) expression. The invasive properties along with epithelial–mesenchymal plasticity of T_TEX-educated CSCs increased metastasis <i>in vivo</i>. T_TEX increased cell surface levels and activation of VEGFR2 in CSCs, and silencing or inhibition of VEGFR2 reversed the CSC-stimulatory effects of T_TEX. LAMP3 and NRP1 on the surface of T_TEX stimulated VEGFR2 in CSCs to promote aggressiveness. Cumulatively, these findings suggest that screening patients with carcinoma for both CD8⁺ TILs and T_TEX 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 T_TEX frequency.</p>Significance:<p>Cross-talk with T_TEX CD8⁺ T cells mediated by the VEGFR2 axis induces aggressive properties in cancer stem cells to promote tumor progression.</p></div>

<p>Contains supplementary table ST1, ST2 and ST3.</p>