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.

Myeloid-derived suppressor cells (MDSCs) suppress antitumor immune functions. We have observed that an immunomodulator, neem leaf glycoprotein (NLGP), inhibits tumor-resident MDSCs and enhances antitumor CD8+ T cell immunity. NLGP inhibits the number as well as functions of tumor-resident MDSCs (Gr1±CD11b±) and enhances antitumor CD8± T cell immunity by downregulating arginase 1 and inducible nitric oxide synthase production in MDSCs. Accordingly, decreased T cell anergy and helper to regulatory T cell conversion have been observed in the presence of NLGP, which ultimately augments T cell functions. Mechanistically, NLGP-mediated rectification of T cell suppressive functions of MDSCs was primarily associated with downregulation of the interleukin (IL)-10/signal transducer and activator of transcription 3 (STAT3) signaling axis within the tumor microenvironment, as confirmed by knockdown of STAT3 (by STAT3-siRNA) and using IL-10−/− mice. Thus, NLGP-mediated suppression of MDSC functions in tumor hosts is appeared to be another associated effective mechanism for the eradication of murine melanoma by NLGP.

Altered RGS5‐associated intracellular pericyte signaling and its abnormal crosstalk with endothelial cells (ECs) result chaotic tumor‐vasculature, prevent effective drug delivery, promote immune‐evasion and many more to ensure ultimate tumor progression. Moreover, the frequency of lethal‐RGS5high pericytes within tumor was found to increase with disease progression, which signifies the presence of altered cell death pathway within tumor microenvironment (TME). In this study, we checked whether and how neem leaf glycoprotein (NLGP)‐immunotherapy‐mediated tumor growth restriction is associated with modification of pericytes' signaling, functions and its interaction with ECs. Analysis of pericytes isolated from tumors of NLGP treated mice suggested that NLGP treatment promotes apoptosis of NG2+RGS5high‐fuctionally altered pericytes by downregulating intra‐tumoral TGFβ, along with maintenance of more matured RGS5neg pericytes. NLGP‐mediated inhibition of TGFβ within TME rescues binding of RGS5 with Gαi and thereby termination of PI3K‐AKT mediated survival signaling by downregulating Bcl2 and initiating pJNK mediated apoptosis. Limited availability of TGFβ also prevents complex‐formation between RGS5 and Smad2 and rapid RGS5 nuclear translocation to mitigate alternate immunoregulatory functions of RGS5high tumor‐pericytes. We also observed binding of Ang1 from pericytes with Tie2 on ECs in NLGP‐treated tumor, which support re‐association of pericytes with endothelium and subsequent vessel stabilization. Furthermore, NLGP‐therapy‐ associated RGS5 deficiency relieved CD4+ and CD8+ T cells from anergy by regulating ‘alternate‐APC‐like’ immunomodulatory characters of tumor‐pericytes. Taken together, present study described the mechanisms of NLGP's effectiveness in normalizing tumor‐vasculature by chiefly modulating pericyte‐biology and EC‐pericyte interactions in tumor‐host to further strengthen its translational potential as single modality treatment.

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