Targeting exhausted CD8+ T-cell (TEX)–induced aggravated cancer stem cells (CSC) holds immense therapeutic potential. In this regard, immunomodulation via Neem Leaf Glycoprotein (NLGP), a plant-derived glycoprotein immunomodulator is explored. Since former reports have proven immune dependent–tumor restriction of NLGP across multiple tumor models, we hypothesized that NLGP might reprogram and rectify TEX to target CSCs successfully. In this study, we report that NLGP’s therapeutic administration significantly reduced TEX-associated CSC virulence in in vivo B16-F10 melanoma tumor model. A similar trend was observed in in vitro generated TEX and B16-F10/MCF7 coculture setups. NLGP rewired CSCs by downregulating clonogenicity, multidrug resistance phenotypes and PDL1, OCT4, and SOX2 expression. Cell cycle analysis revealed that NLGP educated–TEX efficiently pushed CSCs out of quiescent phase (G0G1) into synthesis phase (S), supported by hyper-phosphorylation of G0G1–S transitory cyclins and Rb proteins. This rendered quiescent CSCs susceptible to S-phase–targeting chemotherapeutic drugs like 5-fluorouracil (5FU). Consequently, combinatorial treatment of NLGP and 5FU brought optimal CSC-targeting efficiency with an increase in apoptotic bodies and proapoptotic BID expression. Notably a strong nephron-protective effect of NLGP was also observed, which prevented 5FU-associated toxicity. Furthermore, Dectin-1–mediated NLGP uptake and subsequent alteration of Notch1 and mTOR axis were deciphered as the involved signaling network. This observation unveiled Dectin-1 as a potent immunotherapeutic drug target to counter T-cell exhaustion. Cumulatively, NLGP immunotherapy alleviated exhausted CD8+ T-cell-induced CSC aggravation.

Implications: Our study recommends that NLGP immunotherapy can be utilized to counter ramifications of T-cell exhaustion and to target therapy elusive aggressive CSCs without evoking toxicity.

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