Progenitor-predominant exhaustion in cytokine-induced killers reveals TIM-3 as a superior target for enhancing cytokine-induced killer cytotoxicity.

Precursor exhausted and terminally exhausted T cells (Tpex and Tex) are associated with immune checkpoints; T-cell checkpoint blockade may improve patient survival. We assessed cytotoxic activity and PD-1/TIM-3 expressions of CD3+CD8+CD56+ T cells (NK-like T) in cytokine-induced killer (CIK) cells across exhaustion states. We grouped the NK-like T cells into 3 based on TOX and TCF1 expressions: TOX-TCF1+ (memory-like NK-like T), TOX+TCF1+ (NK-like Tpex), and TOX+TCF1- (NK-like Tex). The proportion of NK-like Tpex cells among the CIK cells was the highest (>90%) after culture. TIM-3 and PD-1 expression were high in NK-like Tpex but low in NK-like Tex cells. However, TIM-3 expression was higher than PD-1 expression in CIK cells, suggesting that combining CIK cells and TIM-3 inhibitors may have better effects. Perforin, granzyme B, DNAM-1, and NKG2D, which are cytotoxicity biomarkers, were more expressed in NK-like Tpex than in NK-like Tex cells, which implies that NK-like Tpex may still has a strong cytotoxic effect. The CD4⁺ T cell counts and TIM-3 expression level in the NK-like Tpex cells were positively correlated. CD4⁺ T cells may actively sustain progenitor exhaustion, but the mechanisms are unknown. In vitro cytotoxicity assays confirmed that CIK cell-mediated tumor cytotoxicity was significantly enhanced by TIM-3 than by PD-1 blockade. Furthermore, tumor cytotoxicity was greater for CIK cell cultures with higher than those with lower CD4⁺ T cell counts following TIM-3 blockade. This work demonstrates CD4⁺T cell modulation of progenitor exhaustion in CIK cells and positions TIM-3 blockade to rescue their antitumor potential.