TOX functions as a cytotoxicity checkpoint in human NK cells and is reversibly regulated by SLAMF7 receptor

The thymocyte selection-associated high mobility group box protein (TOX) is the master regulator of T cell exhaustion, but its function in human NK cell is not well defined. Here, we identified TOX as a cytotoxicity checkpoint in human NK cells and uncovered a previously unknown role for SLAMF7 as a selective and negative regulator of TOX-mediated NK cells dysfunction. Human PBMCs-derived NK cells were stimulated with IL-2, IL-10, IL-15, and IL-18, or cross-linked with agonistic antibodies targeting NKp46, NKp30, NKp44, CD16, 2B4, NKG2D, DNAM-1, 4-1BB, IL-2Rbeta, SLAMF7, or NKG2A, alone or in combination with SLAMF7. Expression of TOX, IFNgamma, CD107a, Granzyme B, PD-1, and SLAMF7 was quantified by flow cytometry. IL-2 and IL-15 induce rapid, dose-dependent TOX upregulation, with IL-15 showing greater potency. IL-10 and IL-18 failed to upregulate TOX. NKp30, NKp46, CD16, and 2B4 stimulation also upregulated TOX alongside IFN-gamma, CD107a, and Granzyme B induction. SLAMF7 alone did not induce TOX but selectively modulated TOX during co-engagement with other activating receptors. Specifically, SLAMF7 + NKp46 and SLAMF7 + NKp30 co-stimulation suppressed TOX to baseline while preserving strong IFNgamma, CD107a, and Granzyme B responses. SLAMF7 did not alter DAP12-, DAP10-, or FcRgamma- dependent signaling, demonstrating that SLAMF7 specifically counteracts CD3ζ-dependent signaling. In contrast, SLAMF4 (2B4) synergistically amplified TOX and PD-1 across NK cytotoxicity receptor (NCR) and NKG2D pathways, implicating it as a strong exhaustion-promoting receptor. Additionally, SLAMF7 + CD16 increased TOX above CD16 alone. Moreover, SLAMF7 co-stimulation with NKp44, NKG2D, DNAM-1, 4-1BB, IL-2Rbeta, or NKG2A did not alter TOX levels. Functionally, TOX low NK cells represented the dominant IFNgamma⁺, CD107a⁺, and Granzyme B⁺ subset , confirming TOX as a negative regulator of NK cell cytotoxicity. Consistent with these findings, SLAMF7 + NKp46 co-stimulation produced synergistic killing of MDA-MB-231 cells, whereas SLAMF7 + CD16 co-stimulation, which augments TOX, reduced cytotoxicity compared to CD16 alone. Importantly, tumor-induced TOX modulation strongly correlated with NK susceptibility. We observed that NK-sensitive MDA-MB-231 cells induced strong TOX downregulation, resulting in robust effector responses, whereas NK-resistant PANC-1 cells failed to downregulate TOX and elicited weak activation. Together, these results identify TOX as a checkpoint regulating NK cytotoxicity and define a previously undescribed SLAMF7-CD3ζ axis that modulates NK cell exhaustion. Targeting TOX or modulating SLAMF7 signaling may enhance NK-based immunotherapies for resistant solid tumors.