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For reasons not completely clear, natural killer (NK) cells from tumor patients displayed multiple exhaustion features and could not be completely restored even when the inhibitory signals from the intratumoral environment had ceased to exist. Here, we found that the circulating NK cells from bladder cancer patients presented significantly reduced cytotoxicity than the circulating NK cells from healthy volunteers. This impairment in cytotoxicity resulted in part from an overrepresentation of Tim-3+ NK cells in bladder cancer patients. Interestingly, patients with higher frequency of Tim-3+ NK cells tended to present higher frequency of Gal-9+ cells in tumor. Exogenous Gal-9 significantly reduced the cytotoxicity of Tim-3+, but not Tim-3-, NK cells. Patients with better prognosis presented lower levels of Tim-3+ NK cells and Gal-9+ tumor cells. We then attempted to improve the cytotoxicity of NK cells using a combination of exogenous cytokines. IL-2 + IL-15 and IL-2 + IL-21 significantly enhanced, but could not completely restore, the cytotoxicity of NK cells in bladder cancer patients. Notably, when the cytokine concentration increased from intermediate levels to high levels, the cytotoxicity of NK cells from healthy volunteers significantly increased with a strong upward trend, whereas the cytotoxicity of NK cells from bladder cancer patients plateaued at intermediate levels. Further examination revealed that high cytokine concentration significantly increased the Tim-3 expression in NK cells from bladder cancer patients. Blocking Tim-3 not only improved the cytotoxicity of NK cells from bladder cancer patients, but also eliminated the plateauing effect when the NK cells were stimulated with high concentrations of cytokines. Together, these data suggested that proinflammatory cytokines could moderately improve NK cell cytotoxicity in bladder cancer patients. However, the effect was limited due to a concurrent upregulation of Tim-3.
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