Human CD96 Correlates to Natural Killer Cell Exhaustion and Predicts the Prognosis of Human Hepatocellular Carcinoma.

Immune checkpoint blockade has become a promising therapeutic approach to reverse immune cell exhaustion. Coinhibitory CD96 and T-cell immunoglobulin and ITIM domain (TIGIT), together with costimulatory CD226, bind to common ligand CD155. The balancing between three receptors fine-tunes immune responses against tumors. In this study, we investigated the expression of CD96, TIGIT, and CD226 in 55 fresh human hepatocellular carcinoma (HCC) samples, 236 paraffin-embedded HCC samples, and 20 normal human livers. The cumulative percentage, absolute count, and mean fluorescence intensity (MFI) of CD96+ NK cells are significantly increased in the intratumoral tissues of HCC and break the balance between three receptors. Human CD96+ NK cells are functionally exhausted with impaired interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production, high gene expression of interleukin (IL)-10 and transforming growth factor-beta 1 (TGF-ß1), and low gene expression of T-bet, IL-15, perforin, and granzyme B. In addition, blocking CD96-CD155 interaction specifically increases lysis of HepG2 cells by NK cells. HCC patients with a high level of CD96 or CD155 expression within tumor are strongly associated with deteriorating disease condition and shorter disease-free survival (DFS) and overall survival times. Patients with a higher cumulative percentage of CD96+ NK cells within tumor also exhibit shorter DFS. High plasma level of TGF-ß1 in HCC patients up-regulates CD96 expression and dynamically shifts the balance between CD96, TIGIT, and CD226 in NK cells. Blocking TGF-ß1 specifically restores normal CD96 expression and reverses the dysfunction of NK cells. Conclusion: These findings indicate that human intratumoral CD96+ NK cells are functionally exhausted and patients with higher intratumoral CD96 expression exhibit poorer clinical outcomes. Blocking CD96-CD155 interaction or TGF-ß1 restores NK cell immunity against tumors by reversing NK cell exhaustion, suggesting a possible therapeutic role of CD96 in fighting liver cancer.