IL-21 selectively protects CD62L+ NKTs from activation-induced cell death during ex vivo expansion and enhances antitumor activity of NKT cell therapy in vivo

Abstract

Abstract Vα24-invariant natural killer T cells (NKTs) have potent antitumor properties that can be exploited for use in cancer immunotherapy. This requires rigorous protocols for ex vivo expansion of primary NKTs that preserve their longevity and function. We recently reported that CD62L+ NKTs persist longer and have better antitumor activity in vivo than CD62L− counterparts. However, the conditions governing preservation of CD62L expression in NKTs during expansion remain largely unknown. Comparative gene expression analysis of CD62L+ and CD62L− NKTs revealed significantly higher IL-21R expression in the former subset, which was confirmed at the protein level by flow cytometry. Hence, we hypothesized that IL-21 preferentially supports CD62L+ NKTs. We expanded primary human peripheral blood NKTs by stimulating in vitro with cognate antigen, α-galactosylceramide, and supplemented the culture with IL-2, IL-21, or both cytokines. In contrast to IL-2, IL-21 alone failed to support NKT cell expansion, but combined treatment with IL-2 and IL-21 produced more NKTs and preserved a significantly higher frequency of CD62L+ cells. We further demonstrated that IL-21 selectively downregulates BIM in CD62L+ NKTs and protects the subset from activation-induced cell death. Functionally, IL-2/IL-21-expanded NKTs secreted more TH1 cytokines and were more cytotoxic against lymphoma cells, which was associated with granzyme B upregulation by IL-21. Following adoptive transfer to NSG mice, IL-2/IL-21-expanded NKTs persisted significantly longer and had higher therapeutic efficacy in a lymphoma model compared with IL-2-expanded NKTs. Our results instruct inclusion of IL-21 in NKT-cell expansion protocols for cancer immunotherapy applications.