IL-21 Selectively Protects CD62L⁺ NKT Cells and Enhances Their Effector Functions for Adoptive Immunotherapy

T cells expressing CD19-specific chimeric Ag receptors (CARs) produce high remission rates in B cell lymphoma, but frequent disease recurrence and challenges in generating sufficient numbers of autologous CAR T cells necessitate the development of alternative therapeutic effectors. Vα24-invariant NKTs have intrinsic antitumor properties and are not alloreactive, allowing for off-the-shelf use of CAR-NKTs from healthy donors. We recently reported that CD62L⁺ NKTs persist longer and have more potent antilymphoma activity than CD62L^- cells. However, the conditions governing preservation of CD62L⁺ cells during NKT cell expansion remain largely unknown. In this study, we demonstrate that IL-21 preserves this crucial central memory-like NKT subset and enhances its antitumor effector functionality. We found that following antigenic stimulation with α-galactosylceramide, CD62L⁺ NKTs both expressed IL-21R and secreted IL-21, each at significantly higher levels than CD62L^- cells. Although IL-21 alone failed to expand stimulated NKTs, combined IL-2/IL-21 treatment produced more NKTs and increased the frequency of CD62L⁺ cells versus IL-2 alone. Gene expression analysis comparing CD62L⁺ and CD62L^- cells treated with IL-2 alone or IL-2/IL-21 revealed that the latter condition downregulated the proapoptotic protein BIM selectively in CD62L⁺ NKTs, protecting them from activation-induced cell death. Moreover, IL-2/IL-21-expanded NKTs upregulated granzyme B expression and produced more T_H1 cytokines, leading to enhanced in vitro cytotoxicity of nontransduced and anti-CD19-CAR-transduced NKTs against CD1d⁺ and CD19⁺ lymphoma cells, respectively. Further, IL-2/IL-21-expanded CAR-NKTs dramatically increased the survival of lymphoma-bearing NSG mice compared with IL-2-expanded CAR-NKTs. These findings have immediate translational implications for the development of NKT cell-based immunotherapies targeting lymphoma and other malignancies.