CD62L⁺ NKT cells have prolonged persistence and antitumor activity in vivo

Vα24-invariant natural killer T cells (NKTs) localize to tumors and have inherent antitumor properties, making them attractive chimeric antigen receptor (CAR) carriers for redirected cancer immunotherapy. However, clinical application of CAR-NKTs has been impeded, as mechanisms responsible for NKT expansion and the in vivo persistence of these cells are unknown. Here, we demonstrated that antigen-induced expansion of primary NKTs in vitro associates with the accumulation of a CD62L⁺subset and exhaustion of CD62L^- cells. Only CD62L⁺ NKTs survived and proliferated in response to secondary stimulation. When transferred to immune-deficient NSG mice, CD62L⁺ NKTs persisted 5 times longer than CD62L^- NKTs. Moreover, CD62L⁺cells transduced with a CD19-specific CAR achieved sustained tumor regression in a B cell lymphoma model. Proliferating CD62L⁺ cells downregulated or maintained CD62L expression when activated via T cell receptor alone or in combination with costimulatory receptors. We generated HLA^null K562 cell clones that were engineered to express CD1d and costimulatory ligands. Clone B-8-2 (HLA^nullCD1d^medCD86^high4-1BBL^medOX40L^high) induced the highest rates of NKT expansion and CD62L expression. B-8-2-expanded CAR-NKTs exhibited prolonged in vivo persistence and superior therapeutic activities in models of lymphoma and neuroblastoma. Therefore, we have identified CD62L as a marker of a distinct NKT subset endowed with high proliferative potential and have developed artificial antigen-presenting cells that generate CD62L-enriched NKTs for effective cancer immunotherapy.