Ex vivo expansion reshapes invariant natural killer T-cell state, function, and therapeutic fitness

Invariant natural killer T cells are uniquely positioned at the interface of innate and adaptive immunity, combining CD1d-restricted antigen recognition with rapid, diverse cytokine responses and relatively low HLA-driven alloreactivity. These properties make them attractive candidates for adoptive cellular immunotherapy, yet clinical translation has remained limited by inconsistent potency and persistence. This review considers ex vivo expansion as a determinant of product state rather than a passive scale-up step. Cytokine support, antigen and co-stimulatory signals, restimulation schedule, and culture duration can alter iNKT cell subset composition, survival, phenotype, and effector function, thereby influencing therapeutic fitness. We further discuss how human iNKT cell developmental biology and subset heterogeneity provide a framework for interpreting donor variability and culture-induced drift. We also compare peripheral-blood, hematopoietic stem/progenitor cell (HSPC)-derived, and induced pluripotent stem cell (iPSC)-derived manufacturing platforms and propose critical quality attributes (CQAs) for identity, potency, persistence, survival, and manufacturing safety. Progress will require manufacturing strategies that achieve clinically relevant expansion while preserving a defined and functionally validated cell state. • Ex vivo expansion acts as iNKT cell-state engineering. • Cytokines reshape iNKT phenotype, potency, and persistence. • CD62L + memory-like iNKT cells may improve durability. • CAR-iNKT products require preserved CAR and TCR function. • Developmental state should guide iNKT manufacturing.