Impact of IL-21 on CAR T-cell manufacturing: phenotypic, metabolic, and functional outcomes.

INTRODUCTION: Cytokines used during chimeric antigen receptor (CAR) T-cell manufacturing can influence product differentiation and functional fitness. METHODS: We investigated whether adding IL-21 to an IL-7 + IL-15 expansion regimen modulates CD84-directed CAR T cells (CART84) targeting CD84+ acute myeloid leukemia (AML), using IL-7 + IL-15 alone as the primary comparator and IL-2 as a preclinical benchmark. RESULTS: In healthy donor-derived CART84 cells, IL-21 supplementation was linked to an enrichment of less differentiated T-cell subsets, increased LAG-3+ CD8+ T-cell frequency, enhanced expansion following repeated antigen stimulation, and increased CAR expression under G-Rex-based expansion conditions more closely resembling GMP-compatible manufacturing. However, these effects were not accompanied by consistent improvements in cytotoxicity or sustained tumor control upon serial rechallenge in vitro. In AML patient-derived CART84 cells, IL-21 supplementation did not confer a clear advantage in expansion, T-cell memory phenotype, or cytotoxic activity. Furthermore, IL-21 did not enhance CART84 efficacy or persistence in a MOLM-13 AML xenograft model. DISCUSSION: Overall, our results do not support routine incorporation of IL-21 into IL-7 + IL-15-based CART84 manufacturing protocols and highlight the need to evaluate cytokine supplementation strategies within the context of each CAR construct and disease setting.