Short-term IL-21 and GSK-3β inhibition enriches stem-like CD8⁺ T cells and boosts CD19 CAR T-cell effector function

Abstract The success of adoptive cell transfer therapy (ACT) hinges on T cell quality, particularly differentiation state, which governs in vivo persistence and long-term anti-tumor efficacy. CD8⁺ T cells with a stem cell memory (SCM) phenotype exhibit enhanced self-renewal and are associated with durable immune responses. However, inducing such phenotypes without impairing T cell expansion or function remains challenging. Cytokine conditioning and signaling pathway modulation have been explored to steer T cell fate toward less-differentiated states. Here, we investigate the combined effects of IL-21 and Wnt/β-catenin pathway activation—via GSK-3β inhibition—on human CD8⁺ T cell programming, and extend these findings to the generation and functional testing of CD19 CAR T cells. Peripheral blood CD8⁺ T cells from healthy donors were stimulated with anti-CD3/CD28 beads for 48 hours, followed by an 8-day expansion phase in IL-7 and IL-15. IL-21 was added in some conditions, alone or in combination with the GSK-3β inhibitor TWS119 to activate Wnt/β-catenin signaling. Exposure durations were varied to identify the critical window for phenotype modulation and optimize treatment conditions. CD8⁺ T cells were transduced with a CD19 CAR incorporating a 4-1BB costimulatory domain. Functional assessments on day10, included immunophenotyping, proliferation and apoptosis assays, metabolic profiling, and repeated antigen-stimulation cytotoxicity assays using CD19⁺ Nalm6 leukemia cells. IL-21 and TWS119 synergized with IL-7/15 to promote an SCM-like phenotype in CD8⁺ T cells, characterized by elevated expression of memory markers, suppression of effector differentiation, and an IL-2–dominant cytokine profile. However, continuous 10-day exposure impaired T cell quality—marked by reduced expansion, decreased viability, attenuated metabolic activity, and increased apoptosis upon restimulation. In contrast, abbreviated exposure—during days 2-4—mitigated these effects while preserving the memory-enriched phenotype, revealing a critical temporal window for optimal programming. When applied to CAR T-cell manufacturing, short-term IL-21/TWS119 conditioning preserved expansion capacity while increasing the frequency of CD8⁺ CD19 CAR T cells with an SCM-like phenotype and an IL-2–dominant cytokine production. In addition to phenotypic changes, lentiviral mediated CAR transduction was enhanced by IL-21/TWS119 treatment. Following repeated antigen challenge with CD19⁺ Nalm6 leukemia cells, conditioned CAR T cells exhibited superior proliferative capacity and sustained cytotoxic activity, in contrast to conventional CAR T cells. Short-term combinatorial conditioning with IL-21 and GSK-3β inhibition (TWS119) effectively biases human CD8⁺ T cells toward a less-differentiated SCM-like phenotype while avoiding the fitness and viability deficits induced by prolonged exposure. This temporal modulation strategy enhances the durability and anti-tumor potency of CD19 CAR T cells under chronic antigen stress. These findings support transient IL-21/Wnt pathway modulation as a viable enhancement to ACT manufacturing pipelines aimed at improving therapeutic T-cell quality and persistence.