In vitro inhibition of STAT5 signaling limits CD8⁺ T-cell exhaustion and improves outcomes in adoptive T-cell therapy models.

T-cell exhaustion remains a barrier to adoptive cell therapies. Although γc cytokines are widely used to expand T cells, their impact on T-cell states remains incompletely defined. Using an in vitro model of repetitive TCR stimulation, we found that high-dose IL-2 or IL-15 promoted exhausted CD8⁺ T-cell (TEX) differentiation, marked by increased inhibitory receptor expression and loss of stem-like features. During chronic LCMV Clone 13 infection, virus-specific CD8⁺ T cells expressing high IL2Rβ displayed features of terminally exhausted TEX cells, whereas IL2Rβ deficiency favored progenitor-like populations. In TEX cells, IL-2 and IL-15 preferentially induced STAT5 phosphorylation relative to other signaling pathways. Genetic disruption of STAT5 similarly supported progenitor subsets, implicating STAT5 in the progression toward more differentiated TEX states under chronic stimulation. We therefore asked whether transient attenuation of STAT5 signaling during in vitro T-cell expansion could bias differentiation toward progenitor-like populations without compromising functional competence. Transient JAK3 or STAT5 inhibition enriched for TCF1⁺Ly108⁺ progenitor-like cells while preserving cytokine production and degranulation capacity. Following transfer into tumor-bearing mice, cells expanded under STAT5-inhibited (STAT5i) conditions mediated superior tumor control and prolonged survival. Likewise, transient STAT5 inhibition preserved memory-progenitor phenotypes of human CD22 CAR T cells without diminishing cytotoxicity. RNA sequencing of STAT5i-expanded CD8⁺ T cells confirmed reinforcement of stem-like transcriptional programs and reduced enrichment of effector and exhausted signatures. Together, these findings identify the IL-2Rβ–STAT5 axis as a regulator of CD8⁺ T-cell differentiation and support transient STAT5 modulation during ex vivo expansion as a strategy to improve therapeutic T-cell products.