STAT3 isoform dynamics reveal robust splice ratio maintenance across cytokine-activated human immune cells

Alternative splicing of STAT3 produces two principal isoforms, STAT3α and STAT3β, that differ in transactivation capacity and DNA-binding behavior. Whereas STAT3α is thought to be a transcriptional activator, STAT3β - due to the lack of the transactivation domain - is thought to be a transcriptional repressor. Therefore, the relative abundance of STAT3α and STAT3β molecules within a leukocyte will be critical for immune homeostasis and for gene-therapeutic strategies targeting STAT3. This study investigates whether short-term exposure to IFN-α, IL-6/sIL-6Rα, or IL-10 alters STAT3α/STAT3β stoichiometry in purified human CD4⁺, CD8⁺, CD14⁺ and CD19⁺ cells. We evaluated in healthy donors PBMCs the STAT3α and STAT3β mRNA and protein levels after stimulation with IFN-α, IL-6/sIL-6Rα or IL-10. For mRNA analysis, twelve candidate reference genes were assessed for stability across subsets and stimuli, with UBE2D2 identified as the most stable reference gene. We demonstrate that at mRNA level, cytokine treatment induced STAT3α and STAT3β mRNA in a subset-dependent manner. STAT3β mRNA largely paralleled STAT3α, and crucially, the STAT3α/STAT3β mRNA ratio remained constrained within a narrow range. At protein level, STAT3α predominated markedly, yielding a pronounced transcript-protein decoupling. Together, these data indicate that ex vivo short-term cytokine signaling with IFN-α, IL-6/sIL-6Rα, and IL-10 co-induces STAT3 isoforms without broadly reprogramming their splice balance in primary human leukocytes and underscore the importance of preserving physiological isoform ratios in gene-therapeutic strategies targeting STAT3. These findings suggest that short-term inflammatory signals alone are insufficient to shift STAT3 isoform production, supporting the idea that additional or more prolonged regulatory inputs are required to modulate STAT3 splicing in vivo and that physiologically, the STAT3α/STAT3β ratio may be modulated at protein level.