Cytokine-Mediated Preconditioning of Naïve CD4 T Cells Biases Subsequent Effector Differentiation 2329947

Abstract Introduction Naïve CD4 T cells are activated in secondary lymphoid organs, where integration of T cell receptor, co-stimulatory, and cytokine signals directs effector differentiation. Previous studies have shown that exposure to individual cytokines, such as IL-27 or IL-6, or helminth infection—associated environments can alter transcriptional programs and functional potential of naïve CD4 T cells. However, it remains poorly defined which cytokine receptors are constitutively expressed on naïve CD4 T cells and whether cytokine sensing at this stage broadly biases subsequent effector fate decisions. Methods Single-cell RNA sequencing was performed on murine splenic naïve CD4 T cells to profile cytokine receptor expression. Selected receptors were validated by quantitative RT-PCR and flow cytometry. Cytokine responsiveness was assessed by short-term cytokine stimulation followed by phospho-flow analysis of STAT signaling. Cytokine-conditioned naïve CD4 T cells were then activated via T cell receptor stimulation to evaluate downstream functional outcomes. In parallel, systemic inflammation was induced in vivo using poly I:C, and naïve CD4 T cells were isolated to determine whether inflammatory conditioning biased their functional potential. Results Naïve CD4 T cells constitutively expressed IL-4R, IL-6R, IL-7R, IL-21R, IL-27R, and IFNAR. Exposure to these cytokines induced distinct STAT phosphorylation patterns, indicating functional signaling without altering CD62L or CD44 expression. Cytokine conditioning by IL-6, IL-7, and IL-27 favored Th1-like responses, IL-4 enhanced Th2-associated programs, and Th17 differentiation was broadly suppressed. Consistently, in vivo Poly I:C exposure induced Ly6C expression in naïve CD4 T cells and promoted enhanced Th1 commitment. Conclusion Together, our findings demonstrate that cytokine-mediated conditioning at the naïve stage induces biased effector potential without overt phenotypic activation, thereby preprogramming subsequent T cell receptor—driven differentiation. Funding Source RS-2025-25422376, RS-2024-00334289, RS-2025-02232977 Topic Categories Cytokines and Chemokines and their Receptors (CCR)