Tissue-specific clonal selection and differentiation of CD4+ T cells during infection 2306383

Abstract Introduction Pathogen-specific CD4+ T cells expand and contract during infection, giving rise to memory clones that shape future immunity. How distinct tissue environments influence the differentiation and clonal selection of polyclonal CD4+ T cells remains unclear, due in part to the difficulty of tracking pathogen-specific T cells in vivo. Methods To resolve tissue-dependent T cell fate decisions, we generated TRACK mice, a dual-recombinase fate-mapping system enabling precise temporal labeling of recently activated CD4+ T cells. TRACK mice were infected with influenza, and fate-mapped cells from lungs, mediastinal lymph nodes (medLNs), and spleen were analyzed by single-cell RNA-seq and paired TCR sequencing across effector and memory phases. Results Fate-mapping of influenza-specific CD4+ T cells in the TRACK mice revealed pronounced early clonal segregation across tissues, driven by organ-specific antigen availability and recruitment. Early TCR repertoires showed reduced clonal overlap across organs, indicating distinct local antigenic landscapes during the effector immune response. Effector CD4+ T cells exhibited tissue-specific differentiation, with spleen-derived cells acquiring a stem-like migratory phenotype, while medLN-derived cells preferentially became Tfh cells. Over time, tissues showed progressive clonal equilibrium, with increased overlap between lung- and medLN-derived clones. Memory CD4+ T cells also demonstrated converging antigen specificity, focusing on a narrower set of antigens despite maintaining early clonal distinctions. Conclusion These findings reveal that tissue environments impose early antigen-driven clonal segregation and guide distinct differentiation pathways, while memory formation promotes clonal redistribution and convergence of antigen specificity. TRACK mice provide a powerful system to resolve spatial regulation of T cell fate in vivo. Funding Source NIH, CZI, SNF, HHMI Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)