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

Pathogen-specific CD4⁺ T cells undergo dynamic expansion and contraction during infection, ultimately generating memory clones that shape the subsequent immune responses. However, the influence of distinct tissue environments on the di[erentiation and clonal selection of polyclonal T cells remains unclear, primarily because of the technical challenges in tracking these cells in vivo. To address this question, we generated Tracking Recently Activated Cell Kinetics (TRACK) mice, a dual-recombinase fatemapping system that enables precise spatial and temporal labeling of recently activated CD4⁺ T cells. Using TRACK mice during influenza infection, we observed organ-specific clonal selection and transcriptional di[erentiation in the lungs, mediastinal lymph nodes (medLNs), and spleen. T cell receptor (TCR) sequencing revealed that local antigenic landscapes and clonal identity shape repertoire diversity, resulting in a low clonal overlap between tissues during acute infection. During the e[ector phase, spleen-derived CD4⁺ T cells preferentially adopted a stem-like migratory phenotype, whereas those activated in the medLNs predominantly di[erentiated into T follicular helper (Tfh) cells. Memory formation was associated with increased clonal overlap between lung and medLN-derived cells, whereas splenic clones retained a distinct repertoire. Additionally, memory CD4⁺ T cells displayed converging antigen specificity across tissues over time. These results highlight the tissue-dependent mechanisms driving clonal selection and functional specialization during infection and underscore how memory development facilitates clonal redistribution and functional convergence.