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

Pathogen-specific CD4⁺ T cells expand and contract during infection, generating memory clones that shape subsequent immune responses. How distinct tissue environments influence differentiation and clonal selection of polyclonal T cells remains unclear. We developed Tracking Recently Activated Cell Kinetics (TRACK) mice, a dual-recombinase fate-mapping system enabling spatial and temporal labeling of recently activated CD4⁺ T cells. Using TRACK mice during influenza infection, we observed organ-specific transcriptional differentiation and clonal selection in lung, mediastinal lymph nodes (medLNs), and spleen. During the effector phase, spleen-derived CD4⁺ T cells adopted a stem-like migratory phenotype, whereas medLN-activated cells differentiated into T follicular helper cells. T cell receptor sequencing showed low clonal overlap between tissues during the effector response, consistent with distinct antigenic landscapes. During memory formation, overlap increased between lung- and medLN-derived cells, while splenic clones retained a distinct repertoire. These findings define tissue-dependent mechanisms that shape CD4⁺ T cell fate and clonal architecture.