Tissue-resident memory CD4+ T cells are sustained by site-specific levels of self-renewal and continuous replacement.

Tissue-resident memory T cells (T_RM) protect from repeat infections within organs and barrier sites. The breadth and duration of such protection are defined at minimum by three quantities: the rate at which new T_RM are generated from precursors, their rate of self-renewal, and their rate of loss through death, egress, or differentiation. Quantifying these processes individually is challenging. Here we combine genetic fate mapping tools and mathematical models to untangle these basic homeostatic properties of CD4⁺ T_RM in the skin and gut lamina propria (LP) of healthy adult mice. We show that CD69⁺CD4⁺ T_RM in skin reside for ∼24 days and self-renew more slowly, such that clones halve in size approximately every 5 weeks, and approximately 2% of cells are replaced daily from precursors. CD69⁺CD4⁺ T_RM in LP have shorter residencies (∼14 days) and are maintained largely by immigration (4-6% per day). We also find evidence that the continuous replacement of CD69⁺CD4⁺ T_RM at both sites derives from circulating effector-memory CD4⁺ T cells, in skin possibly via a local CD9^- intermediate. Our approach maps the ontogeny of CD4⁺ T_RM in skin and LP and exposes their dynamic and distinct behaviours, with continuous seeding and erosion potentially impacting the duration of immunity at these sites.