Overview of the fate-reporter approach to quantifying the homeostasis of skin- and LP-resident CD4+ T cells.

A Ki67 and CD4 reporter constructs. B Schematic of a simple mathematical model of TRM homeostasis at steady state. New cells enter a TRM subset in skin or LP (the ‘target’ population) from a precursor population at rate θ (cells per unit time). YFP and mTom expression among immigrant cells is assumed to be that of the precursor population in bulk. We considered three possibilities for the Ki67 expression levels among new immigrants; a ‘quiescent’ mode (all Ki67low), ‘neutral’ recruitment (Ki67high frequency identical to that of the precursor). or ‘division-linked’ (all recruited cells Ki67high). The target cells are assumed to be a kinetically homogeneous population that self-renews at average rate ρ, such that their mean interdivision time is 1/ρ. Cells are also lost through death, differentiation or tissue egress at rate δ, implying a mean residence time of 1/δ. The combination of these processes determines the timecourses of frequencies of YFP+ and mTom+ cells with the target population. C Experimental design.

Modelling fate reporter dynamics among CD4+ T cell subsets in skin and lamina propria.

A,B Numbers of CD4+ effector-memory (EM) phenotype and CD4+CD69+T cells recovered from (A) ear skin, and (B) lamina propria in the small intestine, against mouse age. P-values are derived from Spearman rank correlation on data from the Cd4-FR and Ki67-DIVN strains combined. C Observed (points) and best-fit model trajectories (black lines) of the frequencies of mTom+ and YFP+ CD4+ T cell subsets within skin and LP, and the proportions of YFP+ and YFP− cells that expressed Ki67, with time since tamoxifen treatment. D Frequencies of mTom+ and YFP+ cells among lymph node CD4+ T cell subsets, and within CD4+CD44+CD69− T cells in skin and LP. Black lines show the best empirical fitted descriptions of these trajectories (Text S1). Shaded areas indicate the 5-day period over which reporter expression was being induced; data from these periods were not used in the fitting.

Parameters governing the homeostasis of antigen-experienced CD4+ T cells localised within skin and lamina propria (LP) in adult mice.

Violin plots indicate the posterior distributions of parameters. Black points and bars; best (maximum a posteriori) estimates and 95% credible intervals. For each population (target) in skin or LP, potential precursors were; from lymph nodes, CD4+ naive, central memory and effector memory T cells (N, EM and CM); and for CD4+ TRM, CD69+ TRM within the same tissue. For each precursor/target pair, we considered three potential levels of Ki67 expression on immigrant cells (L-R; orange, red, and blue violin plots). ‘High’, the division-linked model (new cells enter as Ki67high); ‘Int’, neutral model (Ki67 expression among new cells mirrors that of the precursor); and ‘Low’, the quiescent model (new cells enter as Ki67low). Shaded regions highlight the parameter estimates derived from the favoured model (precursor and Ki67 levels on immigration) for each target (Fig. 4A).

Candidate ontogenic pathways of CD4+ CD69+ TRM in skin and lamina propria.

A For each target population with skin and LP (CD4+ EM bulk, and CD4+CD69+ T cells), we calculated the relative support for alternative pathways and modes of recruitment, using LOO-IC weights (Methods). For potential precursors, N, CM, EM refer to naive, TCM and TEM in lymph nodes, and we also considered CD69− cells within each tissue. As described in the text, for each precursor, there were three potential levels of Ki67 on newly recruited cells. Model weights sum to 1 for each target population, with the width of each arrow reflecting the degree of support. B Schematics of the most strongly supported pathways of development of CD4+CD69+ TRM in skin and LP, with approximate values of key kinetic parameters (Fig. 3 and Table S1).