Figure 4. Treg cells facilitate HFSC migration and differentiation during epidermal regeneration.

(a) Schematic showing DT and tamoxifen (Tam) administration in Lgr5-tdTom-FoxP3^DTR mice during skin barrier recovery for experiments described in panels b-f. Mice are compared to tamoxifen-labeled, non-DT treated littermates and harvested 4 days after skin barrier disruption.

(b) Representative images demonstrating the patterns of stem cell progeny (tdTomato⁺ cell) localization in the skin of tamoxifen-labeled, Lgr5 stem cell lineage tracing mice after injury. Mice are either sufficient (-DT) or depleted (+DT) of Treg cells. (c) Quantification of Lgr5-derived cell (tdTomato⁺) localization in the upper hair follicle (isthmus and infundibulum; left panel) and IFE (right panel). tdTomato⁺/ HF and tdTomato⁺/hpf represent cell numbers normalized per hair follicle and per high power field respectively. See also Supplementary Figure 4.

(d) FACS purified tdTom⁺ cells from DT-treated (i.e. Treg cell-depleted) or untreated Lgr5-tdTom-FoxP3^DTR (i.e. Treg cell-sufficient) mice were analyzed by RNA-sequencing. Comparison plots of normalized gene expression of tdTomato⁺ cells isolated from Treg cell-sufficient and Treg cell-depleted Lgr5-tdTom-FoxP3^DTR mice. Blue dots represent genes with a p-adjusted value < 0.05 and > 2-fold difference in gene expression between groups.

(e) Volcano plot comparing the p-adjusted value versus fold change for tdTomato⁺ cells isolated from Treg cell-sufficient and Treg cell-deficient Lgr5-tdTom-FoxP3^DTR mice. Blue dots represent genes with a p-adjusted value < 0.05 and > 2 fold differences in gene expression between groups.

(f) Heat map of gene transcripts of tdTomato⁺ cells isolated from Treg cell-sufficient and Treg cell-depleted Lgr5-tdTom-FoxP3^DTR mice focusing on differentially expressed genes with a padj. value < 0.05, fold change differences > 2 and predominant expression in either the bulge or IFE/upper hair follicle in adult mice.(Joost et al., 2016)

(g) Cntrl and FoxP3^DTR mice were treated as diagramed in Figure 1a and compared to uninjured WT mice. Representative plots of CD34⁺ cells in the epidermis 11 days after epidermal injury. Plots are pre-gated on live, CD45-negative epidermal cells.

(h) Percentage of CD34⁺ epidermal cells in uninjured cntrl mice compared to barrier-disrupted cntrl and Treg cell-depleted mice 5 and 11 days after injury. See also Supplementary Figure 5.

For all relevant panels, error bars are +/− SEM. *p < 0.05; **p < 0.01; **** p<0.0001 by Student’s t-test or by one-way ANOVA for panel h. Results in b-c are representative of > 8 independent experiments (n=2 to 4 mice per group); d-f are representative of 2 independent experiments (n=2 mice per group) Results of h (right panel) are pooled data from 3 independent experiments. (n=2–4 mice per group) and representative of 2 experiments (left panel). Scale bar in b is 100 µm; Red-tdTomato⁺ Lgr5 stem cell progeny; Blue – DAPI.