Neonatal regulatory T cells persist into adulthood across multiple tissues with high enrichment in the skin

Abstract

Foxp3⁺ regulatory T cells (T_regs) reside in both lymphoid and nonlymphoid organs, where they play a crucial role in immune tolerance and tissue homeostasis. In mice, T_regs begin colonizing these tissues shortly after birth, contributing to long-term immune response regulation therein. However, the kinetics of T_reg generation across different tissues remains unclear. Here, we investigate T_reg ontogeny from birth to adulthood in various tissues. In lymphoid organs, the adult T_reg pool is continuously replenished with cells generated at different ages. In contrast, the skin retains a large fraction of T_regs that colonize the tissue during the neonatal period, with minimal turnover in adulthood. The liver, lungs, and colon exhibit intermediate T_reg renewal dynamics. Notably, neonatal T_regs that persist into adulthood display a more activated phenotype and express markers associated with tissue-resident T_regs and type 2 immunity. Our findings reveal tissue-specific differences in T_reg generation kinetics and highlight a major phenotypic shift between neonatal and adult-derived T_regs.

Fig. 1.. Dynamics of the generation of the T_reg pool in 8-week-old mice.

Foxp3^iCre R26^Tom mice were treated with tamoxifen either at week (W) 1 or 2 or 3 or 4 or 5 or 6 of life and were analyzed by flow cytometry at 8 weeks of age (A, C, and D) or 7 days after the beginning of tamoxifen treatment (B). (A) Experimental design showing the different mouse groups. (B) Proportion of TagT_regs among Foxp3⁺ cells at day 7 posttreatment from the spleen, LNs, lungs, liver, colon, and skin. The histogram represents the gating strategy used to discriminate unTagT_regs (Foxp3⁺ tdTomato⁻) and TagT_regs (Foxp3⁺ tdTomato⁺). (C) Proportion of TagT_regs among Foxp3⁺ cells in the indicated tissues in 8-week-old mice treated with tamoxifen at weeks 1 to 6 (graphs, each symbol represents one mouse). These values enabled the calculation of the proportion of T_regs generated at week 1 to week 6 among the T_reg pool of 8-week-old mice (10 × 10 dot plots). The pool of T_regs generated before 2 weeks of life was outlined, and their proportion was indicated. (D) To highlight differences between organs, the different graph curves (without symbols) shown in (C) were superimposed on one another in a single graph. Data were from multiple independent experiments with four mice per group (weeks 2, 3, and 4) or eight mice per group (weeks 1, 5, and 6).

Fig. 2.. Neonatal T_regs expressed more GATA3.

(A) Foxp3^iCre R26^Tom mice were treated with tamoxifen at weeks 1 to 6 and were all analyzed at 8 weeks of age as in Fig. 1. Proportion of TagT_regs among GATA3⁺, RORγt⁺, and GATA3⁻RORγt⁻ T_regs in the colon (graphs, each symbol represents one mouse). These values enabled the calculation of the proportion of T_regs generated at week 1 to week 6 among the three subtypes of colonic T_regs of 8-week-old mice (10 × 10 dot plots). The pool of T_regs generated before 2 weeks of life was outlined, and their proportion was indicated. (B) Foxp3^iCre R26^Tom mice were treated with tamoxifen at week 1 and analyzed at 8 weeks of age to compare the proportion of GATA3⁺ and RORγt⁺ among unTag and TagT_regs in the seven analyzed tissues. Representative dot plots from the colon are shown. Data were from multiple independent experiments with four mice per group (weeks 2, 3, and 4) or eight mice per group (weeks 1, 5, and 6) (B) mice per group. Statistical significance was calculated using a nonparametric Wilcoxon test. Each symbol represents one mouse, and the bars represent the medians. *P < 0.05 and **P < 0.01. ns, not significant.

Fig. 3.. T_regs generated during the first week of life that persist into adulthood exhibit an activated phenotype.

Foxp3^iCre R26^Tom mice were treated with tamoxifen at week 1 (A to C) or at weeks 1 to 6 (D) and analyzed by flow cytometry at 8 weeks of age to compare the expression of activation markers in unTagT_regs (blue) and TagT_regs (purple). (A) PCA showing unTag and TagT_regs clusters in the lungs. Activation markers are in red. PERMANOVA, permutational multivariate analysis of variance. (B and C) Proportion of the indicated activation markers among unTag and TagT_regs in the seven analyzed tissues (B) and in the lungs (C). Representative density plots and histogram are shown. MFI, mean fluorescence intensity. (D) Proportions of CD44 and CD62L among TagT_regs in mice treated with tamoxifen at week 1 and among unTagT_regs in mice treated at weeks 1 to 6 as in Fig. 1 and analyzed in the lungs at 8 weeks of age. Experimental design is shown in the left. D0, day 0. Data were from four to eight mice per group pooled from two independent experiments. Statistical significance was calculated using a nonparametric Wilcoxon test. Each symbol represents one mouse, and the bars represent the medians. *P < 0.05 and **P < 0.01.

Fig. 4.. T_regs generated during the first week of life that persist into adulthood express tissue T_regs markers.

Foxp3^iCre R26^Tom mice were treated with tamoxifen at week 1 (A to C) or at weeks 1 to 6 (D) and analyzed by flow cytometry at 8 weeks of age to compare the expression of tissue T_reg markers in unTagT_regs (blue) and TagT_regs (purple). (A) PCA showing unTag and TagT_reg clusters in the VAT and skin. Tissue T_reg markers (KLRG1 and ST2) are colored in red. (B) Representative density plots showing ST2 versus KLRG1 expression among unTag and TagT_regs in the VAT and skin. (C) Proportion of cells expressing ST2 or KLRG1 among the unTag and TagT_regs in the seven analyzed tissues. (D) Proportions of ST2⁺ or KLRG1⁺ cells among TagT_regs in mice treated with tamoxifen at week 1 and among unTagT_regs in mice treated at different ages as in Fig. 1 and analyzed in the VAT at 8 weeks of age. Experimental design is shown in the left. Data were from four to eight mice per group pooled from two independent experiments. (E) Gene set enrichment analysis (GSEA) was performed to analyze whether the transcriptome of splenic neonatal T_regs, compared to the one of adult T_regs (9), was enriched in a tissue T_reg signature (14) or a precursor T_reg signature (15, 17). FDR, false discovery rate; NES, normalized enrichment score. Statistical significance was calculated using a nonparametric Wilcoxon test. Each symbol represents one mouse, and the bars represent the medians. *P < 0.05 and **P < 0.01.

Fig. 5.. Persistence of neonatal T_regs in aged mice and T_reg renewal in adults.

(A and B) Foxp3^iCre R26^Tom mice were treated with tamoxifen at week 1 and analyzed at 8 or 28 weeks of age by flow cytometry. (A) Experimental design. (B) Proportion of TagT_regs among Foxp3⁺ cells in the spleen, LNs, lungs, liver, VAT, and skin (top) and in the GATA3⁻RORγt⁻, GATA3⁺, and RORγt⁺ T_reg populations of the colon (bottom). (C and D) Foxp3^iCre R26^Tom mice were treated with tamoxifen at 10 weeks of age and analyzed at 11, 16, 20, and 28 weeks of age by flow cytometry. (C) Experimental design. (D) Proportions of TagT_regs among Foxp3⁺ cells in the spleen, LNs, lungs, liver, VAT, skin (top), and in the whole, GATA3⁻RORγt⁻, GATA3⁺, and RORγt⁺ T_reg populations of the colon (bottom). Data were from five to eight mice per group pooled from two independent experiments. Statistical significance was calculated using a nonparametric Wilcoxon test. Each symbol represents one mouse, and the bars represent the medians. **P < 0.01 and ***P < 0.001.

Fig. 6.. Proliferation and residency of neonatal skin T_regs.

(A) Proportion Ki67⁺ cells among T_regs or Tconvs in the spleen, LNs, lungs, liver, colon, adipose tissue (AT), and skin in 11- to 45-day-old mice. Top left: Representative density plots in the skin; shown values are the proportion of Ki67⁺ cells among T_reg or Tconv. (B) HO-Tam was applied locally on the skin of 3-week-old Foxp3^iCre R26^Tom mice, and the proportion of TagT_regs was analyzed 1 or 5 weeks later. Experimental approach (top), representative density plots (bottom left), and proportion of TagT_regs among Foxp3⁺ cells in the skin and LNs (bottom right). (C and D) Foxp3^iCre R26^Tom mice were treated with tamoxifen at 1 to 2 weeks of age, and the phenotype of TagT_regs was analyzed at 3, 8, or 28 weeks of age by flow cytometry. (C) PCA depicting the evolution of neonatal T_reg phenotype. (D) Representative density plots (top) and proportions (bottom) of CD62L, CD44, KLRG1, ST2, Ki67, and CD69 expression among neonatal T_regs analyzed at 3 (black), 8 (pink), or 28 (green) weeks of age. Data were from two (A and C) or three (B) independent experiments. Statistical significance was calculated using a nonparametric Wilcoxon test. Each symbol represents one mouse, and the bars represent the medians. *P < 0.05.