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

Morgane Hilaire, Angélina Mimoun, Léonie Cagnet, Rémy Villette, Aristeidis

Roubanis, Hugo Sentenac, Benoît L Salomon

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Morgane Hilaire, Angélina Mimoun, Léonie Cagnet, Rémy Villette, Aristeidis Roubanis, et al.. Neonatal regulatory T cells persist into adulthood across multiple tissues with high enrichment in the skin. Science Advances, 2025, 11 (40), pp.eadx8037. 10.1126/sciadv.adx8037. hal-05369671

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IMMUNOLOGY

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

Morgane Hilaire<fnref n="1" />,²*, Angélina Mimoun², Léonie Cagnet<fnref n="1" />,², Rémy Villette<fnref n="1" />, Aristeidis Roubanis<fnref n="1" />, Hugo Sentenac², Benoît L. Salomon<fnref n="1" />,²*

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

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INTRODUCTION

Mice are born lymphopenic, and T cells begin to emerge from the
thymus and progressively colonize peripheral tissues only after birth.
Mice are thus an ideal model for studying T cell ontogeny and the
progressive establishment of immune homeostasis throughout the
first few weeks of life. During this critical window, the immune sys-
tem must rapidly establish effective defenses against pathogens while
simultaneously promoting tolerance to self-antigens, the microbiota,
and food antigens.

Neonatal and adult T cell immunity differs quite remarkably (1). Hallmark features of neonates include lymphopenia-induced proliferation, a T helper cell 2 (T<sub>H2</sub>)-skewed bias, and a T cell receptor repertoire that is both restricted and broadly cross-reactive. This period is also highly permissive for the generation of Foxp3<sup>+</sup> regulatory T cells (T<sub>regs</sub>), which arise from both thymic and peripheral origins (2, 3). Studies have explored the kinetics of T<sub>reg</sub> colonization in various tissues, revealing distinct spatial and temporal patterns. In secondary lymphoid organs such as the spleen and lymph nodes (LNs), T<sub>regs</sub> of neonates are present in low proportions (4, 5). Higher T<sub>reg</sub> proportions are observed in the liver and lungs, where they peak at 15 to 30% of CD4<sup>+</sup> T cells 2 weeks after birth (4–6). In the liver, early T<sub>reg</sub> colonization limits local activation of conventional T cells (Tconv5) (6). Notably, the skin undergoes a particularly high level of T<sub>reg</sub> accumulation, with these cells constituting up to 80% of CD4<sup>+</sup> T cells by 1 to 2 weeks of age, followed by a gradual decline (5, 7, 8).

A subset of T<sub>regs</sub> generated in neonates (neonatal T<sub>regs</sub>) persists in the adult spleen, where they exhibit higher activation, superior suppressive capacity, and an autoreactive-biased repertoire compared to those generated in adulthood (9). Neonatal T<sub>regs</sub> play an essential role in preventing autoimmunity in adulthood (9, 10). In the skin,

neonatal T<sub>regs</sub> establish long-term tolerance to commensal microbes and dampen local T<sub>H2</sub> responses in adults (7, 11). However, whether their sustained immunoregulatory effects are due to their prolonged persistence or the induction of infectious tolerance remains unknown.

Despite their critical role in immune homeostasis, the precise ontogeny and generation kinetics of the adult T<sub>reg</sub> pool across tissues remains undetermined. While it has been shown that neonatal T<sub>regs</sub> persist in the spleen, their proportion in the adult T<sub>reg</sub> compartment remains poorly quantified (9). Here, we addressed these questions by exploiting a fate-mapping system to track T<sub>reg</sub> generation across different developmental stages and tissues. We demonstrate that, in lymphoid organs, the adult T<sub>reg</sub> pool is continuously replenished throughout life. In contrast, most T<sub>regs</sub> in adult skin originate from cells generated during the neonatal period, with early-life colonization shaping the adult T<sub>reg</sub> pool. Furthermore, neonatal T<sub>regs</sub> exhibit a distinct phenotypic signature, characterized by elevated expression of activation markers and molecules associated with tissue residency and type 2 immunity.

RESULTS

Experimental approach

To investigate T<sub>reg</sub> ontogeny, we used a fate-mapping strategy by crossing *Foxp3<sup>tm9(EGFP/cre/ERT2)</sup>A<sub>yr</sub>/J (*Foxp3<sup>iCre</sup>) mice with B6.Cg-Gt(ROSA)26Sor<sup>tm9(CAG-tdTomato)Hze1</sup> (*R26<sup>Tom</sup>) mice. In this system, the *Foxp3<sup>iCre</sup> construct ensures T<sub>reg</sub>-specific expression of the *Cre-ERT2* recombinase, which remains cytoplasmic until tamoxifen administration triggers its nuclear translocation. The *R26<sup>Tom</sup>* allele contains a loxP-flanked STOP cassette upstream of the *tdTomato* reporter. Upon tamoxifen treatment, Cre-mediated recombination excises the STOP cassette, leading to stable tdTomato expression in existing T<sub>regs</sub>, termed TagT<sub>regs</sub>. T<sub>regs</sub> arising posttreatment, which lack tdTomato expression, are designated as unTagT<sub>regs</sub>. To determine the generation kinetics of the adult T<sub>reg</sub> pool, we administered tamoxifen at various ages from birth to 6 weeks. All mice were analyzed at 8 weeks across multiple tissues, including the spleen, LNs, lungs, liver, visceral adipose tissue (VAT), colon, and skin (Fig. 1A).

To validate this approach, we confirmed specificity of tdTomato
labeling to T<sub>regs</sub> upon tamoxifen induction (fig. S1A). In addition,

<footnote n="1">¹Sorbonne Université, INSERM U1135, CNRS, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), F-75013, Paris, France. ²Toulouse Institute for Infectious and Inflammatory Diseases (Infinity), INSERM UMR1291-CNRS UMR5051-University Toulouse III, Toulouse, France.
*Corresponding author. Email: benoit.salomon@inserm.fr (B.L.S.); hilaire.morgane77@gmail.com (M.H.)</footnote>
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<figure><img src="image_7.png" /><figcaption>**Fig. 1.** Dynamics of the generation of the T<sub>reg</sub> pool in 8-week-old mice. Foxp3<sup>Ire</sup> R26<sup>Tom</sup> 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<sub>regs</sub> among Foxp3<sup>+</sup> cells at day 7 posttreatment from the spleen, LNs, lungs, liver, colon, and skin. The histogram represents the gating strategy used to discriminate unTagT<sub>regs</sub> (Foxp3<sup>+</sup> tdTomato<sup>-</sup>) and TagT<sub>regs</sub> (Foxp3<sup>+</sup> tdTomato<sup>+</sup>). (C) Proportion of TagT<sub>regs</sub> among Foxp3<sup>+</sup> 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<sub>regs</sub> generated at week 1 to week 6 among the T<sub>reg</sub> pool of 8-week-old mice (10 × 10 dot plots). The pool of T<sub>regs</sub> 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).</figcaption></figure>
we assessed the efficiency of tdTomato $T_{reg}$-tagging across tissues by analyzing mice 7 days post-tamoxifen administration, which represents the optimal timing (fig. S1B). The proportion of Tag$T_{regs}$ was consistently high, more than 90% in most conditions and nearly 100% in the skin (Fig. 1B). Last, we evaluated the duration of tamoxifen activity by transferring $T_{regs}$ from *Foxp3*<sup>iCre</sup> R26<sup>Tom</sup> mice into $T_{reg}$-depleted recipients (fig. S1C). Analysis revealed peak recombination activity 1 to 5 days post-tamoxifen injection, with no more activity after 8 days (fig. S1D). Thus, our system robustly labels $T_{regs}$ while ensuring transient tamoxifen exposure.

## Tissue-specific kinetics of $T_{reg}$ pool generation

To determine the temporal dynamics of $T_{reg}$ generation, *Foxp3*<sup>iCre</sup> R26<sup>Tom</sup> mice were treated with tamoxifen during the first to the sixth weeks of life, and $T_{regs}$ were analyzed at 8 weeks across seven tissues (Fig. 1A). This protocol enabled retrospective investigation of the ontogeny of $T_{regs}$ from birth to 8 weeks of age. Tamoxifen treatment during the first week of life marked $T_{regs}$ generated at week 1. Treatment during the second week of life marked $T_{regs}$ generated at weeks 1 and 2. By difference, we could then calculate the proportion of $T_{regs}$ generated at week 2. Similarly, treatments carried out the third, fourth, fifth, and sixth weeks of life identified $T_{regs}$ generated in weeks 3, 4, 5, and 6, respectively (Fig. 1A). We represented the composition of the $T_{reg}$ pool of 8-week-old mice in two ways: with graphs showing the progressive accumulation of newly generated $T_{regs}$ (Fig. 1C, left) and with 10 × 10 dot plots depicting the composition of the $T_{reg}$ pool according to their generation stage (Fig 1C, right). In lymphoid tissues (spleen and LNs), newly generated $T_{regs}$ accumulated gradually. Approximately 25% of LN $T_{regs}$ and 35% of spleen $T_{regs}$ were generated by week 2, rising to 50 and >60% by week 4, respectively (Fig. 1C). Nonlymphoid tissues exhibited distinct kinetics. In the lungs, $T_{regs}$ accumulated rapidly, with 50% generated by week 2. In liver and colon, $T_{reg}$ generation increased sharply, with ~70% Tag$T_{regs}$ by week 4. $T_{regs}$ in VAT and skin exhibited the most pronounced neonatal bias; 75% of VAT $T_{regs}$ and 85% of skin $T_{regs}$ were generated by week 2, with 60% of skin $T_{regs}$ arising within the first week of life (Fig. 1C). The comparison of $T_{reg}$ pool generation kinetics between tissues is highlighted in Fig. 1D. Collectively, these findings highlight tissue-specific differences in $T_{reg}$ ontogeny: Lymphoid tissues maintain continuous $T_{reg}$ renewal, whereas VAT and skin predominantly establish their $T_{reg}$ pool during early life.

To determine whether $T_{reg}$ ontogeny correlates with tissue colonization, we analyzed the proportion of $T_{regs}$ among CD4⁺ T cells across ages (fig. S2). In lymphoid tissues, $T_{reg}$ proportions remained relatively stable (10 to 18%) between 1.5 and 6.5 weeks of age. In the liver, an early peak (30% at 1.5 weeks) was followed by stabilization at 15 to 18%. Lungs exhibited a similar pattern but with a milder early peak. In skin, $T_{regs}$ constituted 70 to 80% of CD4⁺ T cells between 1.5 to 3.5 weeks of age, dropping to 20 to 30% after 4.5 weeks. This kinetics align with our $T_{reg}$ ontogeny data, suggesting that kinetics of tissue $T_{reg}$ colonization dictates cell composition.

## Neonatal $T_{regs}$ persisting in adults preferentially express GATA3

$T_{regs}$ in the colon comprise Helios/GATA3⁺, RORγt⁺, and double-negative (GATA3⁻RORγt⁻) subsets (12). Neonatal $T_{regs}$ preferentially differentiate into GATA3⁺ $T_{regs}$, while adult-derived $T_{regs}$ exhibit a bias toward RORγt⁺ differentiation (13). Using our fate-mapping approach, we assessed whether neonatal $T_{regs}$ would persist into

adulthood and retain their identity. At 8 weeks, more than 60% of GATA3⁺ $T_{regs}$ were generated before 2 weeks of age, whereas only 12% of RORγt⁺ $T_{regs}$ originated from this early life period (Fig. 2A). The development kinetics of the double-negative $T_{reg}$ pool was comparable to GATA3⁺ $T_{regs}$ (Fig. 2A). Beyond the colon, GATA3⁺ $T_{regs}$ constitute 20 to 60% of the $T_{reg}$ pools in various lymphoid and nonlymphoid tissues in adults. In contrast, RORγt⁺ $T_{regs}$ remain largely restricted to the colon, where they represent 50% of the $T_{reg}$ population (fig. S3A). We thus investigated whether the preferential expression of GATA3 by neonatal $T_{regs}$ observed in the colon would be also present in other tissues. This trend was consistent across all tissues except the skin, where neonatal Tag$T_{regs}$ were significantly enriched for GATA3 expression compared to unTag$T_{regs}$ (Fig. 2B). Conversely, RORγt expression was lower in Tag$T_{regs}$ across all tissues where it was detectable. We also identified a poorly described population of GATA3⁺RORγt⁺ $T_{regs}$. They represented less than 10% of $T_{regs}$ in the skin and colon and were almost undetectable in other tissues. As for RORγt⁺ $T_{regs}$, their proportion was increased in unTag compared to Tag$T_{regs}$ (fig. S3B). These data indicate that neonatal $T_{regs}$ preferentially adopt a GATA3⁺ fate, which persists into adulthood, shaping tissue specific $T_{reg}$ phenotypes.

## Neonatal $T_{regs}$ persisting in adults exhibit an activated phenotype

To further characterize neonatal $T_{regs}$, we performed spectral flow cytometry using a 25-marker panel optimized for $T_{reg}$ subset identification and functional profiling. Principal components analysis (PCA) revealed tissue-specific $T_{reg}$ signatures, with skin and colon $T_{regs}$ clustering separately due to differential expression of RORγt, CCR2, CD44, and CD69 (fig. S4A). We then treated *Foxp3*<sup>iCre</sup> R26<sup>Tom</sup> mice with tamoxifen at week 1. For each of the seven analyzed tissues, unTag and Tag$T_{regs}$ clustered separately, exhibiting different phenotypes (fig. S4B). Neonatal Tag$T_{regs}$ exhibited an activated phenotype across all analyzed tissues. Specifically, in spleen, LNs, liver, and lungs, Tag$T_{regs}$ preferentially expressed CD44, CD69, CD73, CTLA4, ICOS, PD1, and TNFR2, whereas unTag$T_{regs}$ were enriched for CD62L, Bcl2, and TCF1, markers associated with quiescence (Fig. 3, A to C, and fig. S5, A to C). A similar but less pronounced trend was observed in colon, VAT, and skin, where most $T_{regs}$ exhibited an activated phenotype (Fig. 3B). We also compared CD44 and CD62L expression in Tag$T_{regs}$ generated at week 1 with unTag$T_{regs}$ generated at weeks 2 to 6 in the lungs. Again, neonatal $T_{regs}$ had a more activated phenotype than $T_{regs}$ generated later on (Fig. 3D). Similar findings were obtained with Bcl2, PD1, and CTLA4 (fig. S5D). Our data show that neonatal $T_{regs}$ that persist into adulthood exhibit an activated phenotype in various lymphoid and nonlymphoid tissues.

## Neonatal $T_{regs}$ persisting in adults express tissue $T_{reg}$ markers

We then assessed whether unTag and Tag$T_{regs}$ differently express markers of tissue $T_{regs}$. KLRG1, ST2, and CCR2 are well-described tissue $T_{reg}$ markers (14), and we confirm here their preferential expression in nonlymphoid tissue $T_{regs}$ (fig. S3C). In lymphoid tissues, they have been described as markers of tissue $T_{reg}$ precursors (15, 16). In *Foxp3*<sup>iCre</sup> R26<sup>Tom</sup> mice treated with tamoxifen at week 1, Tag and unTag$T_{regs}$ of VAT and skin have distinct phenotype with preferential expression of KLRG1, ST2, and CCR2 in neonatal $T_{regs}$ (Fig. 4A). In the VAT, colon, and skin, 60 to 80% of Tag$T_{regs}$ expressed these markers versus only 10 to 30% for unTag$T_{regs}$ (except 50% for ST2 in the skin) (Fig. 4, B to D). Although attenuated, the same tendency
<figure><img src="image_8.png" /><figcaption>**Fig. 2. Neonatal T<sub>regs</sub> expressed more GATA3.** (A) *Foxp3<sup>ICre</sup> R26<sup>Tom</sup>* 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<sub>regs</sub> among GATA3<sup>+</sup>, RORγt<sup>+</sup>, and GATA3<sup>-</sup>RORγt<sup>-</sup> T<sub>regs</sub> in the colon (graphs, each symbol represents one mouse). These values enabled the calculation of the proportion of T<sub>regs</sub> generated at week 1 to week 6 among the three subtypes of colonic T<sub>regs</sub> of 8-week-old mice (10 × 10 dot plots). The pool of T<sub>regs</sub> generated before 2 weeks of life was outlined, and their proportion was indicated. (B) *Foxp3<sup>ICre</sup> R26<sup>Tom</sup>* mice were treated with tamoxifen at week 1 and analyzed at 8 weeks of age to compare the proportion of GATA3<sup>+</sup> and RORγt<sup>+</sup> among unTag and TagT<sub>regs</sub> 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.</figcaption></figure>

was observed in other tissues. In the liver and lungs, 30 to 60% of TagT<sub>regs</sub> expressed KLRG1 and ST2, values that dropped to 10 to 20% for unTagT<sub>regs</sub>. In the spleen and LNs, 10 to 20% of TagT<sub>regs</sub> and less than 5% of unTagT<sub>regs</sub> expressed these two markers (Fig. 4C). CCR2 expression was also higher in TagT<sub>regs</sub> generated at week 1 compared to T<sub>regs</sub> generated later on in most tissues (fig. S6). These data show that, in 8-week-old mice, neonatal T<sub>regs</sub> expressed tissue T<sub>reg</sub> markers at higher levels compared to T<sub>regs</sub> generated later in life. To confirm these observations, we investigated whether neonatal T<sub>regs</sub> were enriched for a tissue T<sub>reg</sub> signature using published transcriptomic data. We analyzed differential transcriptomic data comparing neonatal and adult splenic T<sub>regs</sub> (9), alongside a tissue T<sub>reg</sub> signature established by comparing skin and VAT T<sub>regs</sub> to LN T<sub>regs</sub> in

adult mice (14). This analysis revealed a strong enrichment of the tissue T<sub>reg</sub> signature in neonatal T<sub>regs</sub> compared to adult T<sub>regs</sub> (Fig. 4E). Since tissue T<sub>reg</sub> precursors have been identified in secondary lymphoid organs (15, 17), we also examined whether neonatal T<sub>regs</sub> expressed a signature enriched for these precursors. Again, we observed a strong enrichment of tissue T<sub>reg</sub> precursor signatures in neonatal T<sub>regs</sub> compared to adult T<sub>regs</sub> (Fig. 4E). Thus, neonatal T<sub>regs</sub> preferentially acquire tissue-resident characteristics and signatures and persist long-term in nonlymphoid compartments.

## T<sub>reg</sub> turnover in aging mice

To assess whether neonatal T<sub>regs</sub> persist beyond young adulthood, we examined the proportion of TagT<sub>regs</sub> in 8-week-old and 28-week-old
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** *T<sub>regs</sub>* generated during the first week of life that persist into adulthood exhibit an activated phenotype. *Foxp3<sup>ICre R26Tom</sup>* 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<sub>regs</sub> (blue) and TagT<sub>regs</sub> (purple). (A) PCA showing unTag and TagT<sub>regs</sub> 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<sub>regs</sub> 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<sub>regs</sub> in mice treated with tamoxifen at week 1 and among unTagT<sub>regs</sub> 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.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** *T<sub>regs</sub>* generated during the first week of life that persist into adulthood express tissue *T<sub>regs</sub>* markers. *Foxp3<sup>ICre</sup> R26<sup>Tom</sup>* 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<sub>reg</sub>* markers in unTag*T<sub>regs</sub>* (blue) and Tag*T<sub>regs</sub>* (purple). (A) PCA showing unTag and Tag*T<sub>reg</sub>* clusters in the VAT and skin. Tissue *T<sub>reg</sub>* markers (KLRG1 and ST2) are colored in red. (B) Representative density plots showing ST2 versus KLRG1 expression among unTag and Tag*T<sub>regs</sub>* in the VAT and skin. (C) Proportion of cells expressing ST2 or KLRG1 among the unTag and Tag*T<sub>regs</sub>* in the seven analyzed tissues. (D) Proportions of ST2+ or KLRG1+ cells among Tag*T<sub>regs</sub>* in mice treated with tamoxifen at week 1 and among unTag*T<sub>regs</sub>* 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<sub>regs</sub>* compared to the one of adult *T<sub>regs</sub>* (9), was enriched in a tissue *T<sub>reg</sub>* signature (14) or a precursor *T<sub>reg</sub>* 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.</figcaption></figure>
Foxp3<sup>iCre</sup> R26<sup>Tom</sup> mice that had been labeled at week 1 (Fig. 5A). In spleen, LNs, lungs, and liver, neonatal T<sub>regs</sub> constituted 10 to 15% of the T<sub>reg</sub> pool at both ages. In contrast, in the skin, neonatal T<sub>regs</sub> represented 60% at 8 weeks and still 35% at 28 weeks. In the VAT, neonatal T<sub>regs</sub> dropped from 35 to 40% to 10% between 8 and 28 weeks (Fig. 5B). In the colon, the proportion of neonatal T<sub>regs</sub> decreased among the GATA3<sup>+</sup> T<sub>reg</sub> subset and increased among the RORγt<sup>+</sup> T<sub>reg</sub> subset as the mice aged (Fig. 5B). To assess T<sub>reg</sub> renewal in adults, we then treated Foxp3<sup>iCre</sup> R26<sup>Tom</sup> mice with tamoxifen at 10 weeks and analyzed persistence of TagT<sub>regs</sub> at 11, 16, 20, and 28 weeks (Fig. 5C). In lymphoid tissues, lungs, liver, and RORγt<sup>+</sup> colonic T<sub>regs</sub>, we observed a gradual decrease of TagT<sub>regs</sub> as the mice aged. In contrast, the proportion of TagT<sub>regs</sub> stayed at a high level among VAT, skin, and GATA3<sup>+</sup> colonic T<sub>regs</sub> (Fig. 5D). These findings reinforce a model where some tissue T<sub>reg</sub> niches are stably occupied by neonatal-derived cells, while others undergo continuous renewal throughout life.

## High proliferation of neonatal T<sub>regs</sub> and residency of skin T<sub>regs</sub>

Our findings demonstrate that in certain tissues, such as the skin and VAT, T<sub>regs</sub> generated during the neonatal period persist long-term and constitute a substantial fraction of the adult T<sub>reg</sub> pool. To further investigate the biological properties of neonatal skin T<sub>regs</sub>, we analyzed their proliferation, colonization, and phenotypic evolution. While it is well known that T cells proliferate more during the neonatal period than in adulthood, the specific dynamics of T<sub>regs</sub> compared to Tconvs remain less understood. To address this, we

compared the proliferation of T<sub>regs</sub> and Tconvs in lymphoid and nonlymphoid tissues during the first 2 months of life (Fig. 6A). In nonlymphoid tissues (including the skin, VAT, lungs, liver, and colon) as well as in LNs, the proportion of Ki67<sup>+</sup> T<sub>regs</sub> reached 80% or higher in most mice at 11 days of age. However, this proportion declined sharply in the skin, dropping to ~20% by 3 weeks of age (Fig. 6A). Other tissues exhibited similar declines, although at a more gradual pace, with Ki67<sup>+</sup> T<sub>regs</sub> reaching 20% by 6 to 8 weeks of age. In contrast, Tconvs proliferation followed a different trajectory: At 11 days of age, only 40% of Tconvs were Ki67<sup>+</sup> across most tissues, and this proportion remained relatively stable or decreased only slightly in older mice. In the spleen, Ki67 expression was initially comparable between T<sub>regs</sub> and Tconvs at 11 days of age, but after this stage, T<sub>regs</sub> exhibited higher proliferation rates than Tconvs (Fig. 6A). To confirm the high proliferation rate of neonatal T<sub>regs</sub> using a more direct technique, we measured incorporation of 5-ethynyl-2'-deoxyuridine (EdU) that was administered in vivo in 1-week-old- and 3-week-old mice. In both ages, T<sub>regs</sub> proliferated substantially more than Tconvs in lymphoid and nonlymphoid tissues (fig. S7). T<sub>reg</sub> proliferation was higher in 1-week-old mice compared to 3-week-old mice in the skin, confirming data obtained with the Ki67 staining. Collectively, these data reveal that neonatal T<sub>regs</sub> proliferate notably more than Tconvs across all analyzed tissues before declining with age, particularly in the skin.

Our results reveal that by 8 weeks of age, 85% of the skin T<sub>reg</sub> pool originates from T<sub>regs</sub> generated before 2 to 3 weeks of age. However, whether these cells had already colonized the skin at the time of tamoxifen treatment remained unknown. Alternatively, TagT<sub>regs</sub>

<figure><img src="image_11.png" /><figcaption>**Fig. 5. Persistence of neonatal T<sub>regs</sub> in aged mice and T<sub>reg</sub> renewal in adults.** (A and B) Foxp3<sup>iCre</sup> R26<sup>Tom</sup> 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<sub>regs</sub> among Foxp3<sup>+</sup> cells in the spleen, LNs, lungs, liver, VAT, and skin (top) and in the GATA3<sup>-</sup>RORγt<sup>-</sup>, GATA3<sup>+</sup>, and RORγt<sup>+</sup> T<sub>reg</sub> populations of the colon (bottom). (C and D) Foxp3<sup>iCre</sup> R26<sup>Tom</sup> 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<sub>regs</sub> among Foxp3<sup>+</sup> cells in the spleen, LNs, lungs, liver, VAT, skin (top), and in the whole, GATA3<sup>-</sup>RORγt<sup>-</sup>, GATA3<sup>+</sup>, and RORγt<sup>+</sup> T<sub>reg</sub> 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.</figcaption></figure>
A

<figure><img src="image_12.png" /></figure>

B

<figure><img src="image_13.png" /></figure>

C

<figure><img src="image_14.png" /></figure>

D

<figure><img src="image_15.png" /><figcaption>**Fig. 6.** Proliferation and residency of neonatal skin T<sub>regs</sub>. (A) Proportion Ki67<sup>+</sup> cells among T<sub>regs</sub> or Tconv 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<sup>+</sup> cells among T<sub>reg</sub> or Tconv. (B) HO-Tam was applied locally on the skin of 3-week-old Foxp3<sup>ICre</sup> R26<sup>Tom</sup> mice, and the proportion of TagT<sub>regs</sub> was analyzed 1 or 5 weeks later. Experimental approach (top), representative density plots (bottom left), and proportion of TagT<sub>regs</sub> among Foxp3<sup>+</sup> cells in the skin and LNs (bottom right). (C and D) Foxp3<sup>ICre</sup> R26<sup>Tom</sup> mice were treated with tamoxifen at 1 to 2 weeks of age, and the phenotype of TagT<sub>regs</sub> was analyzed at 3, 8, or 28 weeks of age by flow cytometry. (E) PCA depicting the evolution of neonatal T<sub>reg</sub> phenotype. (D) Representative density plots (top) and proportions (bottom) of CD62L<sup>+</sup>, CD44<sup>+</sup>, KLRG1<sup>+</sup>, ST2<sup>+</sup>, Ki67<sup>+</sup>, and CD69<sup>+</sup> expression among neonatal T<sub>regs</sub> 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.</figcaption></figure>
may derive from precursors tagged in lymphoid tissues that later migrated to the skin or from T<sub>regs</sub> tagged in other tissues that subsequently recirculated to the skin. To address this question, we applied 4-hydroxytamoxifen (HO-Tam) topically to the skin of 3-week-old *Foxp3<sup>iCre</sup> R26<sup>Tom</sup> mice (Fig. 6B). One week posttreatment, ~50% of skin T<sub>regs</sub> were tagged, whereas T<sub>regs</sub> in other tissues (draining LNs, spleen, and colon) remained untagged, confirming that labeling was restricted to the treated skin. To assess the long-term residency of these TagT<sub>regs</sub>, we quantified their proportion 5 weeks after tamoxifen application (Fig. 6B). In 8-week-old mice, TagT<sub>regs</sub> still accounted for 30% of the total skin T<sub>reg</sub> population, indicating that more than half of the skin T<sub>regs</sub> present at 3 weeks persist into adulthood.

Last, we examined the phenotypic evolution of neonatal skin T<sub>regs</sub>
that persist in adulthood, comparing them to T<sub>regs</sub> from 3-week-old
mice. Using an optimized panel of 25 antibodies designed for T<sub>reg</sub>
subpopulation analysis, we found that neonatal skin T<sub>regs</sub> that per-
sist at 8 and even 28 weeks of age maintain a phenotype largely sim-
ilar to that of 3-week-old skin T<sub>regs</sub> (Fig. 6, C and D). In contrast, a
parallel analysis of LN T<sub>regs</sub> revealed a distinct pattern: Neonatal LN
T<sub>regs</sub> that persisted into adulthood exhibited substantial phenotypic
changes compared to their 3-week-old counterparts (Fig. 6C and
fig. S8). Collectively, these findings indicate that most of adult skin
T<sub>regs</sub> have already colonized the skin by 3 weeks of age and that their
phenotype remains stable over time.

DISCUSSION

Our study provides a comprehensive analysis of the kinetics of T<sub>reg</sub>
generation across various lymphoid and nonlymphoid tissues in
adult mice. We observed major differences between tissues. In lym-
phoid organs, the T<sub>reg</sub> pool is progressively constituted and continu-
ously replenished, whereas in certain nonlymphoid tissues, such as
the skin, most of the T<sub>reg</sub> pool is established during the neonatal
period and persists long-term.

In the spleen and LNs, our data show that ~10% of the adult T<sub>reg</sub>
pool is derived from cells generated during the first week of life,
which are stably maintained for up to 28 weeks. The remaining pop-
ulation is progressively replaced by newly generated T<sub>regs</sub> over time.
The continuous renewal of T<sub>regs</sub> in lymphoid organs is further sup-
ported by the gradual decline in TagT<sub>regs</sub> observed in mice treated
with tamoxifen at 10 weeks of age, suggesting ongoing replenish-
ment as mice age.

The liver and lungs exhibit a distinct pattern of T<sub>reg</sub> accumula-
tion. While these tissues also undergo progressive replenishment, a
higher fraction (40 to 50%) of the adult T<sub>reg</sub> pool is generated before
2 weeks of age, compared to 25 to 35% in lymphoid organs. This
early T<sub>reg</sub> colonization may be linked to a peak in T<sub>reg</sub> proportion
between 2 and 3 weeks of age, as previously described (4, 6, 15), a
phenomenon we confirm here. In the liver, early T<sub>reg</sub> colonization is
known to result from thymic output during the first week of life, fol-
lowed by extensive in situ expansion to regulate Tconv activation (6).

The VAT exhibits an early T<sub>reg</sub> generation, an intriguing finding
given that this tissue is minimally developed in neonates. The loca-
tion of the neonatal VAT T<sub>reg</sub> pool remains uncertain, but we pro-
pose three nonmutually exclusive hypotheses: (i) T<sub>regs</sub> may reside in
lymphoid organs during early life before migrating to VAT, as previ-
ous studies have localized VAT T<sub>reg</sub> precursors in the spleen and LNs
of young mice (15, 16); (ii) a small number of T<sub>regs</sub> may initially
colonize the nascent VAT, undergoing high in situ expansion as the

tissue matures, a hypothesis compatible with our data showing a
high rate of VAT T<sub>reg</sub> proliferation in the first 3 weeks of life; (iii)
neonatal T<sub>regs</sub> may first reside in other adipose depots before later
recirculating to the VAT. By 8 weeks of age, T<sub>regs</sub> generated in the
first week of life constitute 35% of the VAT T<sub>reg</sub> pool, but this de-
clines to 10% by 28 weeks. However, in 28-week-old mice treated
with tamoxifen at 10 weeks, TagT<sub>regs</sub> still account for 80% of VAT
T<sub>regs</sub>, indicating a slow renewal between 10 and 28 weeks. A likely
explanation of our results is that the pool of adult T<sub>regs</sub> is mainly
composed of cells generated during the first months of life. These
findings align with reports that VAT T<sub>regs</sub> in young and aged adults
differ phenotypically, possibly due to population replacement over
time (18).

The kinetics of T<sub>reg</sub> generation in the skin is particularly unique.
More than 55 and 85% of skin T<sub>regs</sub> in 8-week-old mice originate
from cells generated during the first week and first 2 weeks of life,
respectively. Even at 28 weeks of age, T<sub>regs</sub> generated in the first week
of life still constitute 35% of the skin T<sub>reg</sub> pool. The slow renewal of
skin T<sub>regs</sub> in adulthood is further confirmed by the sustained pres-
ence of TagT<sub>regs</sub> in mice treated with tamoxifen at 10 weeks and ana-
lyzed 6 and 18 weeks later. Our findings are compatible with a previous
study showing that skin T<sub>reg</sub> precursors peak at 10 days of age in
lymphoid tissues (15). Our findings are also consistent with the ear-
ly and important colonization of the skin by activated T<sub>regs</sub>, which
constitute up to 80% of CD4<sup>+</sup> T cells in 2- to 3-week-old mice before
declining to below 50% later in life [(5, 7) and our data]. This early
migration is driven by microbiota-induced CCL20 expression in
hair follicles, which recruits CCR6<sup>+</sup> T<sub>regs</sub>, highly enriched in
the neonatal thymus (8). Neonatal skin T<sub>regs</sub> are essential for inducing
long-term immune tolerance to microbiota (7), a process that may
be maintained by either the long-term persistence of neonatal T<sub>regs</sub>
or an infectious tolerance mechanism involving another regulatory
cell player. Our data strongly support the former hypothesis, as most
of skin T<sub>regs</sub> in adulthood are of neonatal origin, which retain an
activated phenotype and likely maintain strong suppressive function.

We also observed a systemic and high expansion of T<sub>regs</sub> during
the second week of life across all tissues except the spleen. This phe-
nomenon was not observed in Tconvs. Neonatal T<sub>reg</sub> expansion may
be driven by lymphopenia-induced proliferation, a process previ-
ously described for T cells in neonates, but the differential proliferation
of T<sub>regs</sub> and Tconvs was not analyzed (19). T<sub>regs</sub> have been described
to undergo more robust expansion than Tconvs after adoptive trans-
fer into lymphopenic adult mice (20), maybe explaining the greater
expansion of T<sub>regs</sub> over Tconvs in neonates. Prior studies have de-
scribed high T<sub>reg</sub> expansion in the neonatal liver (4, 6). Two other
studies have investigated T<sub>reg</sub> proliferation in mice aged 2 weeks or
more, i.e., after the stage of massive expansion that we reveal here.
One showed slightly greater T<sub>reg</sub> proliferation in skin than in lym-
phoid tissues (5), and the other slightly greater proliferation of T<sub>regs</sub>
than Tconvs in small intestine (21). Our study provides a longitudi-
nal, multitissue analysis of this phenomenon. The systemic and mas-
sive expansion of neonatal T<sub>regs</sub> may favor their tissue implantation
and acquisition of their activated phenotype.

Another important finding is the phenotypic switch between
neonatal-derived and later-generated T<sub>regs</sub>. Neonatal T<sub>regs</sub> persisting
into adulthood exhibit an activated phenotype, a phenomenon more
visible in tissues with mixed populations of resting and activated
T<sub>regs</sub>, such as the spleen, LNs, and lungs. This phenotype is less pro-
nounced in the liver and VAT and difficult to observe in the colon
and skin, where resting $T_{regs}$ are rare. Similar observations have been described, but the reliability of the results was uncertain since only a fraction of neonatal $T_{regs}$ was tagged, thus traced cells may not be representative of the entire population (9, 22). The activated phenotype of neonatal $T_{regs}$ persisting in adults may be driven by early lymphopenia-induced proliferation or their intrinsic higher self-reactivity compared to later-generated $T_{regs}$ (9). To determine which of these two mechanisms is involved, one could compare the activation phenotype following adoptive transfer of either weakly self-reactive or strongly self-reactive $T_{regs}$ into neonatal versus adult recipients. This activation state may be crucial for long-term immune regulation, particularly in controlling autoimmunity. Along these lines, it was shown that neonatal $T_{regs}$ had a higher suppressive activity than adult $T_{regs}$ in an in vitro assay and that neonatal $T_{regs}$ that persist in adults were more efficient than adult $T_{regs}$ to control an autoimmune syndrome after adoptive transfer in neonates (9).

In addition, we demonstrate that neonatal $T_{regs}$ exhibit a type 2 immune profile, characterized by increased expression of GATA3 and ST2 compared to later-generated $T_{regs}$, which could be related to the known type 2 immunity bias of the neonatal period (1). This finding may have important implications. VAT $T_{regs}$, for example, are known to exhibit a $T_{H2}$-like profile ($GATA3^+ST2^+$) (23), suggesting that their differentiation may be optimal in neonates because of its type 2 immune environment. This hypothesis is further supported by our data showing reduced GATA3 and ST2 expression in VAT $T_{regs}$ generated after the neonatal period. A similar scenario may apply to skin $T_{regs}$, which are predominantly generated in neonates and exhibit a $T_{H2}$ profile (24). We observed a strong correlation between the proportion of $GATA3^+ T_{regs}$ and neonatal-derived $T_{regs}$ across multiple tissues, reinforcing the hypothesis that the neonatal type 2 immune environment drives the differentiation of $T_{regs}$ expressing markers of type 2 immunity (fig. S9).

The preferential differentiation of $GATA3^+ T_{regs}$ in neonates likely plays a crucial role in shaping the colon's $T_{reg}$ landscape. This tissue contains two well-characterized $T_{reg}$ subsets, $GATA3^+$ (also characterized by Helios expression) and RORγt$^+$, which have distinct functions. Typically, these subsets have been attributed to thymic and peripheral origins, respectively, although this lineage segregation has recently been debated (12). Notably, studies have shown that in neonates, peripherally derived colonic $T_{regs}$ express GATA3 and Helios but lack RORγt, a pattern not observed in older mice (13). As a result, the $GATA3^+/Helios^+$ subset predominates in the neonatal colon. Our findings support this, demonstrating that the neonatal period preferentially drives $GATA3^+ T_{reg}$ differentiation, affecting both thymic $T_{regs}$ (across multiple tissues) and peripherally derived $T_{regs}$ (specifically in the colon). Consistently, in the colon, we found that more than 60% of the $GATA3^+ T_{reg}$ pool in adult mice originates before 2 weeks of age, whereas only 10% of the RORγt$^+ T_{reg}$ pool is generated within this timeframe. These results suggest that the $T_{H2}$ bias characteristic of neonates is critical for establishing the appropriate balance between these two $T_{reg}$ subsets in the adult colon.

Our findings may have clinical relevance, particularly in the context of immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, where $T_{reg}$ deficiency results in severe $T_{H2}$-skewed immunopathology (25), for which drugs neutralizing interleukin-4 (IL-4) and IL-13 are effective (26). In neonates, the preferential differentiation of $GATA3^+ T_{regs}$ known to better control type 2 immunity (27), may explain why patients with IPEX, who typically present in early childhood, exhibit type 2 immunopathology (1). Furthermore, our findings align with previous reports showing that neonatal $T_{reg}$

depletion results in a $T_{H2}$-biased immune response in adults (11, 28), emphasizing the long-term impact of neonatal $T_{regs}$ on immune homeostasis. Also, the capacity of skin $T_{regs}$ to preferentially control local type 2 immunity has been reported (24, 29), which is compatible with the long-term persistence of neonatal type 2 skin $T_{regs}$ that we show here. All these experiments strongly suggest that neonatal $T_{regs}$ persisting into adulthood preferentially regulate type 2 immunity. It would be difficult to further support this hypothesis using in vitro experiments because regulation of type 2 immunity by $T_{regs}$ has been mostly documented in vivo.

Last, our skin-specific $T_{reg}$ fate-mapping experiments confirm that skin $T_{regs}$ exhibit high residency in young adults (3 to 8 weeks of age). A similar experiment performed in older adults indicate lower residency, with approximately a 50% decline in Tag$T_{regs}$ 3 weeks postlabeling (30). This suggests that skin $T_{reg}$ residency is more pronounced in young adulthood, followed by increased replenishment. The transient residence of skin $T_{regs}$ in adult mice was confirmed using parabiosis or specific markers (31–33). In the VAT, the residence of $T_{regs}$ in adults would be of several weeks to months, as demonstrated in parabiosis and adoptive transfer experiments (33–35). Our findings challenge the recent concept of tissue-agnostic $T_{regs}$, which posits that tissue-resident $T_{regs}$ freely recirculate independently of their tissue of origin (33). However, $T_{reg}$ recirculation from or to the skin or VAT was not analyzed in this report. Here, we show that $T_{regs}$ resident in the skin and VAT are not tissue-agnostic since, if they were so, we would have the same proportion of Tag$T_{regs}$ between these tissues and the liver or the lungs, which is clearly not the case. Collectively, our experiments suggest a prolonged residence of tissue $T_{regs}$ in young adults, at least in the skin. Then, the pool of skin and VAT $T_{regs}$ is slowly replenished, either by $T_{reg}$ recirculation between skin and VAT (plus possibly other unidentified tissues) or by precursors generated during the perinatal period.

In conclusion, our study provides a comprehensive analysis of the $T_{reg}$ generation kinetics across multiple tissues. Our study underscores the critical role of the neonatal period in establishing long-lived $T_{reg}$ populations specifically in the skin. These neonatal-derived $T_{regs}$ exhibit an activated phenotype and a type 2 immune profile, suggesting specialized functions in long-term immune regulation. Understanding the ontogeny of $T_{regs}$ provides key insights into immune tolerance and tissue-specific immune homeostasis in adulthood.

## MATERIALS AND METHODS

### Mice

Foxp3<sup>tm9(EGFP/cre/ERT2)Ayr/J</sup> (Foxp3<sup>iCre</sup>), B6.Cg-Gt(ROSA)26Sor<sup>tm9(CAG-tdTomato)Hze/J</sup> (R26<sup>Tom</sup>), and Foxp3<sup>tm3(DTR/GFP)Ayr/J</sup> (Foxp3<sup>DTR</sup>) mice were purchased from the Jackson Laboratory. All mice were on a C57BL/6J background. Experiments were performed in both males and females, and since no differences were observed between sexes, the results were pooled and are presented together in the figures. They were housed under specific pathogen-free conditions. All experimental protocols were approved by our local Institutional Animal Care and Use Committees under the license numbers 02811.03, APAFIS#26677-202007211707430, and APAFIS#48261-2024031218097130. Mouse experimentation was performed in compliance with European Union guidelines.

### Tamoxifen and EdU administration

The tamoxifen (MP Biomedical, Ref: 11435161) was reconstituted at a final concentration of 40 mg/ml in peanut oil, obtained after 4 hours
of vortex at 37°C. For mice older than 6 weeks, 8 mg of tamoxifen was administered orally by gavage. For mice younger than 6 weeks, tamoxifen was administered by two intraperitoneal injections, 3 days apart. They received a dose adapted to their age (0.1, 0.2, 1, and 2 mg for 1-, 2-, 3-, and 4- to 6-week-old mice, respectively). For the local treatment of the skin, HO-Tam (Sigma-Aldrich, Ref: T176) was resuspended at the final concentration of 20 µg/ml in acetone. Fifty microliters of the solution was applied directly on the ears during 4 consecutive days. EdU was administered intraperitoneally [at 5 mg/ml in phosphate-buffered saline (PBS)] three times on the day before tissue collection (at 7 a.m. and 1 and 7 p.m.). One-week-old mice received 50 µl per injection, whereas 3-week-old mice received 100 µl per injection. EdU incorporation was detected using the Click-iT Plus EdU Pacific Blue Flow Cytometry Assay Kit (Thermo Fisher Scientific, Ref: C10636), according to the manufacturer's instructions.

## Preparation of cell suspensions

After cervical dislocation, mice were perfused intracardially with PBS to eliminate blood circulating cells. Lymphoid tissues (spleen and brachial/axillary LNs) and liver were collected in PBS complemented with 3% fetal calf serum (PBS-3%FCS). A single-cell suspension was obtained by mechanical dilaceration with a 70-µm cell strainer. The single-cell suspension obtained from the liver was resuspended in 40% Percoll, laid on an 80% Percoll, and centrifuged for 20 min at 2000 rpm at 20°C. Cells were collected at the 40%:80% Percoll interface and washed

with PBS-3%FCS. The entire colon was collected, washed to remove feces, and cut longitudinally and then into small pieces. The tissues were subjected to three consecutive baths in order to remove the epithelium and collect the lamina propria: twice with PBS-3%FCS EDTA 5 mM and once with PBS-3%FCS, each lasting 20 min. After each bath, the tubes were vortexed for 10 s, and the pieces of tissue were transferred into the next tube. For the spleen and lungs, the red blood cells were lysed with ammonium chloride potassium for 1 min.

The VAT, lungs, and skin were collected in PBS and cut into small pieces before enzymatic digestion. For mice under 1 month old, the adipose tissue was collected subcutaneously. Digestion kits specific to each tissue from Miltenyi were used. The gentleMACS Dissociator was used for the lungs (Ref: 130-095-927) and the VAT (Ref: 130-105-808) in accordance with the instructions provided by the supplier. For the skin (Ref: 130-110-201) and the lamina propria (Ref: 130-097-410), the protocol was adapted, and the cells were digested using 12-well plates at 37°C with constant agitation for 105 and 30 min, respectively. After the digestion, the tissues were filtered with a 70-µm cell strainer. Enzymatic digestions had minimal impact on expression of surface molecules, as suggested from studying CD8a expression that is very sensitive to such treatment.

## Antibodies and flow cytometry

Cells were stained in 96-well plates with the antibodies and fluorescent reagent listed in Table 1. The viability of the cells was determined

<table><caption>**Table 1. Antibodies used in flow cytometry.** PE, phycoerythrin; Cy7, Cyanine 7; AP, allophycocyanin; PerCP, peridinin-chlorophyll-protein complex.</caption><thead><tr><td>Target</td><td>Clone</td><td>Fluorochrome</td><td>Supplier</td><td>Reference</td></tr></thead><tbody><tr><td>Bcl2</td><td>10C4</td><td>PE-Cy7</td><td>Invitrogen</td><td>25-6992-42</td></tr><tr><td>CCR2</td><td>475301</td><td>BUV661</td><td>BD Biosciences</td><td>750042</td></tr><tr><td>CD25</td><td>PC61</td><td>BV605</td><td>BioLegend</td><td>102036</td></tr><tr><td>CD3</td><td>145-2C11</td><td>BUV563</td><td>BD Biosciences</td><td>749277</td></tr><tr><td>CD4</td><td>GK1.5</td><td>BUV496</td><td>BD Biosciences</td><td>612952</td></tr><tr><td>CD44</td><td>IM7</td><td>PerCP</td><td>BioLegend</td><td>103035</td></tr><tr><td>CD45</td><td>30-F11</td><td>BUV395</td><td>BD Biosciences</td><td>564279</td></tr><tr><td>CD62L</td><td>MEL-14</td><td>BV650</td><td>BD Biosciences</td><td>564108</td></tr><tr><td>CD69</td><td>H1.2F3</td><td>BUV805</td><td>BD Biosciences</td><td>741927</td></tr><tr><td>CD73</td><td>TY/11.8</td><td>PerCP-Cy5.5</td><td>BioLegend</td><td>127214</td></tr><tr><td>CD8</td><td>53-6.7</td><td>PE-Cy5.5</td><td>Invitrogen</td><td>35-0081-80</td></tr><tr><td>c-Maf</td><td>sym0F1</td><td>eF450</td><td>Invitrogen</td><td>48-9855-42</td></tr><tr><td>CTLA-4</td><td>UC10-4F10-11</td><td>PE-CF594</td><td>BD Biosciences</td><td>564332</td></tr><tr><td>EZH2</td><td>11/EZH2</td><td>BV421</td><td>BD Biosciences</td><td>562963</td></tr><tr><td>Foxp3</td><td>FJK-16 s</td><td>FITC</td><td>Invitrogen</td><td>11-5773-82</td></tr><tr><td>GATA3</td><td>TWAJ</td><td>PE-Cy5</td><td>Invitrogen</td><td>15-9966-41</td></tr><tr><td>ICOS</td><td>7E.17G9</td><td>BV711</td><td>BD Biosciences</td><td>740763</td></tr><tr><td>Ki67</td><td>SolA15</td><td>AF532</td><td>Invitrogen</td><td>58-5698-82</td></tr><tr><td>KLRG1</td><td>2F1</td><td>BUV615</td><td>BD Biosciences</td><td>751191</td></tr><tr><td>NRP1</td><td>V46-1954</td><td>BV750</td><td>BD Biosciences</td><td>752452</td></tr><tr><td>PD-1</td><td>j43</td><td>BUV737</td><td>BD Biosciences</td><td>749422</td></tr><tr><td>RORγT</td><td>Q31-378</td><td>AF647</td><td>BD Biosciences</td><td>562682</td></tr><tr><td>ST2</td><td>U29-93</td><td>BV480</td><td>BD Biosciences</td><td>746701</td></tr><tr><td>TCF1</td><td>S33-966</td><td>R718</td><td>BD Biosciences</td><td>567587</td></tr><tr><td>TNFR2</td><td>REA228</td><td>APC</td><td>Miltenyi</td><td>130-123-275</td></tr></tbody></table>
using a fixable live/dead dye (LIVE/DEAD Blue, Invitrogen). Non-specific binding was limited by using the 2.4G2 hybridoma (anti-CD16/32). Membrane staining was performed for 20 min at 4°C in PBS-3%FCS. Fixation and permeabilization of the cells were performed using the Foxp3/Transcription Factor Staining Buffer Set from eBioscience (00-5523-00). Intracellular staining was performed for 45 min at 4°C using the buffer provided. Cells were acquired on the spectral cytometer Aurora from Cytek and analyzed using the FlowJo software. Cells treated with the Foxp3/Transcription Factor Staining Buffer from eBioscience maintained tdTomato expression. Combined with the use of the spectral cytometer Aurora from Cytek, we could clearly discriminate between tdTomato⁺ and tdTomato⁻ T<sub>regs</sub>.

## T<sub>reg</sub> adoptive transfer in Foxp3<sup>DTR</sup> mice

Single-cell suspension obtained from the spleen and LNs of the Foxp3<sup>iCre</sup> R26<sup>Tom</sup> donor mice were stained with an anti-CD25 (7D4) biotin-labeled antibody and were then coated with anti-biotin micro-bead (Miltenyi, Ref: 130-090-485). After magnetic sorting, cells of the CD25⁺ enriched positive fraction were labeled with the carboxyfluorescein diacetate succinimidyl ester (CFSE) CellTrace kit from eBioscience and were intravenously injected into Foxp3<sup>DTR</sup> recipient mice (10⁶ cells per mouse). The recipient mice were treated with tamoxifen (two times, 2 mg) and diphtheria toxin (three times, 1 µg) by intraperitoneal injections before the adoptive transfer. The donor cells were identified by flow cytometry in the spleen, LNs, lungs, liver, and colon based on the CFSE expression 5 days after the adoptive transfer.

## Flow cytometry analysis, GSEA, biostatistic, and data modelization

Raw FCS data from the Aurora were analyzed using FlowJo software to produce data tables. The following analysis was performed using R (4.3.1). PCA was performed using factominer (2.8) and factoextra (1.0.7). Boxplots and line plots were generated using ggplot2 (3.4.3). Statistical analyses were performed using rstatix (0.7.2) and ggpubr (0.6.0). Gene set enrichment analysis (GSEA) was performed using the Webgestalt software (https://www.webgestalt.org/). Normalized gene expression data were used as input, along with a preranked list of genes based on their correlation with the experimental condition [log₂ fold change between neonatal and adult T<sub>regs</sub> obtained from Yang *et al.* (9)]. The analysis was conducted in preranked mode with 1000 permutations, using the gene set of VAT- and skin T<sub>regs</sub> versus LN T<sub>regs</sub> (14) or the gene sets of tissue T<sub>reg</sub> precursors versus other T<sub>regs</sub> in the spleen, identified by their Nfilt³+Klg1⁺ or nonlymphoid like T<sub>reg</sub> phenotype (15, 17).

## Supplementary Materials

This PDF file includes:

Figs. S1 to S9

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**Acknowledgments:** We are grateful to M. Larsen and M. Zahm for help in biostatistical analyses, to C. Benoist and D. Mazzone for critical reading of the manuscript, and to F. I券ert, O. Bregerie, M. Mihoc, and J. Perronnet for the expert care of the mouse colony. **Funding:** This work was supported by the Agence Nationale de la Recherche [ANR-21-CE15-0049 (Trindi) and ANR-22-CE18-0045 (Reality)] to B.L.S., Association de la recherche sur la sclérose en plaques to B.L.S., and Fondation pour la recherche sur le cancer and Ligue contre le cancer to B.L.S. M.H. has received funding from the Fondation pour la recherche médicale, and A.R. has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 955321. **Author contributions:** Conceptualization: M.H. and B.L.S. Investigation: M.H., A.M., L.C., H.S., and B.L.S. Methodology: M.H., A.R., and B.L.S. Formal analysis: M.H. and B.L.S. Visualization: M.H., R.V., and B.L.S. Writing—original draft: M.H. and B.L.S. Writing—review and editing: M.H. and B.L.S. **Competing interests:** The authors declare that they have no competing interests. **Data and materials availability:** All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Submitted 28 March 2025
Accepted 3 September 2025
Published 3 October 2025
10.1126/sciadv.adx8037