Stability and function of regulatory T cells expressing the transcription factor T-bet

Abstract

Adaptive immune responses are tailored to different types of pathogens through differentiation of naive CD4 T cells into functionally distinct subsets of effector T cells (T helper 1 (T_H1), T_H2, and T_H17) defined by expression of the key transcription factors T-bet, GATA3, and RORγt, respectively¹. Regulatory T (T_reg) cells comprise a distinct anti-inflammatory lineage specified by the X-linked transcription factor Foxp3 (refs 2, 3). Paradoxically, some activated T_reg cells express the aforementioned effector CD4 T cell transcription factors, which have been suggested to provide T_reg cells with enhanced suppressive capacity^4,5,6. Whether expression of these factors in T_reg cells—as in effector T cells—is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here we demonstrate that expression of the T_H1-associated transcription factor T-bet in mouse T_reg cells, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss of function or elimination of T-bet-expressing T_reg cells—but not of T-bet expression in T_reg cells—resulted in severe T_H1 autoimmunity. Conversely, following depletion of T-bet⁻ T_reg cells, the remaining T-bet⁺ cells specifically inhibited T_H1 and CD8 T cell activation consistent with their co-localization with T-bet⁺ effector T cells. These results suggest that T-bet⁺ T_reg cells have an essential immunosuppressive function and indicate that T_reg cell functional heterogeneity is a critical feature of immunological tolerance.

Main

Whether T_reg cells expressing the T_H1-associated transcription factor T-bet represent a stable sub-lineage of cells with unique function or a transient activation state remains unknown. To address this question, we assessed the stability of T-bet expression in T_reg cells using a novel Tbx21^{tdTomato-T2A-creERT2} knock-in allele combined with the R26Y recombination and Foxp3^Thy1.1 reporters. The resulting Tbx21^RFP-creERT2 mice showed a range of red fluorescent protein (RFP) expression and CreERT2 activity, which faithfully reflected endogenous T-bet protein levels in major lymphocyte subsets (Fig. 1a, Extended Data Fig. 1a, b). RFP⁺ T_reg cells comprised between 30–70% of CD44^hiCD62L^lo effector T_reg cells in lymphoid organs and non-lymphoid tissues; interestingly, intestinal T_reg cells exhibited prevalent co-expression of T-bet and RORγt, but not T-bet and GATA3 (Extended Data Fig. 1d–i).

Figure 1: Stable T-bet expression in a subset of peripheral T_reg cells.

a, Splenic cells in Tbx21^RFP-creERT2 mice 3 weeks after tamoxifen gavage on days −2 and 0. Numbers on graph indicate the mean. Data are mean ± s.e.m. b, Schematic of tamoxifen administration to Tbx21^RFP-creERT2 mice (top) and flow cytometry (bottom) of splenic CD4 Thy1.1⁺ and Thy1.1⁻ cells. c, Upper, RFP⁺ (left axis, squares) and YFP⁺ (right axis, circles) T_reg cells. Lower, percentage of RFP⁺ of YFP⁺ T_reg cells 3 weeks (white symbols), 3 months (grey symbols), and 7 months (black symbols) after tamoxifen gavage. d, Upper, schematic of tamoxifen treatment and N. brasiliensis infection. Lower, percentage of RFP⁺ among YFP⁺ T_reg cells in mice challenged with PBS (white circles) and N. brasiliensis (Nb; black circles); (bottom, right) RFP expression in T_reg (shaded histograms) or YFP⁺ T_reg (open histograms) cells from spleens of mice challenged with PBS (black) or N. brasiliensis (red). Data are mean. Two-tailed t-test (NS, not significant). All data are representative of 2 experiments, n ≥ 3 mice per group each.

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Three weeks after tamoxifen administration we found—in contrast to a previous report⁷—that the vast majority of both yellow fluorescent protein (YFP)-labelled T_reg and effector CD4 T cells continued to express RFP (Fig. 1b, c, Extended Data Fig. 1j). The percentage of YFP⁺ cells expressing RFP was similarly high at three and seven months after tamoxifen administration, although percentages of YFP⁺ cells declined, indicating that continual T_reg cell recruitment into the T-bet⁺ subset balances out cell turnover over time (Fig. 1b, c, Extended Data Fig. 1j). Indicative of intrinsic stability of T-bet⁺ T_reg cells typical of a differentiated cell state, treatment of Tbx21^RFP-creERT2 mice with tamoxifen 3 weeks before infection with the helminth Nippostrongylus brasiliensis did not result in loss of RFP expression among YFP⁺ T_reg (or effector CD4) T cells despite robust T_H2 activation and cytokine production in the spleens and lungs of infected mice (Fig. 1d, Extended Data Fig. 3a, data not shown).

The presence of small percentages of YFP⁺RFP⁻ cells 3 weeks after gavage (Fig. 1b, c) suggested that some T_reg cells might have experienced transient unstable T-bet expression at the time of tamoxifen administration. Such a scenario would reconcile the above result with an earlier study⁷. Indeed, in Tbx21^RFP-creERT2 mice we observed RFP^lo T_reg cells that lacked the T-bet-dependent expression of chemokine receptor CXCR3, in addition to RFP^hiCXCR3⁺ cells (Extended Data Fig. 2a). The former exhibited slightly lower CD44 and slightly higher CD62L expression than the latter and RNA sequencing (RNA-seq) analysis suggested that CD44^hiRFP^loCXCR3⁻ T_reg cells were differentiation intermediates between CD44^hiRFP⁻ cells and CD44^hiRFP^hiCXCR3⁺ cells (Extended Data Fig. 2b–d). Around 40% of FACS-sorted RFP^loCXCR3⁻ (but not RFP^hiCXCR3⁺) T_reg cells lost RFP expression following transfer into lymphoreplete hosts, whereas some became RFP^hiCXCR3⁺ (Extended Data Fig. 2e, f). Notably, populations of RFP^loCXCR3⁻ and YFP⁺RFP⁻ cells were also observed within the CD4 non-T_reg cell population (Extended Data Fig. 2a). Thus, the observed instability of a low level of T-bet expression is not unique to T_reg cells but is indicative of the gradual process of peripheral T cell effector differentiation^8,9.

In addition to steady state cues, T_H1-polarizing infection can drive increases in T-bet⁺ T_reg cells¹⁰. To determine whether infection expands T-bet⁺ T_reg cells present at steady state, or rather induces T-bet expression in T-bet⁻ cells, we administered tamoxifen to Tbx21^RFP-creERT2 mice 3 weeks before challenge with the intracellular bacteria Listeria monocytogenes. Upon L. monocytogenes challenge, RFP⁺ T_reg and effector CD4 T cell subsets increased markedly; however, YFP⁺ subsets did not (yielding a decreased YFP⁺/RFP⁺ ratio) (Fig. 2a, b, Extended Data Fig. 3b). This pattern was indicative of de novo differentiation of T-bet⁺ cells from T-bet⁻ T_reg precursors in parallel with differentiation of T_H1 cells. Following transfer, both CD44^loCD62L^hi RFP⁻ and CD44^hiRFP⁻ T_reg cells upregulated RFP in response to L. monocytogenes infection (Extended Data Fig. 3c). Notably, upon L. monocytogenes infection, preformed T-bet⁺ T_reg cells tagged with YFP prior to infection increased expression of T-bet and CXCR3, but not IL-10, an important suppressor molecule¹¹. The latter was demonstrated by fate mapping experiments in Tbx21^RFP-creERT2Il10^eGFP/WT mice, which revealed no increase in IL-10 (eGFP⁺) among YFP⁺ T_reg cells, whereas bulk T-bet (RFP⁺) IL-10⁺ cells increased around threefold (Extended Data Fig. 3d–g). Similar results were obtained during lymphocytic choriomeningitis virus infection (data not shown).

Figure 2: Stable differentiation of T-bet⁺ T_reg cells in response to L. monocytogenes infection.

a, Schematic of experiment shown in b combining tamoxifen (TX) treatment and L. monocytogenes (Lm) infection in Tbx21^RFP-creERT2 mice. b, Percentage of RFP⁺, YFP⁺, and YFP⁺/RFP⁺ ratio in CD4⁺ Thy1.1⁺ (left) and Thy1.1⁻ (right) cells in spleens and livers of mice challenged with PBS and L. monocytogenes. c, Schematic of experiments shown in d, e and f; 1° and 2° indicate primary and secondary challenge, respectively. d, Data presented as in b. e, Percentage of RFP⁺ of YFP⁺ T_reg cells. f, Data presented as in b. Bars, mean. Two-tailed t-test (***P < 0.001, **P < 0.01, and *P < 0.05, respectively; NS, not significant). All data are representative of ≥ 2 experiments, n ≥ 4 mice per group each.

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We next assessed the persistence and recall response of T-bet⁺ T_reg cells induced by L. monocytogenes infection. To preferentially label infection-induced T-bet⁺ cells, tamoxifen was administered at the peak of the primary L. monocytogenes response (days 7 and 9). Mice were assessed 8 weeks later, at which time the percentage of splenic and liver T_reg cells that were RFP⁺ had returned to roughly pre-infection levels (Fig. 2c, d). Given the turnover rate of T-bet⁺ cells (Fig. 1c), we reasoned that by day 60 after infection YFP⁺ cells would be relatively enriched for infection-induced T_reg cells compared to the bulk RFP⁺ cell pool. Reinfection increased bulk RFP⁺ T_reg and effector CD4 T cells and even more prominently increased the corresponding cell subsets tagged with YFP (Fig. 2d, Extended Data Fig. 3h–j). On day 65 after primary infection, more than 90% of YFP⁺ T_reg cells continued to express T-bet, as did uninfected control cells (Fig. 2e). Furthermore, mice infected with L. monocytogenes that were administered tamoxifen on days 37 and 39 after resolution of the primary response and re-infected on day 60 exhibited a parallel increase in bulk RFP⁺ and YFP⁺ T_reg cell subsets on day 65, suggesting cells that acquired T-bet expression during primary infection remained T-bet-positive and expanded upon reinfection (Fig. 2f). Together, these studies demonstrate that bacterial infection caused de novo differentiation of T-bet⁻ T_reg cells into stable T-bet⁺ cells uniquely suited for reactivation under conditions that drove their initial acquisition of T-bet.

Although the stability of T-bet⁺ T_reg cells suggested a particular function presumably imparted by T-bet itself, we found that 12-week-old Foxp3^YFP-creTbx21^fl/fl mice were clinically indistinguishable from littermate controls, consistent with previous studies^7,12,13. Foxp3^YFP-creTbx21^fl/fl mice did exhibit mild T_H1 (but not CD8 T cell) activation, indicating that T-bet expression in T_reg cells moderately potentiated suppression of T_H1 autoimmunity (Extended Data Fig. 4a). We considered the possibility that T-bet deficiency might not fully impair the function of T-bet⁺ T_reg cells. As T_reg cell suppressor function requires continuous expression of the Foxp3 gene¹⁴, we ablated Foxp3 in T-bet⁺ T_reg cells using a novel Tbx21^{tdTomato-T2A-cre} allele (Extended Data Fig. 5a). Loss of Foxp3 expression in T-bet⁺ T_reg cells in 8-week-old Tbx21^RFP-creFoxp3^fl mice resulted in deceased weight gain, lymphadenopathy, T cell activation, and marked immune infiltration in the lung; with age, loss of hair pigmentation and rectal prolapse were evident (Fig. 3a–d, Extended Data Fig. 5).

Figure 3: Foxp3 ablation in T-bet⁺ T_reg cells results in spontaneous T_H1 autoimmune disease.

a, Body weights of 8–10-week-old Tbx21^RFP-creFoxp3^WT (grey circles), Tbx21^RFP-creFoxp3^fl (red circles), Tbx21^RFP-creFoxp3^WT/WT (blue circles), and Tbx21^RFP-creFoxp3^fl/WT (white circles) mice. b, Haematoxylin and eosin staining (left) and histology scores (right) of lungs from Tbx21^RFP-cre mice combined with indicated Foxp3 alleles, treated or not with antibiotics (ABX). Tbx21^RFP-creFoxp3^fl mice show moderate perivascular and peribronchiolar inflammation, mild respiratory epithelial hyperplasia and mucus metaplasia with hyalinization (arrows). Pulmonary arterioles are contracted with thickened media, reactive endothelia and marginating leukocytes (arrowheads). Original magnification, 20×. c, d, Lymph node cell numbers (c) and characterization of T cell populations in spleens (d). e, Flow cytometry of splenic cells in Tbx21^RFP-creFoxp3^WT (left) and Tbx21^RFP-creFoxp3^fl (right) mice, gated on fixed CD4⁺ (top) and live CD4⁺CD25⁻ (bottom) cells. f, Quantification of RFP⁻ and RFP⁺ CD4 T cells, as shown in e (bottom). g, RFP expression (left) and cytokine production (right) in splenic CD8 T cells. h, Cytokine production by splenic CD4⁺Foxp3⁻ T cells. i, Representative images (left) and insets (right) of spleen sections from Tbx21^RFP-cre mice with CD4 (green, top) or CD8 (green, bottom), RFP (red), Foxp3 (blue) and CD44 (grey). Inset, arrowheads indicate CD4⁺CD44^hiRFP⁺Foxp3⁻ (top) or CD8⁺CD44^hiRFP⁺ (bottom) cells and arrows indicate CD4⁺CD44^hiRFP⁺Foxp3⁺ cells. j, k, Nearest distances between cells as shown in i. Foxp3⁺ denotes CD4⁺CD44^hiFoxp3⁺; Foxp3⁻ (j) denotes CD4⁺CD44^hiFoxp3⁻ and CD8⁺ (k) denotes CD8⁺CD44^hiRFP⁺. Each circle (j, k) represents the distance between cells on imaged sections from three mice. Data are mean ± s.e.m. Two-tailed t-test (***P < 0.001, **P < 0.01; NS, not significant). All data are representative of several experiments.

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Indicative of T_H1-type inflammation, the majority of expanded effector CD4 and CD8 T cells in Tbx21^RFP-creFoxp3^fl mice expressed RFP (Fig. 3e–g, Extended Data Fig. 5). Additionally, IFNγ and IL-2 but neither IL-4 nor IL-17 production by T cells were increased compared to controls (Fig. 3g, h, Extended Data Fig. 5). Antibiotic treatment did not mitigate autoimmunity in Tbx21^RFP-creFoxp3^fl mice, excluding microbial antigens as the drivers of T_H1 inflammation (Fig. 3b, data not shown). We considered whether induction of a robust non-T_H1 immune response in Tbx21^RFP-creFoxp3^fl mice might reveal a potential function for T-bet⁺ T_reg cells in its control. However, the T_H2 response to N. brasiliensis infection was not increased in Tbx21^RFP-creFoxp3^fl mice compared to control mice, in contrast to the exacerbated T_H2 response observed upon pan-T_reg-cell depletion during helminth infection^15,16 (Extended Data Fig. 6). Notably, whereas CXCR3⁺ T_reg cells were significantly depleted neither total nor effector T_reg cell numbers were diminished, and analysis of Tbx21^RFP-creR26Y mice confirmed that a significant proportion of effector T_reg cells had not undergone Cre-mediated recombination (Fig. 3d, Extended Data Fig. 5c, f). These results suggested that immune activation could not be attributed to non-specific loss of effector T_reg cells.

Cells that are likely to represent ex-T_reg cells, which have lost Foxp3 expression, but continue to express high CD25, CD39, CTLA4 and GITR levels^2,17,18 were readily found in male Tbx21^RFP-creFoxp3^fl and, to a lesser extent, female Tbx21^RFP-creFoxp3^fl/WT mice (Fig. 3e, Extended Data Fig. 5g, h). The lack of autoimmunity in Tbx21^RFP-creFoxp3^fl/WT females—in which only half of T-bet⁺ T_reg cells lose Foxp3 owing to X-inactivation—indicated that ex-T_reg cells were efficiently controlled by remaining T-bet⁺ T_reg cells (Fig. 3). Moreover, upon adoptive transfer into T-cell-deficient hosts, ex-T_reg cells induced no more pathology and expanded less than CD4 effector T cells (Extended Data Fig. 7a–c). These results indicate that ex-T_reg cells were unlikely drivers of (although it is possible that they may play some role in) the observed autoimmunity.

To determine whether punctual ablation of T-bet⁺ T_reg cells would similarly unleash T_H1 inflammation, we generated bone marrow chimaeric mice with a 1:1 mix of either CD45.1⁺ Foxp3^WT or Foxp3^KO with CD45.2⁺ Tbx21^RFP-cre/WTR26^iDTR haematopoietic precursor cells (Fig. 4). In Foxp3^KO:Tbx21^RFP-cre/WTRosa26^iDTR mixed chimaeras, all T-bet⁺ T_reg cells expressed diphtheria toxin receptor (DTR) and were susceptible to diphtheria-toxin-mediated ablation, whereas the rest of the T-bet-expressing cell types and subsets represented a 1:1 mix of DTR-expressing and non-expressing cells. Before treatment with diphtheria toxin, both sets of mixed chimaeras were healthy with similar basal levels of T cell activation (data not shown). Administration of diphtheria toxin over 2 weeks resulted in weight loss, T cell activation, and a selective increase in IFNγ production by CD4 and CD8 T cells in Foxp3^KO:Tbx21^RFP-cre/WTR26^iDTR chimaeric mice ablated of T-bet⁺ T_reg cells compared to Foxp3^WT:Tbx21^RFP-cre/WTR26^iDTR controls (Fig. 4). T_reg cell percentages in experimental mice were only very modestly decreased (from 13 ± 0.53 to 11 ± 0.82, P = 0.022) and, as in Tbx21^RFP-creFoxp3^fl mice, percentages of CD44^hiCD62L^lo T_reg cells were undiminished compared to controls (Fig. 4b, c). This experimental model is not confounded by generation of ex-T_reg cells, providing additional evidence that the latter were not the sole drivers of pathology in the absence of T-bet⁺ T_reg cells. Finally, weight loss was not observed in Tcrb^KO:Tbx21^RFP-cre/WTR26^iDTR mixed chimaeras, in which T-bet⁺ T_reg and effector T-bet⁺ TCRαβ⁺ cells were simultaneously ablated, implicating the latter in driving disease (Extended Data Fig. 7d, e).

Figure 4: Acute ablation of T-bet⁺ T_reg cells results in T_H1 immune activation.

Bone marrow chimaeric mice were injected with 0.5 μg diphtheria toxin (DT) on day 0, then treated daily with 0.1 μg of diphtheria toxin until day 15. a, Weight loss in the indicated mice. b, Flow cytometry of splenic CD4 (top) and T_reg (bottom) cells in the indicated mice. c, Activation status of CD45.1⁺ and CD45.2⁺ T_reg cell compartments in spleens of indicated mice. d, e, T cell activation (d) and cytokine production (e) in control (white circles) and T-bet-depleted (black circles) chimaeras. Data are mean ± s.e.m. Two-tailed t-test (**P < 0.01, *P < 0.05; NS, not significant). Data are representative of 2 experiments, n ≥ 6 mice per group.

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RNA-seq analysis revealed that 561 genes, including Tbx21, Cxcr3, Gzmb, Ebi3, Fgl2, and Il10, were more highly expressed in CD44^hiRFP⁺ cells compared to CD44^hiRFP⁻ T_reg cells (Extended Data Fig. 2c). Expression of this gene set was increased upon loss of Foxp3 in ex-T_reg cells, suggesting that Foxp3 opposes the transcriptional signature of T-bet⁺ T_reg cells to prevent full T_H1 differentiation¹⁰ (Extended Data Fig. 4b). Notably, the T_H1-associated chemokine receptor CCR5 and adhesion molecule β1-integrin (CD29) were expressed in T-bet⁺ T_reg cells independently of T-bet (Extended Data Fig. 4c, d) indicating that some functional redundancy of homing molecules may in part explain the mild phenotype of Foxp3^YFP-creTbx21^fl/fl mice. Moreover, we found that the TCR repertoires of CD44^hiCXCR3(T-bet)⁺ and CD44^hiCXCR3(T-bet)⁻ T_reg subsets in DO11.10 TCRβ⁺ Tcra^+/− mice were distinct, suggesting that antigenic specificity of T-bet⁺ T_reg cells may also contribute to distinct localization and suppressor capacity, as recent studies revealed TCR-dependent spatial proximity of T_reg and IL-2-producing self-reactive T cells¹⁹ (Extended Data Fig. 4e).

Therefore, we sought to determine the relative spatial positioning of T-bet⁺ and T-bet^– T_reg and effector T cells in secondary lymphoid organs of Tbx21^RFP-cre mice. Immunofluorescence imaging revealed pronounced preferential proximity of CD44^hiT-bet⁺ versus CD44^hiT-bet^– T_reg cells to CD44^hiT-bet⁺ T_H1 and CD8 T cells (Fig. 3i–k, Extended Data Fig. 8a–d). In contrast, CD44^hiT-bet⁺ T_reg cells were no nearer to T-bet⁻ CD4 effectors than were CD44^hiT-bet⁻ T_reg cells (Fig. 3j, Extended Data Fig. 8c). Notably, the CD44^hiT-bet⁻ T_reg cells remaining in Tbx21^RFP-creFoxp3^fl mice were no nearer to T_H1 or CD8 T cells than were CD44^hiRFP⁻ T_reg cells in healthy Tbx21^RFP-creFoxp3^WT mice (Extended Data Fig. 8e, f). This result suggests that failure of non-T-bet⁺ T_reg cells to approximate T_H1 cells may at least in part account for their inability to suppress T_H1 inflammation.

Lastly, to complement T-bet⁺ T_reg cell ‘loss-of-function’ experiments we sought to selectively eliminate T-bet⁻ T_reg cells. We generated a Foxp3^fl-DTR allele by inserting a loxP-flanked IRES-DTReGFP DNA sequence into the 3′ UTR of the Foxp3 gene (Extended Data Fig. 9a) and generated Foxp3^fl-DTRTbx21^RFP-creERT2 mice (Fig. 5a). After 9 days of diphtheria toxin treatment, T_reg cells in mice pre-treated with tamoxifen (day −5 and −3) were present in undiminished percentages and were exclusively T-bet⁺ and CXCR3⁺ (Fig. 5a–c). Compared to vehicle (oil)-treated mice, tamoxifen-treated Foxp3^fl-DTRTbx21^RFP-creERT2 mice displayed robustly suppressed CD8 T cell activation and selective suppression of IFNγ production by CD4 and CD8 T cells, but unrestrained T_H2 and T_H17 cytokine production (Fig. 5d–f). T-bet⁺ T_reg cells similarly suppressed pre-established T_H1, but not T_H2 or T_H17, activation induced by depletion of T_reg cells before tamoxifen treatment (Extended Data Fig. 9b–g). Selective T_H1 suppression was not simply a feature of activated T_reg cells rebounding after depletion, as partial depletion and recovery of T_reg cells in Foxp3^DTR mice resulted in prominently inhibited T_H2 responses (Extended Data Figs 9h–l, 10).

Figure 5: T-bet⁺ T_reg cells suppress T_H1 and CD8⁺ T cells, but not T_H2 or T_H17 responses.

a, Schematic for tamoxifen administration and depletion of non-T-bet-expressing T_reg cells in Foxp3^fl-DTRTbx21^RFP-creERT2 mice. b, Flow cytometry of splenic CD4 T cells in the indicated mice on day 9, as outlined in a. c–f, T_reg cell percentages (c), and activated (d) and cytokine-producing (e, f) T cells in spleens of tamoxifen-treated Foxp3^Thy1.1Tbx21^RFP-creERT2 (open circles), oil-treated Foxp3^fl-DTRTbx21^RFP-creERT2 mice (black circles) and tamoxifen-treated Foxp3^fl-DTRTbx21^RFP-creERT2 (grey circles) mice. Data are mean ± s.e.m. Two-tailed t-test (***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant). Data are representative of 2 experiments, n ≥ 2 mice per group each.

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Our studies suggest that T-bet expression in T_reg cells denotes a differentiated cell state with unique T-bet-dependent and -independent gene expression and TCR specificity, capable of driving potent immunosuppression limited to circumstances of T_H1 and CD8 T cell activation. It is possible that GATA3- and RORγt-expressing T_reg cells may play analogous roles in suppression of T_H2 and T_H17 responses. Such division of anti-inflammatory labour among T_reg cells, arising at steady state and during infection, may enable focused regulation of specific T helper cell responses without incurring undesired bystander suppression.

Methods

Animals

Tbx21^{tdTomato-T2A-creERT2} mice were generated by insertion of a targeting construct into the Tbx21 locus by homologous recombination in embryonic stem cells on the C57BL/6 background; the targeting construct was generated by inserting sequence containing exons 2–5 of the Tbx21 gene from BAC RP23-237M14 (BACPAC Resources Center) into a plasmid backbone containing a PGK promoter driving expression of diphtheria toxin A subunit (DTA) followed by BGHpA sequence (modified PL452 plasmid). A SalI restriction enzyme site was simultaneously engineered into the Tbx21 3′ UTR between the stop codon and the polyadenylation site. The Clontech Infusion HD Cloning system was used to generate in the pUC19 plasmid backbone sequence containing (in order from 5′ to 3′) encephalomyocarditis virus IRES; tandem dimer (td) Tomato; T2A self-cleaving peptide from Thosea asigna virus; Cre recombinase fused to the oestrogen receptor ligand binding domain (ER); followed by a frt site-flanked PGK-Neomycin resistance gene (NEO)-BGHpA cassette. The IRES-tdTomato-T2A-CreERT2-frt-NEO-BGHpA-frt sequence was PCR-amplified and inserted into the SalI site in the Tbx21 3′ UTR in the modified PL452 backbone. The resulting plasmid was linearized with the restriction enzyme NotI before electroporation into embryonic stem cells. Tbx21^{tdTomato-T2A-cre} mice were generated similarly, with Cre recombinase containing a nuclear localization sequence replacing the CreERT2 sequence. Tbx21^{tdTomato-T2A-creERT2} and Tbx21^{tdTomato-T2A-cre} mice were bred to FLPeR mice to excise the NEO cassette and backcrossed to C57BL/6 mice to remove the FLPeR allele.

Foxp3^fl-DTR mice were similarly generated by insertion of a targeting construct into the Foxp3 locus by homologous recombination in embryonic stem cells on the C57BL/6 background; the targeting construct was generated by inserting sequence containing exons 8–13 of the Foxp3 gene from a 30.8-kb cosmid containing the complete Foxp3 gene into the plasmid backbone containing a PGK promoter driving expression of diphtheria toxin A subunit (DTA) followed by BGHpA sequence (modified PL452 plasmid). The Clontech Infusion HD Cloning system was used to generate in the pUC19 plasmid backbone sequence containing (in order from 5′ to 3′) a loxP site; encephalomyocarditis virus IRES; diptheria toxin receptor (DTR) enhanced green fluorescent protein (eGFP) fusion protein; a triple SV40 polyA (STOP); a second loxP site; encephalomyocarditis virus IRES; Thy1.1; followed by a frt site-flanked PGK-Neomycin resistance gene (NEO)-BGHpA cassette. The loxP-IRES-DTReGFP-STOP-loxP-IRES-Thy1.1-frt-NEO-BGHpA-frt sequence was PCR-amplified and inserted into the BaeI site in the Foxp3 3′ UTR in the modified PL452 backbone. The resulting plasmid was linearized with the restriction enzyme NotI before electroporation into embryonic stem cells. Foxp3^fl-DTR mice were bred to FLPeR mice to excise the NEO cassette and backcrossed to C57BL/6 mice to remove the FLPeR allele.

Foxp3^Thy1.1, R26Y, Foxp3^fl, Foxp3^KO, Rorc^GFP, Foxp3^YFP-cre, IL-10^eGFP, and Tbx21^fl mice have been previously described^{2,20,21,22,23,24,25}. CD45.1, R26^iDTR, and Tcrb^KO mice were purchased from Jackson Laboratories²⁶. Foxp3^Thy1.1Tbx21^CreERT2R26Y (called Tbx21^CreERT2 mice in the text) mice are homozygous at each locus. Tbx21^RFP-creFoxp3^WT, Tbx21^RFP-creFoxp3^fl, Tbx21^RFP-creFoxp3^WT/WT, and Tbx21^RFP-creFoxp3^WT/fl mice described in the text are homozygous for the Tbx21 knock-in allele. Foxp3^fl-DTRTbx21^RFP-creERT2 mice described in the text are homozygous at each locus. Generation and treatments of mice were performed under protocol 08-10-023 approved by the Sloan Kettering Institute (SKI) Institutional Animal Care and Use Committee. All mouse strains were maintained in the SKI animal facility in specific pathogen free (SPF) conditions in accordance with institutional guidelines and ethical regulations.

For tamoxifen administration, 40 mg tamoxifen dissolved in 100 μl ethanol and subsequently in 900 μl olive oil (Sigma-Aldrich) were sonicated 4 × 30 s in a Bioruptor Twin (Diagenode). Mice were orally gavaged with 200 μl tamoxifen emulsion per treatment. For diphtheria toxin (DT) injections, DT (Sigma-Aldrich) was dissolved in PBS and 200 μl of indicated doses (1 μg per mouse unless otherwise indicated) were injected i.p. per mouse. For antibiotic treatment, mice were weaned onto filtered antibiotic-treated water containing ampicillin, kanamycin, vancomycin and metronidazole (0.1% w/v each).

All mice analysed were sex and aged matched (8–12 weeks old) with the exception of some Tbx21^RFP-creFoxp3^WT and Tbx21^RFP-creFoxp3^fl mice used for immunofluorescence imaging that were up to 10 months of age (results were similar to in 8–12-week-old mice).

Isolation of cells

For analysis of YFP-labelled CD4 T cells in Tbx21^RFP-creERT2 mice, CD4 T cells in spleens and lymph nodes were enriched using the Dynabeads CD4 Positive Isolation Kit (Invitrogen). To isolate lymphocytes from tissues, mice were euthanized and immediately perfused with 20 ml PBS. Small and large intestines were removed, flushed with PBS and Peyer’s patches were removed. Subsequently, 0.5-cm-long fragments of intestines were washed in PBS and incubated in PBS supplemented with 5% fetal calf serum, 1% l-glutamine, 1% penicillin–streptomycin, 10 mM HEPES, 1 mM dithiothreitol, and 1 mM EDTA for 15 min. Samples were washed and incubated in digest solution (RPMI supplemented with 5% fetal calf serum, 1% l-glutamine, 1% penicillin–streptomycin, 10 mM HEPES, 1 mg ml⁻¹ collagenase, and 1 U ml⁻¹ DNase I) for 10 min twice. After filtering through a 100-μm strainer, cells were resuspended in 35% Percoll to eliminate debis. Lymphocytes from livers and lungs were isolated by 50–60 min incubation in digest solution, filtered through 100-μm strainers, and after debris removal in 35% Percoll, purified by centrifugation (1,000g, 7.5 min) over a step-wise 44%/67% Percoll gradient at room temperature.

Nippostrongylus brasiliensis and Listeria monocytogenes infections

N. brasiliensis was maintained by passage in 9–10-week-old male Wistar rats as previously described²⁷. In brief, rats were injected subcutaneously (s.c.) with 7000 L3 N. brasiliensis and stool was collected on days 6–9 after infection. Fecal pellets were mixed with 5 × 8 bone charcoal and incubated on moist filter paper in Petri dishes at 26 °C for 7 days. L3 larvae were recovered from the edge of the filter paper and the perimeter of the plates and extensively washed with PBS to eliminate contaminants before infection. Mice infections were carried out using a 23G needle at a concentration of 500 L3 N. brasiliensis in 200 μl. For L. monocytogenes infections, frozen stocks were thawed, resuspended in Brain-Heart Infusion media, and grown at 37 °C to an OD₆₀₀ of 0.1. For primary infections, mice were injected via lateral tail vein with 5–10 × 10³ colony-forming units (cfu) of L. monocytogenes diluted in 200 μl PBS. For secondary infection, mice were injected via lateral tail vein with 10⁵ cfu of L. monocytogenes in 200 μl PBS.

Treatments of rats were performed under protocol 08-10-023 approved by the Sloan Kettering Institute (SKI) Institutional Animal Care and Use Committee. Rats were maintained in the SKI animal facility in Biosafety Level 2 conditions in accordance with institutional guidelines and ethical regulations.

Cell transfer experiments

For cell transfer experiments, pooled spleens and lymph nodes were enriched for CD4 T cells using the Dynabeads CD4 Positive Isolation Kit. Cells were FACS-sorted on an Aria II cell sorter (BD Bioscience), washed 3 times in PBS, resuspended in 200 μl PBS, and transferred into recipients via retro-orbital injection.

Generation of bone marrow chimaeric mice

Tcrb^−/−Tcrd^−/− recipient mice were lethally irradiated with 650 Gy. The following day, bone marrow was isolated from femurs of donor mice and depleted of T cells and RBCs via staining with biotinylated anti-Thy1.2 and anti-Ter119 antibodies followed by magnetic bead negative selection. 5 × 10⁶ total T-cell-depleted bone marrow cells were transferred into recipient mice via retro-orbital injection.

Flow cytometric analysis

Cells were stained with LIVE/DEAD Fixable Yellow Dead Cell Stain (Molecular Probes) and the following antibodies purchased from eBioscience, BioLegend, BD Biosciences, Tonbo, or obtained from the NIH tetramer core facility: anti-CD4 (RM4-5, Biolegend 100548), anti-CD8a (5H10, BD Biosciences 564297), anti-TCRβ (H57-597, eBioscience 47-5961-82), PBS-57-loaded mCD1d tetramer (NIH 26181), anti-Thy1.1 (HIS51, eBioscience 17-0900-82), anti-CD44 (IM7, BioLegend 103026), anti-CD62L (MEL-14, eBioscience 25-0621-82), anti-CXCR3 (CXCR3-173, eBioscience 17-1831-173), anti-CD25 (PC61.5, eBioscience 17-0251), anti-CTLA-4 (UC10-4B9, eBioscience 17-1522-82), anti-GITR (DTA-1, eBioscience 48-5874-82), anti-CD39 (24-DMS1, eBioscience 25-0391-82), anti-CD11b (M1/70, Tonbo Bioscience 25-01120U100), anti-SiglecF (E50-2440, BD Pharmingen 562681), anti-CCR5 (HM-CCR5(7A4) (eBioscience 12-1951-82) and C34-3448 (BD Biosciences 559921), anti-CD29 (eBioHMb1-1, eBioscience 48-0291-80), anti-Foxp3 (FJK-16 s, Tonbo Bioscience 35-5773-U100), anti-T-bet (4B10, BioLegend 644816), anti-RORγt (B2D, eBioscience 12-6981-82), anti-Gata-3 (TWAJ, eBioscience 46-9966-41), anti-DsRed (Living Colours DsRed Polyclonal Antibody, Clontech 632496), anti-IFNγ (XMG1.2, eBioscience 48-7311-80), anti-IL-4 (11B11, eBioscience 51-7041-82), anti-IL-17A (17B7, eBioscience 61-7177-82), anti-IL-13 (eBio13A, eBioscience 12-7133-82), anti-IL-5 (BD Pharmingen, 554396), and anti-IL-2 (JES6-5H4, eBioscience 25-7021-82). Flow cytometric analysis was performed using an LSRII flow cytometer (BD Bioscience) and FlowJo software (Tree Star). Intracellular staining was performed using eBioscience Fixation Permeabilization buffers. For cytokine staining lymphocytes were stimulated with soluble anti-CD3 clone 2C11 (5 μg ml⁻¹) and anti-CD28 clone 37.51 (5 μg ml⁻¹) in the presence of 1 μg ml⁻¹ brefeldin A for 5 h at 37 °C, 5% CO₂. Unless otherwise stated, CD4 T cells were pre-gated as TCRβ⁺ PBS-57-CD1d tetramer⁻ cells.

RNA-seq analysis

Pooled spleens and lymph nodes were enriched for CD4 T cells using the Dynabeads CD4 Positive Isolation Kit. CD4⁺Thy1.1⁺ cells were FACS-sorted on an Aria II cell sorter (BD Bioscience) into four populations (CD62L^hiCD44^loRFP⁻, CD44^hiRFP⁻, CD44^hiRFP^loCXCR3⁻, and CD44^hiRFP^hiCXCR3⁺ cells) and resuspended in Trizol. Three replicates of each cell subset were generated. RNA-sequencing reads were aligned to the reference mouse genome GRCm38 using the Burrows–Wheeler Aligner (BWA)²⁸ and local realignment was performed using the Genome Analysis Toolkit (GATK)²⁹. For each sample, raw count of reads per gene was measured using R, and DESeq2 R package³⁰ was used to perform differential gene expression among different conditions. A cutoff of 0.05 was set on the obtained P values (that were adjusted using Benjamini–Hochberg multiple testing correction) to get the significant genes of each comparison.

TCR sequencing and data analysis

In brief, following isolation of CD4⁺ T cells from spleens and lymph nodes of DO11.10 TCRβ transgenic Tcra^+/−Foxp3^DTR mice using the Dynabeads CD4 Positive Isolation Kit (Invitrogen), CD44^hiCXCR3⁻ and CD44^hiCXCR3⁺eGFP(Foxp3)⁺ T_reg and eGFP⁻ effector CD4 T cells were FACS sorted and stored in Trizol. TCR sequencing and data analysis were performed as previously described³¹. Pearson’s correlation of clonotype frequencies for the shared TCR clones was used for the generation of the dendrogram.

Microscopy

Confocal imaging was done using standard conditions. In brief, mice were perfused in PLP buffer. Lymph nodes and spleens were excised, fixed for 1 h at room temperature in 4% paraformaldehyde, and dehydrated at 4 °C in sucrose (30% in PBS). Tissues were snap-frozen in OCT compound (Sakura Tissue-Tek). 10 μm tissue sections were cut and fixed with Acetone for 20 min at −20 °C, rehydrated in PBS and blocked with 10% normal donkey serum, in PBS with 0.3% Triton X-100, followed by overnight antibody staining at 4°C in a humidified chamber. After antibody staining nuclei were stained with 5 μM Draq7 (Abcam) for 20 min at room temperature. Sections were imaged in Prolong Diamond mounting media (Life Technologies). All images were acquired using a confocal microscope (LSM880; Carl Zeiss) with a 40× oil immersion objective. Images were processed and analysed using ImageJ software (version 2.0.0-rc-54/1.51h; National Institutes of Health). Nearest neighbour analysis was performed using MATLAB (version R2016b, MathWorks).

Statistical analysis

All statistical analyses (excluding RNA-seq and TCR sequence analyses, described above) were performed using GraphPad Prism 6 software. Differences between individual groups were analysed for statistical significance using the unpaired or paired two-tailed t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; NS, not significant. The Kolmogorov–Smirnov test is used to determine the significance between the distributions of signature genes and the rest of expressed genes. One-way ANOVA is used to compare the means of three or more samples. No statistical method was used to predetermine sample size. The number of mice used in each experiment to reach statistical significance was determined on the basis of preliminary data. No animals were excluded from the analyses. No methods of randomization were used to allocate animals into experimental groups. No blinding was used. Data met assumptions of statistical methods used and variance was similar between groups that were statistically compared.

Code availability

The colocalization program (ImageJ software, 2.0.0-rc-54/1.51h, National Institutes of Health) used to find cell positions and the MATLAB program (software R2016b, MathWorks) to calculate nearest cell distance are provided in the Supplementary Information.

Data availability

The RNA-seq data that support the findings of this study have been deposited in the NIH SRA database with the accession code SRP102941.

Extended data figures and tables

Extended Data Figure 1 Analysis of T-bet⁺ cells in Tbx21^RFP-creERT2 reporter mice.

a, Targeting strategy for the Tbx21 locus. b, T-bet protein levels in immune cells in Tbx21^RFP-creERT2 mice. c, T-bet protein levels in Tbx21^RFP-creERT2 mice gavaged with tamoxifen on days −2 and 0 and analysed 3 weeks later. Shaded grey and open histograms represent all and YFP⁺ cells, respectively. d, Flow cytometry of RFP expression in T_reg and non-T_reg CD4 T cells. e, Flow cytometry of splenic T_reg cells. f, Percentage of CD44^hiCD62L^lo among Thy1.1⁺ (top) and RFP⁺ among CD44^hiCD62L^loThy1.1⁺ (bottom) cells in Tbx21^RFP-creERT2 3 weeks (white squares), 3 months (grey squares), and 7 months (black squares) after tamoxifen treatment. g, Flow cytometry of T-bet expression in GATA3⁺ (blue gate, left, and histogram, right) and RORγt⁺ (black gate, left, and histogram, right) T_reg cells isolated from the large intestine laminia propria. h, Percentage RFP⁺ cells among eGFP⁺CD4⁺Thy1.1⁺ (open circles) and Thy1.1⁻ (black circles) cells in Tbx21^RFP-creERT2Rorc^GFP/WT mice. LN, lymph node; SI, small intestine; LI, large intestine. i, Flow cytometry of CD4 T cells in Tbx21^RFP-creERT2Rorc^GFP/WT mice as quantified in h. j, Top, RFP⁺ (left axis, squares) and YFP⁺ (right axis, circles) effector CD4 T cells. Bottom, percentage of RFP⁺ among YFP⁺ effector CD4 T cells 3 weeks (white symbols), 3 months (grey symbols), and 7 months (black symbols) after tamoxifen gavage, as outlined in Fig. 1b. Data are mean ± s.e.m. All data are representative of ≥ 2 experiments, n ≥ 4 mice per group each.

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Extended Data Figure 2 T-bet^lo cells probably represent transient unstable intermediates in the differentiation of stable T-bet^hi T_reg cells.

a, Flow cytometry of the indicated cell subsets. b, CD44 and CD62L expression on RFP⁻CXCR3⁻ (grey shaded histograms, squares), RFP^loCXCR3⁻ (black histograms, squares), and RFP^hiCXCR3⁺ (red histograms, squares) splenic CD4⁺Thy1.1⁺ cells. c, Differential gene expression between CD44^hiRFP⁻ and CD44^hiRFP^hiCXCR3⁺ T_reg cells sorted from pooled spleens and lymph nodes of Tbx21^RFP-creERT2 mice. All genes significantly up- (red) or downregulated (blue) are indicated. d, Expression of the 288 genes up- (≥1.5-fold; left) or 184 genes downregulated (≤1.5-fold; right) in CD44^hiRFP^hiCXCR3⁺ versus CD44^hiRFP⁻ cells. Genes with a mean expression value of <15 were excluded from the analysis. P, paired t-test; adjustments were made for multiple comparisons. e, CD44^loCD62L^hiRFP⁻, CD44^hiRFP⁻, CD44^hiRFP^loCXCR3⁻, and CD44^hiRFP^hiCXCR3^hi CD4⁺Thy1.1⁺ cells were FACS-sorted and transferred into lymphoreplete hosts and analysed in pooled spleens and lymph nodes 14 days after transfer. f, Quantification of data in e using a two-tailed t-test (***P < 0.001). All data are representative of ≥ 2 experiments, n ≥ 2 mice per group each.

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Extended Data Figure 3 Fate mapping T-bet-expressing T_reg cells during infectious challenge.

a, Preferential expansion of CD44^hiRFP⁻ versus CD44^hiRFP⁺ CD4 effector T cells during N. brasiliensis infection. Flow cytometry analysis of splenic (top) and lung (bottom) CD4⁺Thy1.1⁻ cells from mice challenged with PBS (left) and N. brasiliensis (Nb, right). b, Flow cytometry of splenic CD4⁺ Thy1.1⁺ (left) and Thy1.1⁻ (right) cells of mice challenged with PBS (top) and L. monocytogenes (Lm, bottom), as indicated in Fig. 2a. Numbers indicate percentage of RFP⁺ (left) and YFP⁺ (right) cells. c, Top, schematic of experiment. CD44^loCD62L^hiRFP⁻, CD44^hiRFP⁻, and CD44^hiRFP^hiCXCR3^hi CD4⁺Thy1.1⁺ cells were FACS-sorted from pooled spleens and lymph nodes of Tbx21^RFP-creERT2 mice and transferred into lymphoreplete hosts one day before PBS or L. monocytogenes challenge. Bottom, flow cytometry of transferred populations (indicated on left) on day 9 in spleens of mice challenged with PBS (left) or L. monocytogenes (right). d, Representative histograms of RFP and CXCR3 expression on total CD4⁺Thy1.1⁺ (shaded histograms) or Th1.1⁺YFP⁺ (open histograms) cells from spleens of mice challenged with PBS (black) or L. monocytogenes (red), as indicted in Fig. 2a. e–g, eGFP expression in PBS or L. monocytogenes challenged Tbx21^RFP-creERT2Il10^eGFP mice. e, Schematic of tamoxifen (Tx) administration to Tbx21^RFP-creERT2Il10^eGFP/WT mice for data shown in f, g. f, Flow cytometry of T_reg (top) and YFP⁺ T_reg (bottom) cells in spleens of PBS (left) and L. monocytogenes (right) treated mice. g, Left, percentage of RFP⁻eGFP⁺ and RFP⁺eGFP⁺ among T_reg cells, as gated in f (top). Right, percentage of eGFP⁺ cells among YFP⁺ T_reg cells, as gated in f (bottom). h, Schematic of L. monocytogenes reinfection in Tbx21^RFP-creERT2Il10^eGFP/WT mice for data shown in i, j; 1° and 2° indicate primary and secondary challenge, respectively. i, Flow cytometry of cells in Tbx21^RFP-creERT2Il10^eGFP mice on day 65, treated as indicated above. j, Percentage of RFP^–eGFP⁺ and RFP⁺eGFP⁺ cells among Thy1.1⁺ cells, as gated in i. All data are representative of ≥2 experiments, n ≥ 2 mice per group each. Data are mean ± s.e.m. Two-tailed t-test (NS, not significant).

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Extended Data Figure 4 Features of T-bet⁺ T_reg cells.

a, T cell activation, CXCR3 expression, and cytokine production in 12-week-old control Foxp3^YFP-creTbx21^WT/WT and Foxp3^YFP-creTbx21^fl/WT (white circles) and experimental Foxp3^YFP-creTbx21^fl/fl (black circles) mice. Data are mean ± s.e.m. Two-tailed t-test (*P <0.05; NS, not significant). Data are representative of three experiments, n ≥ 7 mice per group. b, Cumulative distribution function plot of the 561 genes up in Thy1.1⁺ CD44^hiRFP^hiCXCR3⁺ versus CD44^hiRFP⁻ cells in Tbx21^RFP-creERT2 mice compared to all genes differentially expressed in CD4⁺CD25⁺ T_reg cells from Tbx21^RFP-creFoxp3^WT mice versus CD4⁺CD25^lo ex-T_reg cells from Tbx21^RFP-creFoxp3^fl mice. P = 0.2 × 10⁻¹⁵, two-sample Kolmogorov–Smirnov test. c, Expression of CCR5 (top) and CD29 (bottom) in CD44^loCD62L^hi naive (blue histogram), CD44^hiCXCR3⁻ (black histogram) and CD44^hiCXCR3⁺ (red histogram) T_reg (left) and CD4⁺Foxp3⁻ (right) T cells from spleens of Foxp3^YFP-creTbx21^WT/WT mice. d, Expression of CXCR3 (left), CCR5 (middle), and CD29 (right), gated on CD4 T cells in spleens of Foxp3^YFP-creTbx21^WT/WT and Foxp3^YFP-creTbx21^fl/fl mice. e, Dendrogram represents cluster analysis of TCR sequences in CD44^hiCXCR3⁺ (red symbols) and CD44^hiCXCR3⁻ (black symbols) T_reg (right) and effector CD4 T (left) cells in spleens (white symbols) and lymph nodes (grey symbols) of DO11.10 TCRβ⁺ Tcra^+/− Foxp3 reporter mice. Sample preparation and statistical analyses are described in the Methods. Pearson’s correlation of clonotype frequencies for the shared TCR clones was used for the generation of the dendrogram.

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Extended Data Figure 5 Characterization of Tbx21^RFP-creFoxp3^fl mice.

a, Targeting strategy for the Tbx21 locus (top) and RFP expression in the indicated cell populations in spleens of homozygous Tbx21^RFP-cre mice (bottom). b, Progressive loss of hair pigmentation in Tbx21^RFP-creFoxp3^fl mice. c, RFP and YFP expression (upper) and CD44 and CD62L expression (lower) in the indicated splenic cell populations in Tbx21^RFP-creR26Y mice. d, Activation and expansion of RFP⁺ T cells in lymph nodes (top) and lungs (bottom) of the indicated mice. e, Cytokine production by CD4⁺Foxp3⁻ and CD8⁺ T cells in lungs of the indicated mice. f, Characterization of lymph node T_reg cells. g, Percentages of ex-T_reg cells in spleens, lymph nodes, and lungs. h, Top, flow cytometry of lymph node CD4 T cells, as quantified in g; numbers indicate the percentage of Foxp3⁻CD25⁺. Bottom, histogram showing expression of T_reg cell signature molecules in CD4⁺Foxp3⁻CD25⁺ cells in lymph nodes of Tbx21^RFP-creFoxp3^WT (open grey histogram), Tbx21^RFP-creFoxp3^fl (open red histogram), Tbx21^RFP-creFoxp3^WT/WT (open blue histogram), and Tbx21^RFP-creFoxp3^fl/WT (open black histogram) mice. CD4⁺Foxp3⁺CD25⁺ cells from a Tbx21^RFP-creFoxp3^WT (shaded grey histogram) mouse are shown as a point of reference. Data are mean ± s.e.m. Two-tailed t-test (***P < 0.001, **P < 0.01 and *P < 0.05, respectively; NS, not significant). Data represent the combined results from several experiments.

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Extended Data Figure 6 The T_H2 response to N. brasiliensis is not exacerbated in Tbx21^RFP-creFoxp3^fl mice.

Tbx21^RFP-creFoxp3^fl and Tbx21^RFP-creFoxp3^WT mice were infected with N. brasiliensis and analysed on day 9 after challenge. a, Flow cytometry of GATA3 expression in CD4⁺Foxp3⁻CD25⁻ T cells in spleens (top) and lungs (bottom) of Tbx21^RFP-creFoxp3^WT (left) and Tbx21^RFP-creFoxp3^fl (right) mice. b, Quantification of data in a. Tbx21^RFP-creFoxp3^WT and Tbx21^RFP-creFoxp3^fl mice are indicated by grey and red circles, respectively. c, Numbers of eosinophils in lungs of the indicated mice. d, Cytokine production by CD4⁺Foxp3⁻ and CD8 T cells in spleens and lungs of the indicated mice. Data are mean ± s.e.m. Two-tailed t-test (*P < 0.05; NS, not significant). Data represents 1 experiment, n ≥ 5 mice per group.

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Extended Data Figure 7 Distinguishing the drivers of autoimmunity in the absence of T-bet⁺ T_reg cells.

a–c, Ex-T_reg cells are no more pathogenic than effector CD4 T cells. a, CD4⁺CD25⁺ (T_reg) cells were sorted from lymph nodes of Tbx21^RFP-creFoxp3^WT mice, and CD4⁺CD25⁻ (effector) and CD4⁺CD25^lo (ex-T_reg) cells were sorted from lymph nodes of Tbx21^RFP-creFoxp3^fl mice for transfer into Tcrb^−/−Tcrd^−/− mice. Intracellular staining for Foxp3 demonstrates purity of cell populations. b, Weights of Tcrb^−/−Tcrd^−/− mice receiving CD4⁺CD25⁺ (white squares), CD4⁺CD25⁻ (black squares), and CD4⁺CD25^lo (grey squares) cells. c, Percentages and numbers of the indicated T cell populations in spleens of mice analysed on day 62 after transfer. d, e, T-bet⁺ effector αβT cells drive disease upon ablation of T-bet⁺ T_reg cells. Lethally irradiated Tcrb^−/−Tcrd^−/− mice were reconstituted with a 1:1 mix of CD45.2+Tbx21^RFP-cre/WTR26^iDTR T-cell depleted bone marrow cells with either CD45.1⁺Foxp3^KO, CD45.1⁺Foxp3^WT, or CD45.2⁺Tcrb^KO T-cell depleted bone marrow cells. Mice were injected with 0.5 μg diphtheria toxin (DT) on day 0, then treated daily with 0.1 μg diphtheria toxin for 22 days before analysis. d, Weight loss in Tbx21^RFP-cre/WTR26^iDTR:Foxp3^KO (red line) versus Tbx21^RFP-cre/WTR26^iDTR:Foxp3^WT (black line) versus Tbx21^RFP-cre/WTR26^iDTR:Tcrb^KO (blue line) reconstituted mice. e, Representative flow cytometry of splenic cell populations (indicated on right) in chimaeric mice (as indicated above). All data represent 1 experiment, n ≥ 3 mice per group.

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Extended Data Figure 8 Co-localization of T-bet⁺ T_reg and T-bet⁺ effector T cells in vivo.

a, b, Representative images (left) and insets (right) of lymph node sections from Tbx21^RFP-cre mice with CD4 (a) or CD8 (b) in green, RFP in red, Foxp3 in blue, and CD44 in grey. In inset, arrowheads indicate CD4⁺CD44^hiRFP⁺Foxp3⁻ (a) or CD8⁺CD44^hiRFP⁺ (b) cells and arrows indicate CD4⁺CD44^hiRFP⁺Foxp3⁺ cells. c, d, Quantification of nearest distances between T_reg cells and CD4 (c) and CD8 (d) T cells, as shown in a, b. Foxp3⁺ denotes CD4⁺CD44^hiFoxp3⁺; Foxp3⁻ (c) denotes CD4⁺CD44^hiFoxp3⁻ and CD8⁺ (d) denotes CD8⁺CD44^hiRFP⁺. e, f, Quantification of nearest distances between T_reg and non-T_reg CD4 (e) and CD8 (f) T cells in spleens of Tbx21^RFP-creFoxp3^WT and Tbx21^RFP-creFoxp3^fl mice. Genotypes of mice are indicated above plots; cell types being analysed are shown below plots, as in c, d. Bars indicate mean. P values were calculated using a two-tailed t-test (c, d) or one-way ANOVA (e, f) (***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant). Data are representative of multiple imaged sections from ≥ 2 mice.

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Extended Data Figure 9 T-bet⁺ T_reg cells suppress pre-established T_H1 but not T_H2 or T_H17 activation induced by depletion of T_reg cells.

a, Targeting strategy for the Foxp3 locus. b, Schematic for experiment shown in c–g depleting all T_reg cells and subsequently depleting all or only non-T-bet-expressing T_reg cells in Foxp3^fl-DTRTbx21^RFP-creERT2 mice. c, Flow cytometry of splenic CD4 T cells in the indicated mice treated with tamoxifen or oil, as indicated. d–g, Percentages of T_reg cells (d) and activation status of (e) and cytokine production by (f, g) splenic CD4⁺Foxp3⁻ and CD8 T cells in tamoxifen-treated Foxp3^Thy1.1Tbx21^RFP-creERT2 (open circles), mock oil-treated Foxp3^fl-DTRTbx21^RFP-creERT2 (black circles), and tamoxifen-treated Foxp3^fl-DTRTbx21^RFP-creERT2 (grey circles) mice. h–l, T_reg cells rebounding post depletion in DT-treated Foxp3^DTRTbx21^RFP-creERT2 mice efficiently suppress T_H2 responses. h, Left, schematic for control experiment shown in i–l. Right, flow cytometry of splenic CD4 T cells in mice treated with high dose diphtheria toxin (DT^hi, 1 μg per mouse), low dose diphtheria toxin (DT^lo, 0.0625 μg per mouse), and PBS. Group 1 (control); group 2 (depletion without T_reg cell recovery); group 3 (depletion with partial recovery); group 4 (depletion with full recovery). i–l, Percentages of T_reg cells (i) and activation status of (j) and cytokine production by (k, l) splenic CD4⁺Foxp3⁻ and CD8 T cells in the indicated groups of mice. Data are mean ± s.e.m. Two-tailed t-test (***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant). Data are representative of ≥ 1 experiment, n ≥ 4 mice per group.

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Extended Data Figure 10 T_reg cells rebounding post transient depletion efficiently suppress T_H2 and T_H17 responses.

a, Experimental schematic. Mice were treated with tamoxifen (tx) or oil (to additionally control for potential effects of tamoxifen) on days −5 and −3 and received PBS on days 0, 1, 3, 5, 7 (control); 1 μg diphtheria toxin (DT^hi) on days 0, 1, 3, 5, and 7 (no T_reg cell recovery); 0.062 μg diphtheria toxin (DT^lo) on days 0, 1, 3, 5, and 7 (partial T_reg cell recovery); or 0.062 μg diphtheria toxin (DT^lo) on day 0 and PBS on days 1, 3, 5, and 7 (full T_reg cell recovery). Mice were analysed on day 9. b, Flow cytometry analysis of CD4 T cells in spleens of the indicated groups of mice. c–e, Percentages of T_reg cells (c) and CD4⁺Foxp3⁻ and CD8 T cell activation (d) and cytokine production (e) in spleens of the indicate mice (group 1, open circles; group 2, black circles; group 3, dark grey circles; group 4, light grey circles). Data are mean ± s.e.m. Two-tailed t-test (***P < 0.001, **P < 0.01; NS, not significant). Data represent the combined results from two experiments, n ≥ 3 mice per group.

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