ARTICLE

DOI: 10.1038/s41467-018-04392-5 OPEN mTOR coordinates transcriptional programs and mitochondrial metabolism of activated Treg subsets to protect tissue homeostasis

Nicole M. Chapman1, Hu Zeng 1, Thanh-Long M. Nguyen1, Yanyan Wang1, Peter Vogel 2, Yogesh Dhungana1, Xiaojing Liu3, Geoffrey Neale 4, Jason W. Locasale 3 & Hongbo Chi1

1234567890():,; Regulatory T (Treg) cells derived from the thymus (tTreg) and periphery (pTreg) have central and distinct functions in immunosuppression, but mechanisms for the generation and acti-

vation of Treg subsets in vivo are unclear. Here, we show that mechanistic target of rapamycin

(mTOR) unexpectedly supports the homeostasis and functional activation of tTreg and pTreg

cells. mTOR signaling is crucial for programming activated Treg-cell function to protect

immune tolerance and tissue homeostasis. Treg-specific deletion of mTOR drives sponta- neous effector T-cell activation and inflammation in barrier tissues and is associated with

reduction in both thymic-derived effector Treg (eTreg) and pTreg cells. Mechanistically, mTOR functions downstream of antigenic signals to drive IRF4 expression and mitochondrial metabolism, and accordingly, deletion of mitochondrial transcription factor A (Tfam) severely

impairs Treg-cell suppressive function and eTreg-cell generation. Collectively, our results show

that mTOR coordinates transcriptional and metabolic programs in activated Treg subsets to mediate tissue homeostasis.

1 Department of Immunology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA. 2 Department of Pathology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS 250, Memphis, TN 38105, USA. 3 Department of Pharmacology & Biology, Duke University School of Medicine, Levine Science Research Center C266, Box 3813, Durham, NC 27710, USA. 4 Hartwell Center for Bioinformatics and Biotechnology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS 312, Memphis, TN 38105, USA. Correspondence and requests for materials should be addressed to H.C. (email: [email protected])

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egulatory T (Treg) cells expressing the transcription factor pathways associated with increased suppressive function of Treg Foxp3 suppress conventional T-cell responses to establish cells, we mined a published dataset of activated Treg cells isolated R DTR self-tolerance, prevent autoimmunity, and maintain tissue from diphtheria toxin (DT)-treated Foxp3 mice (DTR, 1,2 fi 32 homeostasis . Foxp3 de ciency eliminates Treg-cell development diphtheria toxin receptor) . set enrichment analysis and function, leading to autoimmune diseases characterized by (GSEA) revealed that the hallmark mTORC1 and PI3K-Akt- fi excessive T helper 1 (TH1), TH2, or TH17 responses, and germinal mTOR signaling pathways were among the most signi cantly center (GC) B-cell reactions driven by T follicular helper (TFH) (false discovery rate, FDR < 0.05) upregulated gene sets in acti- 3–5 cells . Thymic-derived Treg (tTreg) cells exit the thymus and vated vs. resting Treg cells (Fig. 1a). Thus, increased Treg-cell populate peripheral tissues, where resting Treg cells [also called suppressive activity is correlated with enhanced mTOR signaling. central Treg (cTreg) cells] are activated in response to antigen and To rigorously test the function of mTOR for the suppressive 6–9 inflammatory cues . These activation signals increase effector activity of activated Treg cells, we activated Treg cells in vitro in the molecule expression and induce transcription factors that define presence or absence of the mTOR inhibitor, PP242. We found the selective suppressive functions and tissue localization of that acute inhibition of mTOR diminished the ability of activated activated Treg cells [also known as effector Treg (eTreg) Treg cells to suppress conventional T-cell proliferation (Fig. 1b) 5,10–15 cells] . Peripherally-derived Treg (pTreg) cells are a devel- and to express the immunosuppressive molecule CTLA4 (Fig. 1c), opmentally distinct population of activated T cells that arises indicating a -dependent function of mTOR in T -cell + reg reg from the naive CD4 T-cell pool and inhibit T 2orT17 function. Accordingly, the suppressive activity of T cells iso- – H H fl fl reg responses at mucosal sites6,16 19. The transcription factor inter- lated from Cd4CreMtor / mice was dampened (Fig. 1d). Thus, feron regulatory factor 4 (IRF4) is expressed in both eTreg and mTOR is essential for the suppressive function of Treg cells pTreg cells in vivo and is an essential positive regulator of their in vitro. 7,15,17,20–22 homeostasis and function . IRF4 expression and func- To establish a role for mTOR in Treg-cell function in vivo, we Cre/DTR fl/fl tion are induced by TCR signals in Treg cells by incompletely generated female Foxp3 Mtor mosaic mice. These mice understood mechanisms7,8,22. express a floxed Mtor allele24, whose expression can be deleted by Metabolic rewiring is important for T-cell fate decisions, but Cre recombinase driven under the Foxp3 promoter (denoted as Cre 33 the metabolic programs regulating Treg-cell activation and Foxp3 ) , resulting in the deletion of mTOR within Treg cells specialization remain uncertain23. The activation of the mechan- after they have expressed Foxp3. Acute depletion of DTR- Cre istic target of rapamycin (mTOR) induces metabolic reprogram- expressing Treg cells with DT forces the remaining Foxp3 - ming necessary for conventional T-cell activation expressing Treg cells to become activated, expand, and control and differentiation23,24. In contrast, mTOR appears to antagonize immune homeostasis in adult mice34. Upon DT treatment, fl fl T -cell differentiation and expansion in vitro and suppressive Foxp3Cre/DTRMtor / mosaic mice, but not their respective reg + + + fl activity in vivo23,25,26. Mechanistically, inhibition of mTOR Foxp3Cre/DTRMtor / or / controls, developed inflammation upregulates fatty acid oxidation, which supports mitochondrial associated with increased organ size and cell number, especially respiration important for T -cell differentiation, proliferation, peripheral lymph nodes (Fig. 1e). Further, there were increased reg + and survival in vitro27,28. Moreover, low levels of mTOR activa- frequencies of CD44hiCD62Llo effector/memory CD4 in the tion are needed to prevent excessive that can impair peripheral lymph nodes (Fig. 1f). After DT treatment, Foxp3Cre/ 23 DTR fl/fl Treg-cell survival and lineage stability . Although the prevailing Mtor mice had a profound enrichment for IL-4-producing model is that mTOR activation hinders T -cell function, T and small but significant increase in IL-17A-producing, but not reg reg + cells have higher basal levels of mTORC1 activation than IFN-γ-producing, CD4 T cells in the spleen (Fig. 1g, h). Similar 29,30 conventional T cells , which is essential for Treg-cell function observations were found in peripheral lymph nodes (Fig. 1g). in vivo30. Thus, mTOR-dependent metabolic programming might Therefore, mTOR signaling is essential for the suppressive have context-dependent roles in different Treg-subsets or under function of activated Treg cells, and its acute deletion in Treg distinct physiological conditions. cells leads to loss of immune homeostasis and the activation of Here, we show that mTOR orchestrates activation-induced TH2, and to a lesser extent, TH17 cells. transcriptional and metabolic signatures that are essential fi for Treg-cell activation and function. We nd that either acute or chronic inhibition of mTOR disrupts Treg-cell suppres- Treg cells require mTOR to prevent spontaneous auto- sive activity and leads to uncontrolled conventional T-cell immunity. To determine the effects of long-term deletion of Mtor + activation. In line with this observation, mucosal CD4 T-cell on Treg-cell suppressive function in vivo, we next generated mice responses, including T 2 responses, are increased when T bearing a conditional deletion of Mtor within all committed H reg + fl fl cells lose mTOR, associated with a loss of eT and pT cells Foxp3 T cells (denoted as Foxp3CreMtor / mice). As antici- reg reg reg + in mucosal sites. Mechanistically, mTOR mediates T -cell pated, Mtor was efficiently deleted within Foxp3-YFP T cells reg fl fl reg activation and suppressive activity by promoting IRF4 expression from Foxp3CreMtor / mice (Supplementary Fig. 1a). In contrast and mitochondrial metabolism. Indeed, disruption of mitochon- to their littermate controls that remained healthy, Foxp3Cre fl fl drial metabolism severely impairs the suppressive function Mtor / mice developed an early-onset lymphoproliferative and of activated Treg cells and their homeostasis in tissues. autoimmune disease, indicated by reduced body size and hunched Collectively, our results show that mTOR controls peripheral posture, enlargement of peripheral lymphoid organs, and exten- tolerance by integrating transcriptional and metabolic programs sive lymphocyte and/or myeloid cell infiltration in multiple critical for the homeostasis and suppressive activity of activated organs, such as the skin and lung (Fig. 2a–c). This disease ulti- Cre fl/fl Treg cells. mately led to the early death of Foxp3 Mtor mice (Fig. 2d). These mice had reduced frequencies of CD44loCD62Lhi naive + + CD4 and CD8 T cells and increased frequencies of + + Results CD44hiCD62Llo effector/memory phenotype CD4 and CD8 mTOR promotes activated T -cell suppressive activity.T T cells (Fig. 2e). There were also significant increases in IFN-γ-, reg reg + cells activated in vivo have enhanced suppressive activity critical IL-4-, IL-10-, IL-13-, and IL-17A-producing CD4 T cells and 7,8,31,32 γ + fi for immune homeostasis , yet the molecular events con- IFN- -producing CD8 T cells in mice with mTOR-de cient Treg fi Cre fl/fl trolling Treg-cell activation remain to be fully de ned. To identify cells (Fig. 2f and Supplementary Fig. 1b). Foxp3 Mtor mice

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a b 0.0 ) DMSO mTORC1 signaling Top 20 leading edge PI3K-Akt-mTOR signaling Top 20 leading edge 5 –0.1 2 FDR < 0.001 FDR < 0.05 –0.2 genes: Bub1, Ccnf, genes: Cdk1, Cdkn1a, PP242

–0.3 Aurka, , Lgmn, Il4, E2f1, Prkar2a, No Treg cells –0.4 Rrm2, Dhfr, Fgl2, Dapp1, Hsp90b1, Pten, –0.5 1 –0.6 Cdkn1a, Tmem97, Cab39l, Map2k3, Egln3, Elovl6, Gla, Camk4, Cdk2, Sla, Enrichment score Glrx, Tfrc, Hk2, Uchl5, Enrichment score Mapk8, Gngt1, Hras,

H] TdR uptake (× 10 0 3

Sord, Hmgcr, Asns Pikfyve, Actr2, Prkcb, [ aT > rT 0:1 1:8 1:16 aTreg > rTreg reg reg Cltc Treg:TN ratio

c d Cd4CreMtor +/fl e Foxp3 Cre/DTRMtor +/+ or+/fl (n=4) DMSO 1 2 Cd4CreMtor fl/fl Cre/DTR +fl/fl ) Foxp3 Mtor (n=5) PP242 5 8 No T cells 30915 reg 1.5 6 ns *** )

10746 8 4 1.0 2 0 0.5 H] TdR uptake (× 10 # Cells (× 10

3 0:1 1:2 1:4 1:8 [ 1: Foxp3 Cre/DTRMtor +/+ 0 Treg:TN ratio CTLA4 2: Foxp3 Cre/DTRMtor fl/fl Spleen pLN

f g Foxp3 Cre/DTRMtor +/+ Foxp3 Cre/DTRMtor fl/fl h Cre/DTR fl/fl 58 Foxp3 Cre/DTRMtor +/+ or +/fl (n=4) Foxp3 Mtor (n=5) 0.975 9.07 ns – Cre/DTR +/+ 60 25 ** Foxp3 Mtor Spleen 20 ns Foxp3 22.4 + 40

cells 15 + γ CD4

17.9 + 10 2.44 24.1 20

% IFN- 5 * Foxp3 Cre/DTRMtor fl/fl pLN 0

0 % Cytokine 51.9 + + + + IL-4 CD4 CD8 IL-4 IL-17A CD62L Foxp3– CD44 CD4

Fig. 1 mTOR is essential for activated Treg cell function. a Enrichment plots of the Hallmark mTORC1 (left) and Hallmark PI3K-Akt-mTOR (right) signaling pathways in activated Treg (aTreg) compared to resting Treg (rTreg) cells, identified by gene set enrichment analysis (GSEA). The top 20 enriched genes in each pathway (position indicated by the vertical black line) are listed to the right of each plot. b In vitro suppressive activity of Treg cells activated in the + presence or absence of PP242. TN: naive CD4 T cells. c Flow cytometry analysis of CTLA4 expression in Treg cells activated in the presence or absence of Cre +/+ or +/fl Cre fl/fl PP242. d In vitro suppressive activity of Treg cells isolated from Cd4 Mtor or Cd4 Mtor mice. e Representative image of lymphadenopathy in Foxp3Cre/DTRMtorfl/fl mice after DT treatment (left). Right, cell numbers of the spleen and peripheral lymph nodes (pLN) of Foxp3Cre/DTRMtor+/+ or +/fl or Foxp3Cre/DTRMtorfl/fl mice. f Flow cytometry analysis of naive and effector/memory CD4+Foxp3-YFP− in pLN. g, h Cells from the spleen and pLN of Foxp3Cre/DTRMtor+/+ or +/fl or Foxp3Cre/DTRMtorfl/fl mice that received DT treatments were stimulated using PMA and ionomycin for 4–5h. g Flow cytometry analysis of IL-4-producing CD4+ T cells in the spleen and pLN. h Quantification of IFN-γ+ CD4+Foxp3− and CD8+ T cells or IL-4+ and IL-17A+ CD4+Foxp3− T cells in the spleen. Error bars show mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; unpaired, two-tailed Student’s t- test. Data are representative of three (c) or four (e–g) biological replicates from three (c) or two (e−g) independent experiments. Data are quantified from three technical replicates representative of three independent experiments (b, d) or four or five biological replicates per group as indicated, compiled from two independent experiments (e, h). Numbers indicate percentage of cells in gates

+ − hi + + had 5–10-fold and 10–15-fold increases in the frequencies of cells CD4 Foxp3-YFP CD44 CXCR5 PD-1 TFH cells from Cre fl/fl producing TH2- or TH17-associated cytokines, respectively, while Foxp3 Mtor mice and their littermate controls, and IFN-γ-producing cells were increased by ~5-fold (Supplementary measured the expression of Il4 and Il21.TFH cells from γ Cre fl/fl Fig. 1c). Within Treg cells, the frequency of IFN- -producing cells Foxp3 Mtor mice had increased expression of Il4, but not Cre fl/fl was also increased in Foxp3 Mtor mice (Supplementary Il21, while non-TFH cells had increased expression of both Il4 and Fig. 1d). We also found that the frequencies and total numbers of Il21 (Supplementary Fig. 1g, h). Thus, constitutive depletion of + + + + PD-1 CXCR5 T cells (Fig. 2g) and CD95 GL7 GC B cells mTOR revealed its essential role for T cell-mediated suppres- FH fl fl reg (Fig. 2h) were increased in Foxp3CreMtor / mice. We, therefore, sion of conventional T-cell responses in vivo. performed immunohistochemistry analysis of GCs, B cells, and + T cells in whole tissue sections. This analysis revealed that PNA cells were diffusely distributed in extrafollicular regions, while T mTOR supports Treg-cell suppression of mucosal TH2 and B cells were markedly increased, in mesenteric lymph nodes responses.T cells regulate T-cell responses important for tissue fl fl reg (Supplementary Fig. 1e). Foxp3CreRptor / mice, which have homeostasis, especially at barrier surfaces like the lung, intestines, 30 1,2 Cre fl/fl impaired mTORC1 signaling , also had elevated TFH and GC B- and skin . We found that in the lung of Foxp3 Mtor cell responses (Supplementary Fig. 1f), indicating that mTORC1 mice, there were respective 5–10-fold and 10–15-fold increases of + is essential for the Treg-cell-mediated suppression of spontaneous IL-4- and IL-13-producing CD4 T cells, while the increases in – GC reactions. To determine if TFH cells produce elevated levels TH1 and TH17 responses were less pronounced (2 3-fold 35,36 of IL-4 and/or IL-21 to promote GC reactions , we isolated increased) (Fig. 3a and Supplementary Fig. 2a). TH2 and TH17 + − hi − − CD4 Foxp3-YFP CD44 CXCR5 PD-1 non-TFH cells and responses were also more elevated than TH1 responses in the

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a b c 1 2 1 2 Foxp3 CreMtor +/+ (n=12) Esophagus (20×) Liver (20×) Lung (10×) Skin (20×) Foxp3 CreMtor +/fl (n=12) pLN Foxp3 CreMtor fl/fl (n=24) Foxp3 Cre Mtor +/fl 150

) *

6 ns ** 100 ns ns Spleen ns 50 Foxp3 Cre fl/fl

# Cells (× 10 Mtor 1: Foxp3 CreMtor +/fl 1: Foxp3 CreMtor +/fl 0 Cre fl/fl Spleen pLN 2: Foxp3 Mtor 2: Foxp3 CreMtor fl/fl

d e Cre +/fl Cre fl/fl f Foxp3 CreMtor +/fl (n=14) Foxp3 Mtor Foxp3 Mtor Foxp3 CreMtor +/+ or +/fl Foxp3 CreMtor fl/fl Foxp3 CreMtor fl/fl (n=37) – 100 *** + 40 3 * CD4 78.7 29.9 *** 75 Foxp3 30 + 2 19.6 67.1 cells 50 + CD4

γ ***

20 +

% Survival 25 1 *** 10 0 % IFN- 90.1 27.6 0 25 50 75 100 CD8+ 0 0 % Cytokine Days CD4+Foxp3– CD8+ IL-4+ IL-17A+

CD62L 8.1 67 CD44 g h 3.24 Foxp3 CreMtor +/+ or +/fl Foxp3 CreMtor fl/fl 0.28 Foxp3 CreMtor +/+ or +/fl Foxp3 CreMtor fl/fl Cre Foxp3 Cre Foxp3 30 1.5 +/fl 4 5 * Mtor +/fl ** * Mtor *** ) ) 6 6 4 3 20 1 3

23.0 cells 4.32 2 Foxp3 Cre FH Foxp3 Cre

cells (× 10 2 fl/fl 10 0.5 fl/fl

Mtor FH Mtor % T % GC B cells 1 1 # T # GC B cells (× 10 GL7 CXCR5 0 0 0 0 PD-1 CD95

Cre +/fl Cre fl/fl Fig. 2 Disruption of mTOR in Treg cells results in fatal autoimmunity. a Representative image of 47-day-old Foxp3 Mtor and Foxp3 Mtor littermates. b Representative image of lymphadenopathy in 47-day-old Foxp3CreMtorfl/fl mice (left). Right, cell numbers of the spleen and peripheral lymph nodes (pLN) of Foxp3CreMtor+/+, Foxp3CreMtor+/fl,orFoxp3CreMtorfl/fl mice. The numbers of mice per group are indicated. c Representative hematoxylin and eosin staining of the indicated tissues from 6-week-old Foxp3CreMtor+/fl and Foxp3CreMtorfl/fl mice. The magnifications are indicated above the respective images for each tissue. d Survival curve of Foxp3CreMtor+/fl and Foxp3CreMtorfl/fl mice. The numbers of mice per group are indicated. e Flow cytometry analysis of naive and effector/memory CD4+Foxp3-YFP– (depicted as CD4+) or CD8+ T-cell populations. f Splenocytes from Foxp3CreMtor+/+ or +/fl and Foxp3CreMtorfl/fl mice were stimulated using PMA and ionomycin for 4–5 h. Cytokine production by CD4+ and CD8+ T cells was assessed by + + Cre +/+ or +/fl flow cytometry and quantified. g Flow cytometry analysis of PD-1 CXCR5 TFH cells. Right, frequency and number of TFH cells in Foxp3 Mtor and Foxp3CreMtorfl/fl mice. h Flow cytometry analysis of CD95+GL7+ GC B cells. Right, frequency and number of GC B cells in Foxp3CreMtor+/+ or +/fl and Foxp3CreMtorfl/fl mice. Error bars show mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; unpaired, two-tailed Student’s t-test. Data are representative of at least twelve (a, b, e) or three (c) biological replicates per group. Data are quantified from the numbers of mice as indicated in the legend key (b, d) or from ten or eleven (f, IFN-γ+ and IL-17A+ cells from Foxp3CreMtor+/+ or +/fl or Foxp3CreMtorfl/fl mice, respectively), nine or ten (f, IL-4 + cells from Foxp3CreMtor+/+ or +/fl or Foxp3CreMtorfl/fl mice, respectively), eight (g), or nine (h) biological replicates per group, compiled from more than eight independent experiments (f−h). Numbers indicate percentage of cells in gates colon lamina propria (Fig. 3b). Because we found a consistent also associated with the accumulation of mucosal mast cells fl fl increase of T 2 cytokines in both the lung and colon lamina (MMCs) in the intestines37. Foxp3CreMtor / mice had an H + + propria and acute deletion of mTOR led to a profound increase of increase of MCPT1 interepithelial MMCs and MCPT4 lamina TH2 responses (Fig. 1h), we next performed comprehensive propria MMCs in the large and small intestines (Fig. 3f). Alto- immunohistochemistry analyses of multiple organs in Foxp3- gether, these results underscore an important role for mTOR in Cre fl/fl Mtor mice. Elevated TH2 responses are associated with an mediating Treg-cell-dependent suppression of effector T-cell accumulation of eosinophils, alternatively activated M2 macro- responses, especially T 2-associated events, within mucosal + H phages, and neutrophils in target tissues16. Indeed, MBP eosi- tissues. nophils were increased in the lung (Fig. 3c), as well as the dermis of the skin (Supplementary Fig. 2b) of mice-bearing mTOR- + deficient T cells. Additionally, CD163 macrophages were mTOR enforces mucosal tT - and pT -cell homeostasis. reg + reg reg expanded, including Ym1 M2 macrophages present in the Recent work shows that tTreg cells present in tissues exhibit TH2- alveolar space and interstitium of the lung (Fig. 3d). Increased M2 biased gene signatures and express transcription factors essential 12,38,39 macrophage activation was also evident in the skin (Supple- for the suppression of TH2 responses . Additionally, the mentary Fig. 2c). We also observed an increase of cells positive absence of pT cells drives elevated T 2 responses in mucosal – reg H for iNOS2, which primarily stains for neutrophils, in the lung tissues16 18. Therefore, we hypothesized that reduced abundance fl (Fig. 3e) and skin (Supplementary Fig. 2d). TH2in ammation is of tTreg and/or pTreg cells might account for increased TH2

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a b Lung Colon LP

0.95 0.81 2.5 2.1 4.9 2.9 8.1 38.9 Cre Foxp3 Cre Foxp3 +/fl Mtor +/fl Mtor

6.0 6.6 Cre 21.2 Foxp3 Cre 7.3 9.7 Foxp3 7.8 10.6 36.7 fl/fl Mtor fl/fl Mtor γ γ IL-13 IL-17A IL-4 IFN- IL-4 IL-13 IFN- IL-17A CD4 CD4

c d Lung e Lung (20×) Macrophages (20×) M2 macrophages (20×) Lung (20×)

Foxp3 Cre Foxp3 Cre Foxp3 Cre Mtor +/fl Mtor +/fl Mtor +/fl

Foxp3 Cre Foxp3 Cre Foxp3 Cre Mtor fl/fl Mtor fl/fl Mtor fl/fl

MBP CD163 Ym1 iNOS2

f Large intestine Small intestine ieMMCs (20×) lpMMCs (20×) ieMMCs (20×) lpMMCs (20×)

Cre Foxp3 Cre Foxp3 +/fl Mtor +/fl Mtor

Cre Foxp3 Cre Foxp3 fl/fl Mtor fl/fl Mtor

MCPT1 MCPT4 MCPT1 MCPT4 + Fig. 3 Treg cells require mTOR for the suppression of mucosal TH2 responses. a, b Flow cytometry analysis of cytokine-producing CD4 T cells isolated from the lung (a) or colon lamina propria (LP) (b)ofFoxp3CreMto+/fl or Foxp3CreMtorfl/fl mice. c Representative images of major basic protein (MBP) staining for eosinophils in the lung of Foxp3CreMtor+/fl and Foxp3CreMtorfl/fl mice. d Representative images for M2 macrophages in the lung of Foxp3CreMtor +/fl or Foxp3CreMtorfl/fl mice by CD163 and Ym1 staining. e Representative images for neutrophils, as indicated by inducible nitric oxide synthease 2 (iNOS2) staining, in the lung of Foxp3CreMtor+/fl and Foxp3CreMtorfl/fl mice. f Representative immunohistochemistry of ieMMCs and lpMMCs in the large intestines (left) and small intestines (right) of Foxp3CreMtorfl/+ or Foxp3CreMtorfl/fl mice. Data are representative of four independent experiments (a, b)or three biological replicates per group (c−f). Numbers indicate percentage of cells in gates

Cre fl/fl + γ – responses in the lung and colon of Foxp3 Mtor mice. observations in the lung, pTreg cells, as well as Helios or ROR t Neuropilin-1 (Nrp1) and Helios are expressed at higher levels in tT cells, were reduced in the colon lamina propria of Foxp3- reg fl fl + tT than pT cells40,41. We found that there was a significant CreMtor / mice (Fig. 4d, e). The reduction of RORγt pT cells reg reg + − reg decrease in the frequency of Nrp1 tT and Nrp1 pT cells in may also contribute to the increased T 17 cell activation in the fl fl reg reg flHfl the lung of Foxp3CreMtor / mice (Fig. 4a). Helios staining colon lamina propria of Foxp3CreMtor / mice (Fig. 3b)19. Ana- + − revealed a similar reduction in tT and pT cells in the lung of lysis of Helios and Helios T -cell populations in the colon fl fl reg reg reg Foxp3CreMtor / mice (Fig. 4b). We tested if these effects were lamina propria from mixed bone marrow chimeras verified cell- + cell-intrinsic by adoptively transferring an equal ratio of CD45.1 intrinsic effects (Fig. 4f). Altogether, these results indicate that the + + fl wild-type bone marrow cells and CD45.2 Foxp3CreMtor / or accumulation of mucosal tissue tT and pT cells is disrupted fl fl – – reg reg Foxp3CreMtor / bone marrow cells into irradiated Rag1 / -reci- in the absence of mTOR. pient mice. This inflammation-free system confirmed that the To determine the role for mTOR in pT -cell maintenance − +reg reduction of these lung T -cell populations was cell-intrinsic in vivo, we purified naive Foxp3-YFP CD4 T cells from either reg + fl fl fl (Fig. 4c). We next examined pT and tT cell populations in Foxp3CreMtor / or Foxp3CreMtor / mice and adoptively trans- reg flregfl – – the colon lamina propria of Foxp3CreMtor / mice by staining for ferred these cells into Rag1 / mice (Fig. 4g). In this system, naive either Helios or RORγt, a transcription factor selectively enriched T cells can acquire Foxp3 expression42, and the concomitant 17,19 in pTreg cells isolated from the intestines . Similar to our expression of the Cre transgene induces Mtor deletion in pTreg

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a Foxp3 CreMtor +/+ or +/fl b Foxp3 CreMtor +/+ or +/fl cdCD45.2+Foxp3 CreMtor +/fl chimera Foxp3 CreMtor +/+ or +/fl Foxp3 CreMtor fl/fl Foxp3 CreMtor fl/fl CD45.2+Foxp3 CreMtor fl/fl chimera Foxp3 CreMtor fl/fl 10 6 ** 6 30 *** *** *** cells cells cells cells ** *** *** + + + 4 4 + 20 β β β *** β 5 TCR TCR TCR TCR

+ 10 + 2 2 + + % Cells in % Cells in % Cells in % Cells in CD4 CD4 CD4 0 0 0 CD4 0 Foxp3+ Foxp3+ Foxp3+ Foxp3+ Foxp3+ Foxp3+ Foxp3+ Foxp3+ Nrp1+ Nrp1– Helios+ Helios– Helios+ Helios– Helios+ Helios–

ef+ Cre +/fl g Foxp3 CreMtor +/+ or +/fl CD45.2 Foxp3 Mtor chimera Naive Foxp3-YFP–CD4+ T cells 18.8 17.5 Foxp3 CreMtor fl/fl CD45.2+Foxp3 CreMtor fl/fl chimera from Foxp3 CreMtor +/fl or Foxp3 CreMtor fl/fl Foxp3 Cre 30 12 *** Donor mice Mtor +/fl *** *** cells cells ** + + 20 8 β 61.7 1.92 β Foxp3-YFP+ donor- 12.2 5.33 TCR TCR 4 + Cre + 10 derived pTreg cells % Cells in Foxp3 % Cells in Mtor fl/fl CD4 CD4 0 0 Rag1–/– mouse Foxp3+ Foxp3+ Foxp3+ Foxp3+ 4 weeks + – + – Foxp3 80.1 2.36 RORγt RORγt Helios Helios RORγt

hijk Foxp3 CreMtor+/fl donor (n=3) Foxp3 CreMtor+/fl donor (n=3) Vehicle Torin 1 Foxp3 CreMtor +/+ or +/fl Foxp3 CreMtor fl/fl donor (n=5) Foxp3 CreMtorfl/fl donor (n=5) Cre fl/fl 2.5 * ** Foxp3 Mtor ) )

6 2.0

5 ** 2.5 ** 1.5 8 ns 10 * 1.5 2.0 6 cells

1.0 reg cells 1.0 1.5 ns ns T + reg 4 0.5 5 1.0 cells (× 10

0.5 T cells (× 10 Normalized GATA3 + reg 2 0 % pT 0.5 IL-2: + + – – 0 # pT 0 0 0 % GATA3 # CD4 IL-4: – – + + Butyrate: – + – + + – + Fig. 4 Mucosal tTreg- and pTreg-cell homeostasis is altered in the absence of mTOR. a, b Quantification of the frequencies of Nrp1 and Nrp1 (a) or Helios – Cre +/+ or +/fl Cre fl/fl + and Helios Treg cells (b) in the lung of Foxp3 Mtor and Foxp3 Mtor mice, respectively. c Quantification of the frequencies of Helios and – + + + + Helios Treg cells among the CD45.2 CD4 TCRβ T cells in the lung of mixed bone marrow chimeras. d Quantification of the frequencies of Helios and – Cre +/+ or +/fl Cre fl/fl Helios Treg cells in the colon lamina propria of Foxp3 Mtor and Foxp3 Mtor mice. e Flow cytometry analysis of Foxp3 vs. RORγt expression + – Cre +/+ or +/fl (left) and quantification of the frequencies of RORγt and RORγt Treg cells (right) in the colon lamina propria of Foxp3 Mtor and Cre fl/fl + – + + + Foxp3 Mtor mice. f Quantification of the frequencies of Helios and Helios Treg cells among the CD45.2 CD4 TCRβ T cells in the colon lamina propria of mixed bone marrow chimeras. g Experimental schematic for in vivo pTreg maintenance assay. h, i Quantification of frequency and/or number of + + –/– donor-derived Foxp3-YFP pTreg cells (h) or total CD4 T cells (i)inRag1 mice 4 weeks after adoptive transfer of naive T cells isolated from Cre +/fl Cre fl/fl Foxp3 Mtor and Foxp3 Mtor mice. j Quantification of GATA3 expression in Treg cells stimulated under various conditions (with TGF-β and IL-6 + + + + + included in all the conditions) for 3 days in the presence or absence of Torin 1. k Quantification of GATA3 Treg cells (Foxp3 GATA3 in CD4 TCRβ ) from the colon lamina propria of Foxp3CreMtor+/+ or +/fl and Foxp3CreMtorfl/fl mice. Error bars show mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; unpaired, two-tailed Student’s t-test. Data are quantified from five (a, b), eight (c), ten (d), eleven (e), seven (f), three or five (h, i;as indicated), three (j), or six (k) biological replicates, compiled from five (a, b), four (c, f), eight (d), nine (e), two (h, i), three (j), or six (k) independent experiments. Numbers indicate percentage of cells in quadrants cells generated in vivo. The frequency and number of mTOR- enriched under steady state11,12. These in vitro and in vivo results deficient pT cells were reduced in mesenteric lymph nodes highlight the requirement of mTOR signaling for GATA3 reg + (Fig. 4h), while the numbers of donor-derived total CD4 T cells expression in Treg cells. were comparable (Fig. 4i). These results indicate that mTOR promotes the maintenance of pT cells in vivo. reg mTOR promotes eTreg-cell generation. After thymic develop- Activated T cells express GATA3, which is required to fl reg ment, peripheral cTreg cells undergo antigen and in ammation- suppress T 2 responses1,2,11,12,16,38. Therefore, we next examined H driven activation and differentiate into eTreg cells that are enri- if mTOR regulates GATA3 expression in activated Treg cells. We ched in tissues, including the lung and colon lamina established an in vitro system where T cells from wild-type 1,2,7,8,21,22 reg propria . Although eTreg cells are crucial for immune mice were stimulated with anti-CD3 and anti-CD28 antibodies in homeostasis, the molecular requirements driving their activation β the presence of TGF- and IL-6 to mimic the environmental and function are still poorly defined. Our above data indicated signals at mucosal sites1,2,12. As expected, compared with IL-2 fi that mTOR-de cient tTreg cells were reduced in the lung and stimulation, IL-4 strongly upregulated GATA3 expression under colon. Moreover, unbiased GSEA showed that these conditions12. However, IL-4-induced GATA3 upregulation hi lo mTORC1 signaling was enriched in CD44 CD62L eTreg cells was diminished upon inhibition of mTOR activity (Fig. 4j). The lo hi + compared to CD44 CD62L cTreg cells (Fig. 5a). Given these frequency of GATA3 T cells was also significantly reduced in reg results, we next tested whether mTOR regulates eTreg-cell gen- the colon lamina propria (Fig. 4k), a site where these cells are fl eration. Because Treg cells isolated from in ammatory

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a b c Foxp3 Cre/+Mtor +/+ or +/fl CD45.2+Foxp3 CreMtor +/+ or+/fl chimera 0.45 Cre/+ fl/fl + Cre fl/fl 0.40 Hallmark mTORC1 signaling Foxp3 Mtor CD45.2 Foxp3 Mtor chimera 0.35 0.30 NES = 1.65 80 0.25 Cre/+ * 40 ns 0.20 FDR < 0.001 Foxp3 57.2 * 0.15 1

+/fl ) 0.10

Mtor 4 0.05 60 30 cells 0.8 0.00

* ) –0.05 32.6 reg 4 40 *** 20 T 0.6 + Enrichment score

% Cells 0.4 20 10 (× 10

eT cell > cT cell # Cells (× 10 reg reg Foxp3 Cre/+ 72.4 0.2 +/fl Top 15 leading edge genes: Fgl2, Nfil3, Rrm2, Mtor 0 0 # KLRG1 0 Plod2, Bcat1, Fam129a, Bub1, Bhlhe40, Glrx, 17.1 cTreg eTreg cTreg eTreg Egln3, Gla, Hk2, Dapp1, Tpi1, Map2k3 CD62L CD44 d e CD45.2+Foxp3 CreMtor +/+ or +/fl chimera + Cre +/+ or+/fl + Cre fl/fl CD45.2 Foxp3 Mtor chimera CD45.2 Foxp3 Mtor chimera CD45.2+Foxp3 CreMtor fl/fl chimera ** 3 *** 1.5 1.5 1.5 ns 1.5 ns 10 1.5 *** ** *** 2 1 1 1 1 1 cells 5 FR 1 0.5 0.5 0.5 0.5 0.5 % T Normalized Bcl6 Normalized CD25 Normalized TIGIT Normalized ICOS Normalized Foxp3 0 0 Normalized CTLA4 0 0 0 0 0 f g Foxp3 Cre/+Mtor +/+ or +/fl CD45.2+Foxp3 CreMtor +/fl chimera 12.7 Foxp3 Cre/+ Foxp3 Cre/+Mtor fl/fl + Cre fl/fl 8 ns CD45.2 Foxp3 Mtor chimera Mtor +/fl + 16 *** 1.5 * ) 3 6 ns 12 1 4 cells 8 reg

0.37 cells (× 10 2 Cre/+ 0.5 reg Foxp3 % T 4

Mtor fl/fl % Active caspase-3 # T 0 Foxp3-YFP 0 0 Spleen pLN CD4

h i P = 0.059 αCD3/αCD28 mAb + IL-2 1.5 *** 1.5 *** *** US Vehicle Torin 1 PP242 1 1

0.5 0.5 96.2 27.7 89.1 77.4 CD25 expression

0 FOXP3 expression 0

1.53 63.3 7.82 16.8 US US CD62L VehiclePP242Torin 1 VehiclePP242Torin 1 CD44 αCD3/αCD28 mAb αCD3/αCD28 mAb + IL-2 + IL-2

Fig. 5 mTOR is essential for eTreg cell differentiation. a Enrichment plot of the Hallmark mTORC1 pathway activated in eTreg cells compared to cTreg cells, identified by gene set enrichment analysis (GSEA). The top 15 enriched genes (position indicated by the vertical black line) are listed below the plot. b Flow + + lo hi + + cytometry analysis (left) and quantification of frequencies and cell numbers (right) of CD4 Foxp3-YFP CD44 CD62L cTreg cells and CD4 Foxp3-YFP hi lo Cre/+ +/+ or +/fl Cre/+ fl/fl + CD44 CD62L eTreg cells in Foxp3 Mtor and Foxp3 Mtor mosaic mice. c Quantification of the number of KLRG1 Treg cells in the spleen of mixed bone marrow chimeras. d Quantification of CD25, ICOS, CTLA4, TIGIT, and Foxp3 expression in Treg cells from mixed bone + + + + + marrow chimeras. e Quantification of the frequency of TFR cells (CD4 Foxp3-YFP CXCR5 PD-1 Treg cells, left) and Bcl6 expression in total Foxp3 + Treg cells (right) in mixed bone marrow chimeras. f Flow cytometry analysis (left) and quantification of the frequency and number (right) of Foxp3-YFP Cre/+ +/+ or +/fl Cre/+ fl/fl Treg cells in the colon lamina propria of Foxp3 Mtor and Foxp3 Mtor mosaic mice. g Quantification of active caspase-3 in CD45.2 + + + CD4 Foxp3-YFP Treg cells from mixed bone marrow chimeras. h Flow cytometry analysis of CD44 vs. CD62L expression on cTreg cells activated under the indicated conditions for 3 days. US: unstimulated. i Quantification of CD25 and FOXP3 expression in human CD4+CD25+CD45RA+CD45RO– naive Treg cells activated for 3 days in the presence or absence of Torin 1 or PP242. Error bars show mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; unpaired, two-tailed Student’s t-test. Data are representative of at least six (b), six (f), or three (h) biological replicates per group or are quantified from eleven (b), fifteen (c), seven or eight (d; CD45.2+Foxp3CreMtor+/+ or +/+ chimera or CD45.2+Foxp3CreMtor+/+ or +/fl mice, respectively; CD25 and Foxp3), ten (d; ICOS, CTLA4, and TIGIT; e), six (f), three (g), or five (i) biological replicates, compiled from seven (b), eight (c), four (d, TIGIT; f), five (d; CD25 and Foxp3), six (d; ICOS and CTLA4; e), or two (g, i) independent experiments. Numbers indicate percentage of cells in gates

fi environments could undergo secondary phenotypic changes, we mosaic mice (Fig. 5b). We also con rmed the reduction of eTreg fi analyzed cell-intrinsic effects of mTOR de ciency in cTreg and cells in the spleen of mixed bone marrow chimeras (Supple- eT cells isolated from healthy, female mosaic mice (designated mentary Fig. 3a). Consistent with elevated mTORC1 signaling in reg + as Foxp3Cre/ ). There was an increase in the frequency but not eT cells (Fig. 5a), the frequency and number of eT cells were + reg reg number of Foxp3-YFP CD44loCD62Lhi cT cells and a reduc- reduced in the absence of Rptor (Supplementary Fig. 3b). The reg + + tion in the frequency and number of Foxp3-YFP number of KLRG1 Treg cells was also reduced in absence of hi lo Cre/+ fl/fl CD44 CD62L eTreg cells in the spleen of Foxp3 Mtor mTOR, consistent with a reduction of eTreg cells (Fig. 5c and

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Supplementary Fig. 3c)9. The expression of CD25, a marker Also, the loss of IRF4 expression likely accounted for the 9 fi expressed at higher levels on cTreg cells than eTreg cells , was impairment of mTOR-de cient Treg cells to express ICOS fi increased on mTOR-de cient Treg cells (Fig. 5d and Supple- (Fig. 5d and Supplementary Fig. 3d), which is induced by IRF4- mentary Fig. 3d). Moreover, eTreg-cell-associated molecules like dependent mechanisms to drive eTreg-cell differentiation ICOS and CTLA4 were expressed at lower levels in the absence of in vivo9,15. Altogether, these data indicate that mTOR promotes mTOR, while the expression of TIGIT or Foxp3 was equivalent eTreg-cell differentiation, in part, by modulating IRF4 expression, between the control and mTOR-deficient Treg cells (Fig. 5d and which also helps explain how TH2 responses become elevated in 1,2,9 Cre fl/fl Supplementary Fig. 3d) . Activated Treg cells also differentiate Foxp3 Mtor mice. into specialized or tissue-resident Treg-cell populations, including + + + 5,10,13 CXCR5 PD1 Foxp3 TFR cells that express Bcl6 . Both TFR cells and Bcl6 expression were reduced in the absence of mTOR mTOR orchestrates mitochondrial metabolism in Treg cells. 23 (Fig. 5e and Supplementary Fig. 3e). Consistent with our earlier Metabolism is a crucial determinant of Treg-cell biology , but the analysis of mixed bone marrow chimeras, there was nearly a mechanisms controlling metabolic rewiring required for the Cre fl/fl complete loss of colon Treg cells in Foxp3 Mtor mosaic mice function of activated Treg cells are not clear. GSEA showed that (Fig. 5f). Thus, mTOR is essential for maintaining eTreg cells cTreg cells upregulated mTORC1 signaling and several metabolic in vivo. pathways, including glycolysis, upon activation (Supplementar- Mechanistically, the loss of eTreg cells in the absence of mTOR y Fig. 4a), while mTOR inhibitor-treated cTreg cells had a sig- could be due to defective survival or reduced activation-induced nificant downregulation of genes in the glycolytic pathway differentiation. To test the former, we analyzed the expression of (Fig. 6e and Supplementary Table 1), including Hk2 (Fig. 6d). active caspase-3 in control and mTOR-deficient T cells isolated Because IRF4 also promotes metabolic reprograming of conven- reg + + from mixed bone marrow chimeras and found normal survival of tional CD4 and CD8 T cells45, we determined if mTOR signals fi + mTOR-de cient Treg cells (Fig. 5g). Also, the frequency of 7AAD via IRF4 to regulate Treg-cell metabolism. We performed func- fi lo hi cells was similar or reduced in puri ed CD44 CD62L cTreg cells tional enrichment analysis of IRF4 targets that were differentially activated in the presence of the mTOR inhibitors (Supplementary expressed in cTreg cells activated in the presence of mTOR Fig. 3f), further indicating that mTOR is not essential for cell survival. inhibitors (Fig. 6d). This analysis revealed enrichments for gly- To test the role of mTOR in activation-induced differentiation, colytic and nucleotide metabolism and upstream regulators of fi lo hi 30,46,47 we puri ed CD44 CD62L cTreg cells from wild-type mice and these pathways, including Myc and mTORC1 (Fig. 6f). activated them for 3 days in the presence or absence of the mTOR Thus, the mTOR-IRF4 axis supports the upregulation of glyco- 43 inhibitors, Torin 1 and PP242 . We found that cTreg cells lytic and nucleotide metabolism during cTreg-cell activation. hi lo differentiation into CD44 CD62L eTreg-like cells was impaired Besides glycolysis, cTreg cells upregulated genes in the oxidative by mTOR inhibition (Fig. 5h). Similarly, the frequency of mTOR- phosphorylation pathway in an mTOR-dependent manner deficient T cells in the spleen and peripheral lymph nodes of (Fig. 7a and Supplementary Table 1). Indeed, 366 mitochondrial reg fl fl DT-treated Foxp3Cre/DTRMtor / mice was reduced relative to the genes (identified from the MitoCarta 2.0 database48) were controls (Supplementary Fig. 3g, left panel), but total numbers of differentially expressed in activated cTreg cells (vs. unstimulated fi Treg cells were not signi cantly different (Supplementary Fig. 3g, cells), and 158 of these genes were mTOR targets (Fig. 7b). right panel), likely due to the increased organ size (Fig. 1e). Thus, Twenty-one mitochondrial genes were putative IRF4 gene targets fi 22 mTOR-de cient Treg cells also fail to appropriately respond to (based on IRF4 ChIP-seq analysis ), but only six of these genes activation-induced signals in vivo. We also investigated if these were induced upon cTreg-cell activation. Only one of these IRF4 regulatory pathways applied to human cells. In the presence of targets (Mrps28) was upregulated in an mTOR-dependent hi lo the mTOR inhibitors, activated human CD45RA CD45RO manner during cTreg-cell activation (Fig. 7b). These data, naive Treg cells had impaired upregulation of CD25 and FOXP3 combined with the functional enrichment analysis above, suggest (Fig. 5i), which are expressed more abundantly in human that mTOR promotes mitochondrial in a largely lo hi 44 CD45RA CD45RO activated Treg cells than naive Treg cells . IRF4-independent manner. To further test the effects of mTOR Thus, mTOR activity represents an evolutionarily conserved on the metabolic pathways, we performed metabolomics profiling pathway for driving eTreg-cell generation. using high-resolution mass spectrometry on resting and activated Treg cells. We found that 54 metabolites were differentially expressed between Treg cells activated in the presence of Torin 1 mTOR links activation signals to IRF4 upregulation. IRF4 is vs. DMSO (Fig. 7c). For instance, the expression of the TCA cycle induced by TCR signals to promote eTreg-cell differentiation and intermediates isocitrate/citrate, malate, and succinate and the + fi regulates Treg-cell-mediated suppression of TH2 responses electron acceptor NAD were signi cantly decreased in Treg cells in vivo7,8,15,21,22. To determine if mTOR induces IRF4 expres- activated in the presence of Torin 1 (Fig. 7c). Unbiased metabolite fi fi sion upon activation, we puri ed control and mTOR-de cient set enrichment analysis (MSEA) revealed that activated Treg cells fi cTreg cells and activated them for 24 and 48 h before analyzing signi cantly upregulated metabolic pathways associated with IRF4 expression by flow cytometry. IRF4 expression was reduced mitochondria-dependent energy production and the biosynthesis at 24 and 48 h after activation (Fig. 6a). Acute mTOR inhibition of proteins and nucleotides, such as the , the with Torin 1 or PP242 also significantly reduced activation- mitochondrial , and pyrimidine biosynth- 49 induced upregulation of IRF4 in cTreg cells (Fig. 6b). Mechan- esis (Supplementary Fig. 4b). The upregulation of these istically, mTOR controls IRF4 expression at the post- mitochondria-related metabolic pathways was impaired when transcriptional level, because cTreg cells activated in the pre- Treg cells were activated in the presence of Torin 1 (Fig. 7d). sence of the mTOR inhibitors for 24 or 48 h had increased Irf4 Consistent with this observation, Treg cells activated in the expression compared to the internal controls (Fig. 6c). To show presence of Torin 1 had lower mitochondrial membrane potential that the ~20–30% reduction of IRF4 expression was biologically (TMRM), whereas mitochondria number as indicated by fi important, we analyzed gene expression pro les in cTreg cells Mitotracker staining was comparable (Fig. 7e). Thus, mTOR activated in the presence or absence of Torin 1 or PP242. Among links activation signals to IRF4-dependent and -independent the genes that were consistently altered by both inhibitors were transcriptional programs to induce metabolic reprogramming 8,15,21,22 124 IRF4 target genes, including Ccr8 and Eea1 (Fig. 6d). during Treg-cell activation.

8 NATURE COMMUNICATIONS | (2018) 9:2095 | DOI: 10.1038/s41467-018-04392-5 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04392-5 ARTICLE

abc WT cTreg mTOR-deficient cTreg DMSO Torin 1 PP242 DMSO Torin 1 PP242 1.5 1.5 *** *** 20 P = 0.08 ** ** * * ** * 1 1.0 Irf4 * 10 0.5 0.5 Relative Normalized IRF4 Normalized IRF4 0 0 0 Time (h): 24 48 Time (h): 24 48 Time (h): 24 48 de DMSO 20 h Torin 1 20 h PP242 20 h 0.05 Hallmark glycolysis Top 30 leading edge genes: 123123123 0.00 0.05 Cdk1, Slc25a13, Galk1, Tpi1, Sdcbp2 NES = –1.81 Map4k3 0.10 Cd7 Cep97 0.15 H2k, Lhpp, Gmppb, Me1, Neb FDR < 0.001 Arap2 0.20 Rfx3 Gnpda1, Ak4, Igfbp3, Glrx, Scml4 0.25 2610020H08Rik Fbxo32 0.30 Pgk1, Kif20a, Pdk3, Psmc4, Slc25a45 0.35 D2hgdh Aurka, Tpst1, Sap30, Ldha, Kcna3 0.40 Rbm33 Vps54 Bcas3 Tgfbi, Plod2, Got2, Gusb, Il7r Phlpp1 Chd2 Enrichment score Angptl4, Tmmr, Ecd, Fam162a, Atp10d Tbc1d30 Rars, Arpp19 Adamts10 Mtap Mpp6 Sms Cd226 DMSO 20 h > Torin 1 20 h Farsb Eea1 Il12rb1 Gpr183 Scarb1 Rnaseh2b f Acaca Msto1 Cndp2 Nup93 Pold2 Rrp1b Lrmp Reactome metabolism of nucleotides Psmd1 Slc12a6 Kif21b Nr2c2 Zswim6 Ythdc1 Itpkb Gm8369 2010016I18Rik Arid4b Enrichment_score Vps37b KEGG pyrimidine metabolism Hivep2 Plekhm1 Lyst 10.0 Fam162a Ppat Bzw2 Akap13 Ezh1 Ankrd44 12.5 Utrn Nt5e KEGG purine metabolism Rasa3 Neurl3 Ifngr1 Nars Nme1 15.0 Mrps28 Batf Rbpj Adarb1 Rrp12 Tfrc Hallmark Myc targets v1 17.5 Smyd5 Mms22l Impa2 Lta Prim2 Pus10 Set –Log10(BH_FDR) Cyp51 Bcl2l1 Acot7 3.0 Cpd Hallmark mTORC1 signaling Gemin6 Mfsd2a Lmnb1 2.8 Ptgfrn Gemin6 Abcb1b Gadd45g Galm 2.6 Tigit 1110008L16Rik Setbp1 Hallmark hypoxia Hivep2 2.4 Hk2 Golim4 Nek6 B4galnt4 Tubb6 Fam185a Phactr2 Aurkb Cdca8 Il12rb2 Hallmark glycolysis Ccr8 Dtl Rrm2 Cdk1 Ccnb1 Dgkg Atad5 Spire1 Blrc5 Cobll1 Adap1 Rbpj E2f3

–2 –1 0 1 2 Row Z-score

Fig. 6 mTOR links activation signals to upregulation of IRF4 expression and downstream targets. a Wild-type (WT) and mTOR-deficient CD4+Foxp3-YFP+ lo hi CD44 CD62L cTreg cells were purified from mixed bone marrow chimeras and activated under the indicated conditions for 24 and 48 h. IRF4 expression + + lo hi Cre was assessed by flow cytometry. b CD4 Foxp3-YFP CD44 CD62L cTreg cells were purified from Foxp3 mice and activated as in a in the presence or absence of Torin 1 and PP242. IRF4 expression was assessed by flow cytometry. c cTreg cells were purified and activated as in b and Irf4 mRNA expression was analyzed by qPCR. d Heat map of IRF4 target genes differentially expressed in resting cTreg cells or cTreg cells activated in the presence of DMSO, Torin 1, or PP242 for 20 h. e Enrichment plot of the Hallmark glycolysis pathway in cTreg cells activated for 20 h in the presence of either Torin 1 or DMSO control, identified by gene set enrichment analysis (GSEA). The top 30 enriched genes (position indicated by the vertical black line) are listed. f Functional enrichment of the IRF4 target genes shown in d. Error bars show mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; unpaired, two-tailed Student’s t-test. Data are representative of three independent experiments (b) or are quantified from five (a) and four (b, c) biological replicates, compiled from two independent experiments (a, c)

Tfam is essential for eTreg-cell homeostasis and function.We transcription factor essential for efficient electron transport chain fi 50,51 Cre next genetically de ned the importance of mitochondrial meta- activity ,inTreg cells by breeding Foxp3 transgenic mice bolism in T -cell function in vivo. We conditionally deleted with mice-bearing floxed alleles for Tfam51. Tfam-deficient T reg + fl fl reg mitochondrial transcription factor A (Tfam), a nuclear-encoded cells isolated from Foxp3Cre/ Tfam / mosaic mice had

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ab Hallmark oxidative phosphorylation MitoCarta 2.0 (1158 genes): 0.0 NES = –2.79 –0.1 Top 30 leading edge genes: Idh3a, IRF4 ChIP-seq targets Activated cTreg with mTOR –0.2 FDR < 0.001 Retstat, Mrps22, Nudfc1, (21 genes) inhibitors (166 genes) –0.3 Slc25a4, Uqcrq, Mgst3, Etfb, –0.4 –0.5 Timm17a, Timm50, Ndufb6, Ndufa9, 15 0 8 –0.6 Ndufb2, Mrpl15, Fdx1, Decr1, Ndufab1, Ndufs8, Mtrf1, Ndufs6, Sdhd, 1 5 157

Enrichment score Etfa, Hspa9, Abcb7, Atp6v1h, Ldha, Polr2f, Atp6v1c1, Mrps30, Got2 DMSO 20 h > Torin 1 20 h 203

Activated cTreg cells with DMSO (366 genes)

c d Pathways downregulated in Torin 1-treated activated Treg DMSO Torin 1 RNA transcription 0 h 16 h 16 h –2 –1 0 12 Mitochondrial electron 121212 Row Z-score P < 0.01 C4H9NO2(4) Citric acid cycle Propionyl-carnitine P < 0.05 5,6-dihydrothymine Glutathione metabolism Citrate/isocitrate ATP 2-aminoacrylic acid Ammonia recycling UDP-N-acetyl-α-D-glucos(or galacto)amine UDP-D-glucose/UDP-D-galactose Adipic acid Gluconeogenesis L-alanine Malate L-histidine Glycolysis Nicotinamide Isoputreanine/putreanine Malate-aspartate shuttle G6P/F6P Succinate

2-hydroxyglutarate Rank in MSEA list Glycerol phosphate Icatonate/mesatonate UTP Butyryl carnitine Nucleotide sugars Fumarate N-acetyl-L-glutamate Pyrimidine metabolism UMP NAD+ Cytidine Glucose-alanine cycle 5-methylthioadenosine L-4-hydroxyglutamate semialdehyde L-glutamate Glycerolipid metabolism Aspartate L-threonine (R)-pantothenate 0123 Choline phosphate Taurine (R)-carnitine –Log10 (P -value) Creatine Stearate Glycerol-3-phosphate e N-phosphocreatinate DMSO D-glucosamine-6-phosphate 21064 16100 CDPcholine 15786 16171 Succinyl carnitine Torin 1 ADP-mannose/GDP-L-fucose/ADP-α-D-gluc UDP-α-D-xylose L-2-aminoadipate CDP-ethanolamine GDP-D-mannose CTP UDP-D-glucuronate N(ω)−(L-arginino)succinate Decendyl carnitine Trans-hexadec-2-enoyl carnitine L-cysteinylglycine D-glycerate-3-phosphate/D-glycerate-2-phos Dihydroxyacetone TMRM Mitotracker

Fig. 7 mTOR controls metabolic reprogramming upon Treg-cell activation. a Enrichment plot of the Hallmark oxidative phosphorylation pathway in cTreg cells activated for 20 h in the presence of either Torin 1 or DMSO control, identified by gene set enrichment analysis (GSEA). The top 30 enriched genes (position indicated by the vertical black line) are listed. b Venn-diagram depicting mitochondria-related genes (defined in MitoCarta 2.0 database) that are IRF4 targets (blue circle), or differentially expressed in activated cTreg cells (vs. unstimulated cells; black circle) or activated cTreg cells treated with mTOR inhibitors (vs. those treated with DMSO; red circle). The numbers indicate the shared and independent genes in each category. c Heat map of differentially expressed intracellular metabolites in freshly isolated Treg cells or Treg cells activated for 16 h in the presence of DMSO or Torin 1. d Metabolite set enrichment analysis (MSEA) of KEGG metabolic pathways downregulated in Torin 1-treated activated Treg cells compared to vehicle-treated activated Treg cells. e Flow cytometry of TMRM and Mitotracker staining in Treg cells activated for 20 h in the presence of Torin 1 or vehicle control. Data are representative of three (Mitotracker) or four (TMRM) biological replicates from two (Mitotracker) or three (TMRM) independent experiments (e) comparable mitochondrial content but less mitochondria-derived (Fig. 8g). Only the frequency, not the number, of GC B cells was + reactive oxygen species (ROS) (Fig. 8a), consistent with reduced increased, likely due to a reduction of total B220 B cells in fl fl mitochondrial respiratory chain function. We found that Tfam is Foxp3CreTfam / mice (Fig. 8h and Supplementary Fig. 5b). Thus, Cre fl/fl fi critical for Treg-cell function, as Foxp3 Tfam mice developed Tfam de ciency in Treg cells leads to altered immune homeostasis a severe inflammatory disease associated with smaller body size, and development of autoimmunity. fl skin in ammation, alopecia (Fig. 8b), early lethality (Fig. 8c), and We next analyzed Treg-cell populations in mixed bone marrow enlargement of the peripheral lymph nodes (Fig. 8d). Further, chimeras to determine the cell-intrinsic role of Tfam-dependent there were increased effector/memory T cells (Fig. 8e) and sig- mitochondrial metabolism in eTreg-cell accumulation and fi γ + ni cantly enhanced IFN- -producing CD8 T cell and TH1, TH2, homeostasis. There was a reduction in the frequency and hi lo + and TH17-cell activation (Fig. 8f) in diseased mice-bearing Tfam- number of CD44 CD62L eTreg cells (Fig. 8i) and KLRG1 fi fi fi de cient Treg cells. Tfam-de cient Treg cells also had a propensity Treg cells (Fig. 8j) in the absence of Tfam. Further, Tfam-de cient γ for increased IFN- production (Supplementary Fig. 5a). More- Treg cells had reduced expression of ICOS and CTLA4 (Fig. 8k), over, the frequency and number of TFH cells were increased but not Foxp3 (Supplementary Fig. 5c). However, unlike

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abcd Foxp3 Cre/+Tfam+/fl Foxp3 CreTfam+/+ or +/fl 12 34 100 12 Cre/+ fl/fl Foxp3 Tfam Foxp3 CreTfam+/+ Foxp3 CreTfamfl/fl 75 Cre fl/fl pLN ns Foxp3 Tfam 1.5 * 1.5 1.5 ) * 50 8 1 ns 1 1 *** Spleen % Survival 25 0.5 0.5 0.5 MitoSOX 0 Mitotracker Normalized Normalized 1 and 2: Foxp3 CreTfam +/+ 0 20406080 1: Foxp3 CreTfam+/fl # Cells (× 10 0 0 0 3 and 4: Foxp3 CreTfam fl/fl Days 2: Foxp3 CreTfamfl/fl Spleen pLN

efFoxp3 CreTfam+/fl Foxp3 CreTfamfl/fl g

Foxp3 CreTfam+/+ or +/fl Foxp3 CreTfamfl/fl Foxp3 CreTfam +/+ or +/fl 1.13 Cre fl/fl Foxp3 Cre Foxp3 Tfam CD4+ 72.0 22.3 Tfam+/fl 60 ** 9

– 20 10

+ *** )

19.6 65.1 * 5 cells 40 6 + 15 * γ ** Foxp3

+ ** 10.6 20 3 Cre cells 10 5

Foxp3 FH cells (× 10 % Cytokine + 64.4 22.5 CD4 CD8 % IFN- Tfamfl/fl % T 0 0 5 FH + + + + 9.2 46.5 CD4 CD8 IL-4 IL-17A # T CD62L

PD-1 0 0 CD44 CXCR5

hij Foxp3 CreTfam+/+ or +/fl CD45.2+ Foxp3 CreTfam+/+ chimera CD45.2+Foxp3 CreTfam+/+ chimera 0.4 Foxp3 CreTfamfl/fl CD45.2+ Foxp3 CreTfamfl/fl chimera CD45.2+Foxp3 CreTfamfl/fl chimera Foxp3 Cre Tfam+/fl 3 ) 3 * 5 ns 100 *** 3 10 ) ** 5 cells *** *** ns ) 2 2 2 reg 3

1.96 T

50 + 5 Cre

Foxp3 % Cells 1 1 1 (× 10 fl/fl

Tfam % GC B cells # Cells (× 10 # GC B cells (× 10

0 0 0 0 # KLRG1 0 GL7 cT eT cT eT CD95 reg reg reg reg

klmn CD45.2+Foxp3 CreTfam+/+ chimera CD45.2+Foxp3 CreTfam+/+ chimera Foxp3 CreTfam+/+ or +/fl CD45.2+Foxp3 CreTfam+/+ chimera CD45.2+Foxp3 CreTfamfl/fl chimera CD45.2+Foxp3 CreTfamfl/fl chimera Foxp3 CreTfamfl/fl CD45.2+Foxp3 CreTfamfl/fl chimera

1.5 1.5 1.5 20 * 1.2 * 50 *** 30 *** ** *** *** 1 1 1 0.8 20 cells cells cells 10 25 reg reg FR * 0.5 0.5 0.5 0.4 10 ** % T % T % T

0 0 0 0 Normalized Bcl6 0 0 0

Normalized expression ICOS CTLA4 TIGIT cLP LungcLP Lung

Fig. 8 Mitochondrial metabolism is critical for Treg-cell function in vivo. a Quantification of Mitotracker (left) and MitoSOX (right) in Treg cells from Foxp3Cre/+Tfam+/fl and Foxp3Cre/+Tfamfl/fl mosaic mice. b Image of 8-week-old mice. c Survival curve of Foxp3CreTfam+/+ (n = 14) and Foxp3CreTfamfl/fl mice (n = 19). d Representative image of lymphadenopathy (left) and cell numbers in the spleen and peripheral lymph nodes (pLN) of Foxp3CreTfam+/fl and Foxp3CreTfamfl/fl mice. e Flow cytometry analysis of naive and effector/memory CD4+Foxp3-YFP– (depicted as CD4+) or CD8+ T cells in Foxp3CreTfam+/fl and Foxp3CreTfamfl/fl mice. f Quantification of cytokine production by CD4+Foxp3– and CD8+ T cells. g Flow cytometry analysis (left) and frequency and number (right) of TFH cells. h Flow cytometry analysis (left) and frequency and number (right) of GC B cells. i, j Quantification of frequencies and + numbers of cTreg cells and eTreg cells (i) or number of KLRG1 Treg cells (j) in the spleen of mixed bone marrow chimeras. k Quantification of ICOS, CTLA4, and TIGIT expression in Treg cells from mixed bone marrow chimeras. l Quantification of the frequency of TFR cells (left) and Bcl6 expression + in total Foxp3 Treg cells (right) in mixed bone marrow chimeras. m, n Quantification of frequency of Treg cells in the colon lamina propria and lung of Foxp3CreTfam+/+ or +/fl or Foxp3CreTfamfl/fl mice (m) or mixed bone marrow chimeras (n). Error bars show mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant; unpaired, two-tailed Student’s t-test. Data are representative of nine (b, d, e), twelve (g), or ten (h) biological replicates per group, or are quantified from five (a), eight or nine (d, f; Foxp3CreTfamfl/fl or Foxp3CreTfam+/+ or +/fl mice, respectively), twelve (g), ten (h), nine or ten (i−l, n; CD45.2+Foxp3CreTfam+/+ chimeras and CD45.2+Foxp3CreTfam+/fl chimeras, respectively), five (m, colon), or eight (m, lung) biological replicates per group, compiled from four (a), six (d, f), eight (g), seven (h), four (i−l, n), three (m; colon), or five (m; lung) independent experiments. Numbers indicate percentage of cells in gates

fi mTOR-de cient Treg cells, CD25 expression was not increased on critical for the function and homeostasis of activated Treg cells fi Tfam-de cient Treg cells (Supplementary Fig. 5c), and TIGIT in vivo. expression was reduced (Fig. 8k). Tfam deficiency also reduced TFR-cell generation and Bcl6 expression (Fig. 8l). Further, there was fi a cell-intrinsic reduction of Tfam-de cient Treg cells within the Discussion colon lamina propria and the lung (Fig. 8m, n). Collectively, these Activated tTreg and pTreg cells are crucial for peripheral T-cell results indicate that Tfam-dependent mitochondrial metabolism is tolerance and tissue homeostasis. Here, we show that activated

NATURE COMMUNICATIONS | (2018) 9:2095 | DOI: 10.1038/s41467-018-04392-5 | www.nature.com/naturecommunications 11 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04392-5

Treg-cell populations have increased mTOR signaling necessary suggest a key role for Raptor-mTORC1-induced mitochondrial for Treg-cell activation and tissue Treg-cell homeostasis. metabolism in establishing the fate and function of activated Treg Mechanistically, mTOR tunes IRF4-dependent transcriptional cells in different microenvironments. programming and mitochondrial metabolism. In the absence of Treg cells must adapt to different environmental cues to acquire mTOR, activated tTreg and pTreg cells are severely decreased in unique suppressive functions, and the appropriate balance of mucosal tissues, associated with excessive TH2, and to a lesser mTOR and metabolic signaling appears to be linked to this extent TH1 and TH17 responses, and disrupted tissue home- process. Inactivation of mTORC1 alone or combined with 30 ostasis. Further, the homeostasis and suppressive activity of mTORC2 disrupts Treg-cell suppressive activity ; however, 58 activated Treg cells is impaired by the loss of mitochondrial whether mTORC1-independent functions of Raptor or Raptor/ metabolism and consequently leads to autoimmunity (Supple- Rictor-independent mTOR complexes play roles in Treg-cell mentary Fig. 5d). Thus, our data identify and establish a critical biology remained unclear. Our results here suggest that mTOR mTOR-dependent metabolic node that regulates the homeostasis does act through Raptor and Rictor to promote Treg-cell function. and suppressive function of activated Treg cells in vivo. Compared to Raptor deficiency alone, loss of mTOR or Raptor 30 TCR-dependent signals coupled with co-stimulation and and Rictor in Treg cells leads to similar extensions in lifespan , inflammatory cues drive Treg-cell activation and differentiation which may be due to more limited tissue damage, such as in the 1,2,7–9,21,22 into specialized or tissue-resident Treg-cell subsets . intestines. The balance of mTORC1- and/or mTORC2-induced Several transcriptional programs are essential for the differ- metabolic programs may also tune Treg-cell suppression of 7,20–22,43,52,53 fi entiation and function of activated Treg cells . How- effector T-cell responses. For instance, Raptor-de cient Treg cells 30 ever, the upstream signaling pathways driving eTreg-cell have increased mTORC2-Akt activity , which can upregulate homeostasis are unknown. Here, we show that activation signals glycolysis at the expense of mitochondrial metabolism and lead to 59–61 through mTOR are required for Treg-cell activation and function, inappropriate suppression of TH1 and TFH responses . fi 30,61 thereby establishing the rst kinase pathway, to our knowledge, Despite being dispensable for Treg-cell suppressive activity , fi fl that links TCR signals and transcriptional programs necessary for mTORC2 can promote Treg-cell traf cking to sites of in amma- Treg-cell activation. Mechanistically, mTOR promotes the tion and non-lymphoid tissues via upregulating glycolysis or expression of IRF4 and GATA3, transcription factors that are suppressing Foxo1 activity43,62. Thus, gain of mTORC1 and essential for Treg-cell-dependent suppression of TH2 respon- concomitant loss of mTORC2 activity could reduce Treg-cell 7,11,12,15 fi fl 63–65 ses . Moreover, IRF4 also enforces eTreg-cell differentia- traf cking to TH1 and TH17 in ammatory sites . mTOR may tion and pT -cell homeostasis to limit mucosal T 2 also promote trafficking to sites of T 2inflammation by mod- reg– H H responses16,17,20 22. Therefore, by promoting the expression of ulating Ccr8 expression and/or CCL22/CCR4-induced chemo- 62,66 fi IRF4 and GATA3, mTOR maintains activated Treg-cell popula- taxis . We, therefore, propose that Treg-cell function is nely tions that facilitate tissue homeostasis. The mTOR-dependent tuned by graded nature of mTOR signaling and metabolic pro- fl induction of IRF4 expression in cTreg cells was not tran- grams, which are likely in uenced by local environmental signals. + 54 scriptionally regulated, and, in conventional CD4 T cells , This tunable nature of mTOR signaling in Treg cells may offer a occurs independently of the mTOR-4EBP1 translation axis. therapeutic strategy to modulate Treg-cell responses to selectively Therefore, mTOR likely regulates IRF4 expression at the post- alter the conventional T-cell responses in autoimmunity, infec- translational level, such as via SUMOlyation55. A key question tious diseases, and cancer. that remains is, why are mucosal tissues more sensitive to the 2 responses than secondary lymphoid organs upregulation of TH Methods fi + – – fl fl in mice-bearing mTOR-de cient Treg cells? One possibility is that Mice. C57BL/6, CD45.1 , Cd4Cre, Foxp3DTR, Rag1 / , Mtor , and Tfam mice were these sites are enriched for activated tTreg- and pTreg-cell popu- purchased from The Jackson Laboratory. Foxp3Cre mice, from Dr. Alexander 33 lations and hence their loss more readily increases TH2 responses Rudensky, have been described previously . All genetic models used in this study at these sites than in the peripheral lymphoid organs1,2,56. Fur- were on the C57BL/6 background, and both male and female mice were used for quantification and analysis, except for histological analysis where only male mice ther, recent work shows that tissue Treg cells express high levels of – 38,39 were used. Mice were generally 4 6-weeks-old unless otherwise indicated. The ST2 (IL-33 receptor) , which induces GATA3 activation that number of animals in each group are provided in the figures and/or figure legends. 39 biases Treg cells toward the TH2 suppressive program . Thus, All mice were kept in specific pathogen-free conditions within the Animal mTOR deficiency and other conditions that decrease GATA3 Resource Center at St. Jude Children’s Research Hospital. The animal protocols expression will impair this feed forward loop and disrupt T 2- were approved by the Institutional Animal Care and Use Committee of St. Jude H Children’s Research Hospital. Mixed bone marrow chimeras were generated by + + like Treg-cell suppressive responses. adoptive transfer of CD45.1 bone marrow cells mixed 1:1 with CD45.2 bone + fl fl fl Metabolic reprogramming contributes to cell fate decisions. marrow cells from Foxp3CreMtor / or Foxp3CreMtor / mice into sub-lethally – – irradiated Rag1 / mice as described30. For the pT cell in vivo maintenance However, the metabolic programs promoting the homeostasis + − reg model, CD4 Foxp3-YFP CD44loCD62Lhi naive T cells from the spleens and and function of activated Treg cells are not completely under- Cre +/fl Cre fl/fl 23 peripheral lymph nodes of Foxp3 Mtor or Foxp3 Mtor mice were pur- stood . Despite the previous work suggesting an inhibitory role ified on a Synergy or Reflection fluorescence activated cell sorter (Sony Bio- of mTOR for mitochondrial oxidative metabolism in induced Treg technology). Then, 0.75 × 106 cells were transferred via retroorbital injection into 27 –/– + cells in vitro , we show here that mitochondrial metabolism is sex-matched Rag1 mice. The presence of Foxp3-YFP Treg cells was evaluated in the mesenteric lymph nodes 4 weeks later42. Foxp3Cre/DTR mosaic mice were highly induced during Treg-cell activation in an mTOR- − treated with DT (50 μgkg 1) i.p. three times per week, for a total of four injections. dependent manner, and is essential for activated Treg-cell func- fl fi The mice were euthanized, and tissues were harvested for ow cytometry analysis tion and tissue homeostasis in vivo. Indeed, Treg-speci c deletion 11 days following the first DT treatment. Sample sizes were chosen based upon of Tfam impairs Treg-cell function, leading to the hyperactivation previous data generated within the laboratory and were selected to maximize the of conventional T cells and autoimmunity. Mechanistically, chance of uncovering statistically significant differences of the mean. No animals were excluded from analysis. mitochondrial metabolism could affect eTreg-cell proliferation or survival within tissues as has been reported in vitro27,28. Of note, fi Raptor-de cient Treg cells have reduced mitochondria-related Flow cytometry. Lymphocytes were harvested form the peripheral lymphoid tissues fl fl fl fl 30,67 gene expression30, and Foxp3CreTfam / and Foxp3CreRptor / by manual disruption or the colon lamina propria as previously described . For surface marker analyses, cells were stained in PBS containing 2% (wt/vol) BSA and mice have similar elevations in activated T-cell responses, disease fl 30 fi the appropriate antibodies. The following uorescent-conjugate-labeled antibodies, pathologies, and survival kinetics . Additionally, de ciency of purchased from various commercial sources (Biolegend, BD Biosciences, Thermo 57 fi Tfam and Raptor impairs TFR cell accumulation . Thus, our data Fisher Scienti c, and Sony Biotechnology), were used: anti-CD4 (clone RM4-5),

12 NATURE COMMUNICATIONS | (2018) 9:2095 | DOI: 10.1038/s41467-018-04392-5 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-04392-5 ARTICLE anti-CD8 (clone 53-6.7), anti-B220 (clone RA3-6B2), anti-CD62L (clone MEL-14), UHPLC (Dionex) coupled to Q Exactive Plus-Mass spectrometer (QE-MS, Thermo anti-CD44 (clone IM7), anti-CD95 (clone Jo2), anti-GL7 (clone GL-7), anti-CD279 Fisher Scientific) for metabolite profiling. Detailed methods were previously (PD-1) (Clone J43), and anti-TCRβ (clone H57-597) antibodies. Biotin-conjugated described68, except that mobile phase A was replaced with water containing 5 mM anti-CXCR5 antibody (clone 2G8) and PE-labeled streptavidin from BD Biosciences ammonium acetate (pH 6.8). Differentially expressed metabolites were identified were used for TFH cell staining. Intracellular staining was performed using the by Limma (Bioconductor) and the Benjamini-Hochberg method was used to Foxp3/Transcription Factor Staining buffers (Cat #00-5523-00, Thermo Fisher estimate the false discover rate (FDR). MetaboAnalyst was used to analyze range- Scientific) per the manufacturer’s instructions. The following antibodies were used: scale data and provide KEGG pathway analysis of significantly altered metabolic γ = 49 anti-CD152 (CTLA4) (clone UC10-4B9), anti-Foxp3 (clone NRRF-30), anti-ROR t pathways (log2 0.5) (www.metaboanalyst.ca/) . (clone B2D), anti-GATA3 (clone TWAJ), anti-IRF4 (clone 3E4), anti-Helios (clone 22F6), anti-IL-4 (clone 11B11), anti-IL-10 (clone JES5-16E3), anti-IL-13 (clone Gene expression analysis. For mTOR deletion efficiency in T cells, quantitative eBio13A), anti-IFN-γ (clone XMG1.2), anti-IL-17A (clone TC11-18H10.1), anti- reg real-time PCR analysis was performed using Mtor Taqman probes (Thermo Fisher human CD25 (clone BC96), anti-human CD45RA (clone HI100), anti-human + – Scientific, Cat #4351372). For detection of Il4 and Il21, CD4 Foxp3-YFP CD45RO (clone UCHL1), anti-human CD4 (clone A161A1), and anti-human – − + – + + CD44hiCXCR5 PD-1 non-T cells or CD4 Foxp3-YFP CD44hiCXCR5 PD-1 FOXP3 (clone 236 A/E7). For intracellular cytokine staining, total splenocytes were FH − T cells were stimulated for 4 h using plate bound anti-CD3 (5 μgml 1) and anti- stimulated for 4–5 h with phrobol 12-myristate 13-acetate (PMA) and ionomycin in FH − CD28 (5 μgml 1) antibodies. Quantitative real-time PCR analysis was performed the presence of monensin (BD Biosciences). Surface and intracellular staining was using SyBR Green Real-Time PCR Master Mix (Thermo Fisher Scientific) and then performed as above. For active caspase-3 staining, surface molecules were primers for Il4 (Forward 5’-GGTCTCAACCCCCAGCTAGT-3’; Reverse stained before cells were fixed, permeabilized, and stained for intracellular active 5’-GCCGATGATCTCTCTCAAGTGAT-3’) and Il21 (Forward 5’-GGACCCTTG caspase-3 using the BD Biosciences active caspase-3 apoptosis kit per the manu- TCTGTCTGGTAG-3’; Reverse 5’-TGTGGAGCTGATAGAAGTTCAGG-3’). For facturer’s instructions (Cat # 550914). Staining for mitochondrial dyes (MitoSOX, microarray analysis, RNA samples from unstimulated cT cells or cT cells TMRM, and Mitotracker Deep Red; Thermo Fisher Scientific) was performed as reg reg activated in the presence of vehicle, Torin 1, or PP242 for 20 h as indicated above previously described30. were analyzed with the GeneChip Mouse Gene 2.0 ST Array (Thermo Fisher Scientific). Differentially expressed transcripts in biological triplicate samples were fi Histology and immunohistochemistry. Tissues were xed in 10% (vol/vol) identified by ANOVA (Partek Genomics Suite version 6.5), and the Benjamini- fi neutral buffered formalin solution, embedded in paraf n, section, and stained with Hochberg method was used to estimate the FDR. hematoxylin and eosin. Blinded samples were analyzed by an experienced GSEA of hallmark pathways in resting vs. activated Treg cells from these pathologist (P.V.) for the presence of lesions indicative of autoimmune disease. For microarray samples or published datasets (GSE5575332 or GSE610777) was fi the identi cation of ieMMCs or lpMMCs, tissue sections from the small intestines performed as previously described30. IRF4 targets were identified from ChIP-seq and large intestines were respectively stained with primary rat anti-MCPT1 data22 deposited in GSE98263 and compared against genes that were differentially fi monoclonal antibody (Cat # 14-55303-82, Thermo Fisher Scienti c) or goat anti- expressed in activated cT cells treated with or without mTOR inhibitors as above. 37 reg MCPT4 antibody (LS-B5958, LifeSpan Biosciences) as described previously . GCs IRF4 target genes that were differentially expressed in activated cT cells treated fi reg were identi ed by staining with anti-CD3 antibody and peanut agglutinin as with mTOR inhibitors were subjected to functional enrichment analysis of 61 described . metabolism-related pathways, where significance was determined using the Fisher exact test and Benjamini-Hochberg method (FDR < 0.05). In vitro Treg-cell suppression assays. For analysis of T -cell suppression + + + reg in vitro, CD4 CD25hi T cells or CD4 Foxp3-YFP T cells, isolated from the reg reg Statistics. The results in graphs represent the mean ± s.e.m., with the numbers of lymphoid organs of the respective Cd4Cre-orFoxp3Cre-expressing mice, were co- + mice per group and number of experimental replicates indicated in each figure cultured with naive CD4 T cells and irradiated splenocytes as antigen presenting legend. The P-values were calculated with unpaired, two-tailed Student’s t-test cells as previously described30. For suppression assays using in vitro activated assuming equal variance (GraphPad Prism software), where *P < 0.05; **P < 0.01; T cells, CD25hi T cells were sorted from the lymphoid organs of C57BL/6 reg reg − ***P < 0.001. No specific randomization methods were used in these studies. mice, resuspended in complete Click’s medium containing IL-2 (200 U ml 1), and − − Investigators were not blinded to samples except where indicated for histological activated using anti-CD3 (10 μgml 1) and anti-CD28 (10 μgml 1) antibodies for and immunohistochemical analysis. 3 days in the presence of PP242 (500 nM, Tocris Bioscience) or vehicle control. The live cells were then isolated using Lymphocyte Separation medium and + co-cultured with naive CD4 T cells and irradiated splenocytes for 3 days, and the Data availability. Microarray data that support the findings of this study have incorporation of [3H]-thymidine was assessed as described30. been deposited in the Gene Expression Omnibus with the primary accession code GSE104130. Other data are available from the corresponding author upon request. + + lo hi fi Treg-cell cultures. CD4 Foxp3-YFP CD44 CD62L cTreg cells were puri ed and activated with anti-CD3 and anti-CD28 antibodies in the presence of recombinant Received: 10 September 2017 Accepted: 26 April 2018 IL-2 for various times in the presence of vehicle, Torin 1 (50 nM) or PP242 (500 nM) before total RNA was harvested using the Qiagen RNeasy micro kit per the ’ manufacturer s instructions. Alternatively, cTreg cells were stimulated as above for 1–3 days and analyzed by flow cytometry as previously described43. For analysis of + + GATA3 protein expression, CD4 CD25 T cells were isolated from the + reg mesenteric lymph nodes using the CD25 T -cell enrichment kit (Miltenyi). The reg − − cells were then activated with anti-CD3 (5 μgml 1) and anti-CD28 (5 μgml 1) References antibodies for 3 days in the presence of various stimuli and/or Torin 1 (50 nM) as 1. Li, M. O. & Rudensky, A. Y. 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T.M.N. and Y.W. designed, performed, and analyzed experiments; P.V. performed

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immunohistochemistry analysis and provided histopathology scoring; X.L. and J.W.L. performed metabolomics analysis; Y.D. and G.N. performed bioinformatics analysis; Open Access This article is licensed under a Creative Commons H.C. designed experiments, edited the manuscript, and provided overall direction. Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give Additional information appropriate credit to the original author(s) and the source, provide a link to the Creative Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- Commons license, and indicate if changes were made. The images or other third party ’ 018-04392-5. material in this article are included in the article s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the Competing interests: The authors declare no competing interests. article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from Reprints and permission information is available online at http://npg.nature.com/ the copyright holder. To view a copy of this license, visit http://creativecommons.org/ reprintsandpermissions/ licenses/by/4.0/.

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