Bach2 regulates homeostasis of Foxp3+ regulatory T cells and protects against fatal lung disease in mice.

Bach2 Regulates Homeostasis of Foxp3+ Regulatory T Cells and Protects against Fatal Lung Disease in Mice This information is current as of June 15, 2015.

Eui Ho Kim, David J. Gasper, Song Hee Lee, Erin Hemmila Plisch, John Svaren and M. Suresh

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J Immunol 2014; 192:985-995; Prepublished online 23 December 2013; doi: 10.4049/jimmunol.1302378 http://www.jimmunol.org/content/192/3/985

The Journal of Immunology

Bach2 Regulates Homeostasis of Foxp3+ Regulatory T Cells and Protects against Fatal Lung Disease in Mice Eui Ho Kim,* David J. Gasper,* Song Hee Lee,†,‡ Erin Hemmila Plisch,* John Svaren,x and M. Suresh*

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elf-tolerance, the inability to elicit or sustain an adaptive immune response against a self-Ag, is a critical feature of the adaptive immune system (1–3). Multiple diverse mechanisms are necessary for the establishment and maintenance of self-tolerance, and their individual or collective failure may lead to life-threatening autoimmune disease (2–4). The mechanisms of self-tolerance can be broadly classified as recessive or dominant (2, 4). Recessive mechanisms include clonal deletion of immature self-reactive T cells in the thymus and functional inactivation/ anergy and apoptosis of mature autoreactive T cells in the periphery. Dominant tolerance is primarily mediated by a subset of CD4 T cells termed regulatory T (Treg) cells that express the signature transcription factor Foxp3. These Treg cells not only protect against autoimmunity, they restrain immune responses to foreign Ags to

*Department of Pathobiological Sciences, University of Wisconsin, Madison, WI 53706; †Institute for Molecular Virology, University of Wisconsin, Madison, WI 53706; ‡McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706; and xDepartment of Comparative Biosciences, University of Wisconsin, Madison, WI 53706 Received for publication September 6, 2013. Accepted for publication November 17, 2013. This work was supported by Public Health Service grants from the National Institutes of Health (AI48785 and AI101976 to M.S.). D.J.G. was supported by a training grant from the National Institutes of Health (T32OD010423). The sequences presented in this article have been submitted to National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih. gov/geo/query/acc.cgi?acc=GSE52337) under accession number GSE52337. Address correspondence and reprint requests to Prof. M. Suresh, Department of Pathobiological Sciences, University of Wisconsin, 2015 Linden Drive, Madison, WI 53706. E-mail address: [email protected] Abbreviations used in this article: B6, C57BL/6; BM, bone marrow; BMC, bone marrow cell; CC, control chimera; EC, experimental chimera; ECP, eosinophilic crystalline pneumonia; eTreg, effector regulatory T; IRF, IFN regulatory factor; iTreg, inducible regulatory T; KO, knockout; LP, lamina propria; mLN, mesenteric lymph node; nTreg, natural regulatory T; pTreg, peripherally derived regulatory T; RA, retinoic acid; Treg, regulatory T; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302378

limit inflammation and immune-mediated tissue damage (5). Lossof-function mutations in the Foxp3 gene result in Treg cell deficiency, loss of self-tolerance, altered adaptive immune responses, and the development the devastating autoimmune diseases immune dysregulation, polyendocrinopathy, enteropathy, X-linked in people and scurfy mice (6, 7). Treg cells are a heterogeneous population and have commonly been classified as either natural Treg (nTreg) or peripherally derived Treg (pTreg) cells according to the site at which they acquire their regulatory functions (1, 8). Both classes emerge from CD4 T cells that have successfully navigated thymus-dependent recessive mechanisms of self-tolerance. The development of the nTreg cell lineage proceeds in the thymus, and this class yields the majority of Treg cells in the secondary lymphoid organs and peripheral tissues. In contrast, the pTreg cells develop from conventional CD4 T cells that have disseminated to peripheral tissues such as the gut, and their development proceeds within those tissues under the influence of the local inflammatory and immunological milieu (1, 8). The ability of pTreg cells to differentiate in peripheral tissues greatly augments the regulatory capacity of the nTreg cells. Regardless of origin, normal Treg cell development and acquisition of regulatory function are dependent on the induction and sustained expression of Foxp3 (9–11). Therefore, Foxp3 has been touted as a lineage-specifying master regulator for the establishment and maintenance of the Treg cell transcription program. However, there is mounting evidence that Foxp3 alone might be insufficient for the induction and/or maintenance of the full spectrum of Treg cell characteristics and signature genes (12–14). Genome-wide gene expression profiling and computational network inference studies have suggested that the full induction of the Treg cell transcription program is dependent upon combinatorial association of Foxp3 with a quintet of functionally redundant transcription factors such as IFN regulatory factor 4 (IRF4), Eos, Lef1, Gata1, and Satb1 (12). Several additional transcription factors such as Bach2, Blimp1, Maf, Tcf1, and Xbp1 are also predicted to influence the Treg cell


Variants of the Bach2 gene are linked to vitiligo, celiac disease, and type 1 diabetes, but the underlying immunological mechanisms are unknown. In this study, we demonstrate that Bach2 plays crucial roles in maintaining T cell quiescence and governing the differentiation, activation, and survival of Foxp3+ regulatory T (Treg) cells. Bach2-deficient T cells display spontaneous activation and produce elevated levels of Th1/Th2-type cytokines. Without Bach2, Treg cells exhibit diminished Foxp3 expression, depleted numbers, hyperactivation, enhanced proliferation, and profound loss of competitive fitness in vivo. Mechanistically, reduced survival of Bach2-deficient Treg cells was associated with reduced Bcl-2 and Mcl-1 levels and elevated Bim/Bcl-2 ratio. Additionally, Bach2 deficiency induced selective loss of Helios2Foxp3+ Treg cells and a Treg cell transcriptome skewed toward the Th1/Th2 effector program at the expense of the Treg program. In vitro experiments confirmed that Bach2: 1) is indispensable for TCR/ TGF-b–induced Foxp3 expression; and 2) mitigates aberrant differentiation of Treg cells by repression of the competing Gata3driven Th2 effector program. Importantly, perturbations in the differentiation of induced Treg cells was linked to a fatal Th2-type chronic inflammatory lung disease in Bach2-deficient mice. Thus, Bach2 enforces T cell quiescence, promotes the development and survival of Treg lineage, restrains aberrant differentiation of Treg cells, and protects against immune-mediated diseases. The Journal of Immunology, 2014, 192: 985–995.

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Materials and Methods Mice Bach2 knockout (KO) mice on the C57BL/6 (B6) background were a kind gift from K. Igarashi (Tohoku University, Sendai, Japan) (18). Littermate wild-type (WT) mice were used as controls. B6 and C57BL/6 Ly5.1+ mice were purchased from the National Cancer Institute or The Jackson Laboratory (Bar Harbor, ME). All mice were housed in specific pathogen-free conditions in the animal facilities at the University of Wisconsin-Madison (Madison, WI). Experiments were conducted in accordance with the approved protocols of the institutional animal care committee.

Lymphocyte isolation and flow cytometry Mononuclear cells from thymus and spleen were prepared using standard techniques. Lamina propria (LP) lymphocytes were isolated from the entire small intestine. Following excision of the Peyer’s patches, small intestine was washed, cut into pieces, and incubated in HBSS/HEPES bicarbonate buffer containing 15.4 mg/ml dithioerythritol at 37˚C for 30 min. After washes, the LPs were dissociated in type I collagenase solution at 37˚C for 50 min. LP lymphocytes were purified on a Percoll gradient. Cells isolated from various tissues were stained with Abs diluted in staining buffer (2% BSA in PBS). Fluorochrome-labeled anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD25 (PC61.5), anti-GITR (DTA-1), anti-CD44 (IM7), anti-CD62L (MEL-14), anti-CD69 (H1.2F3), anti-CD127 (A7R34), anti-KLRG1 (2F1), anti-CD45.2 (Ly5.2, 104), anti–IFN-g (XMG1.2), anti–TNF-a (MP6-XT22), anti–IL-4 (BVD4-1D11), anti–IL-13 (eBio13A), anti-Ki67 (B56), antiFoxp3 (FJK-16S), anti-CTLA4 (UC10-4B9), anti–Bcl-2 (3F11), anti-Gata3 (L50-823), and T-bet (eBio4B10) Abs were purchased from BD Biosciences (San Jose, CA) or eBioscience (San Diego, CA). The anti–granzyme B (GB11) Ab was purchased from Invitrogen (Grand Island, NY). Anti-Bim and anti-Tcf1 (C63D9) were purchased from Cell Signaling Technology (Danvers, MA). Anti-Helios (22F6) Ab was purchased from BioLegend (San Diego, CA), and anti-Blimp1 (3H2-E8) Ab was purchased from Novus

Biologicals (Littleton, CA). Intracellular Foxp3 was stained using the Foxp3 Staining Kit (eBioscience). For intracellular cytokine staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) following cell-surface staining. All samples were acquired with FACSCalibur or LSR II (BD Biosciences). Data were analyzed with FlowJo software (Tree Star, Ashland, OR).

Quantitative RT-PCR and DNA microarray CD4 T cells were purified from spleens of WT or Bach2 KO mice by negative selection using MACS (Miltenyi Biotec, Auburn, CA). To obtain Treg cells, purified CD4 T cells were stained with anti-CD4, anti-CD25, and anti-GITR and sorted using an FACSAria II instrument (BD Biosciences). RNAs from purified cells were extracted with the RNeasy kit (Qiagen, Valencia, CA), and cDNA was synthesized with the Superscript III reverse transcription kit (Invitrogen). Quantitative PCR reactions were done with the Power SYBR Green PCR Master Mix (Applied Biosystems, Grand Island, NY), and data were collected by Applied Biosystems 7300 RealTime PCR System (Applied Biosystems). Equivalent amounts of cDNA (as determined by 18S rRNA measurements) were amplified in 40 cycles of PCR. For microarray analysis, the Gene Expression Center at University of Wisconsin-Madison performed BeadChip Mouse Ref-8 V 2.0 (Illumina, San Diego, CA) using 200 ng total RNA extracted as previously described. Gene expression profile was analyzed by using GeneSpring software (Agilent Technologies, Santa Clara, CA). The data discussed in this publication have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus under accession number GSE52337 (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE52337).

Generation of mixed bone marrow chimeras For generating mixed bone marrow (BM) chimeras, BM cells (BMCs) were collected by flushing the marrow from the femurs and humeri from WT and Bach2 KO mice with RPMI 1640 media. A 1:2 mixture of BMCs from WT (Ly5.1; 5 3 106 BMCs) and WT (Ly5.2; 10 3 106 BMCs) or Bach2 KO (Ly5.2; 10 3 106 BMCs) mice were adoptively transferred into lethally irradiated (800 rad) B6/Ly5.1 mice. BM–reconstituted B6 mice were treated with neomycin (0.025 mg/ml) and polymyxin B (0.013 mg/ml; SigmaAldrich, St. Louis, MO) in drinking water for up to 8 wk, and reconstitution of the lymphoid system by the donor BMCs was assessed at 8 wk.

In vitro Treg cell differentiation Splenic CD4+CD252GITR2 T cells sorted by flow cytometry were used at a density of 2 3 105 cells/well in flat-bottom 96-well plates precoated with anti-CD3 (2 mg/ml). Cells were cultured for 72 h with IL-2 (100 U/ml) in the presence or absence of different concentrations of recombinant human TGF-b (PeproTech, Rocky Hill, NJ). Foxp3 expression was assessed by flow cytometry.

Histopathology Representative tissue sections were fixed in 10% neutral buffered formalin. The fixed tissues were paraffin embedded, and 5-mm-thick sections were routinely stained with H&E. Photomicrographs were acquired with cellSens software (Olympus). The GNU Image Manipulation Program, version 2.8.4, was used to improve uniformity of brightness and contrast between individual images and to remove adjacent lung sections from Fig. 6A and 6B.

Statistical analyses Data statistics were determined using SigmaPlot software. Student twotailed t test was used to calculate the statistical significance of differences between groups, and significance was defined at p , 0.05. Error bars represent SEM.

Results Bach2 controls peripheral T cell quiescence Our laboratory reanalyzed previously published T cell microarray data to identify candidate transcription factors predicted to impact T cell differentiation and found strong evidence that Bach2 mRNA was expressed in T cells (23, 24). Additionally, Bach2 gene variants have been linked to autoimmune diseases including celiac disease, vitiligo, and type 1 diabetes (20–22). We investigated the extent to which Bach2 was required for peripheral T cell homeostatic maintenance using Bach2-deficient (Bach2 KO) mice. A striking increase in the percentages and total numbers of CD4 and


gene signature. Further characterization of these additional molecules and their role in the development and maintenance of the Treg cell transcriptional program is necessary for understanding the biology of these important cells and may yield potential targets for therapeutic interventions in cases in which their critical regulatory functions fail. Genome-wide analysis of Foxp3 target genes has suggested that Bach2 is likely a target gene for Foxp3, and Foxp3 is predicted to downregulate Bach2 in both thymic and peripheral Treg cells (10, 15). Bach2 was initially characterized as a B cell–specific transcriptional repressor tasked with opposing plasma cell differentiation and the maintenance of B cell identity (16–18), but evidence now suggests that it is also expressed in T cells. Importantly, in Treg cells, Bach2 has now been identified as a target gene for the transcription factor FoxO1 (19), and Fu et al. (12) predicted that Bach2 expression would potentially influence 14 genes within the Treg signature. Importantly, in humans, genome-wide association studies have linked Bach2 gene variants to immune-mediated diseases such as celiac disease, vitiligo, and type 1 diabetes (20–22). Despite this, the actual role of Bach2 in T cell homeostasis, Treg cell differentiation, or maintenance is yet to be determined. In this manuscript, we report that Bach2 plays a vital role in regulating the homeostasis of conventional T cells and Foxp3+ Treg cells by cell-intrinsic mechanisms. Specifically, we show that Bach2 imparts competitive fitness to Foxp3+ Treg cells and represses the activation and proliferation of Foxp3+ Treg cells. Further, we find that full induction of Foxp3 and TGF-b–induced differentiation of Treg cells requires Bach2. Mechanistically, Bach2 is an integral member of the transcriptional network that promotes the differentiation of Helios2Foxp3+ Treg cells by supporting the Treg transcriptional program at the expense of the effector T cell program. In the absence of Bach2, altered T cell homeostasis and perturbations in the development of induced Treg cells led to a fatal Th2-type immune-mediated disease.

Bach2 CONTROLS REGULATORY T CELLS


Bach2 regulates the homeostasis of Foxp3+ Treg cells Treg cells have been previously reported by the Rudensky group (10, 15) to exhibit dynamic developmental alterations in Bach2 expression, and the Benoist group (12) subsequently predicted that Bach2 expression can potentially influence the transcriptional signature of Treg cells. Therefore, we next investigated whether the dysregulated T cell homeostasis resulting from Bach2 deficiency extended to alterations in the number and phenotype of Foxp3+ Treg cells in the thymus and spleen. As illustrated in Fig. 2A, we identified a small but reproducible and statistically significant reduction in the percentages (1.3-fold,; p , 0.06) and total number (1.7-fold; p , 0.045) of Foxp3+ Treg cells in the thymus

of Bach2 KO mice. Further, these thymic Treg cells exhibited an activated phenotype characterized by elevated levels of CD25, CD44, and GITR expression. Bach2 deficiency did not, however, affect Treg cell expression of CTLA4 in the thymus (Fig. 2B). In contrast to the less dramatic thymic Treg cell alterations, Bach2 deficiency resulted in a marked reduction in the frequency and number of Treg cells in the spleen (Fig. 2C). The frequencies of splenic Treg cells in Bach2 KO mice were only 57% of those exhibited by WT mice. Further, Foxp3+ Treg cells in spleen of Bach2 KO mice displayed enhanced levels of CD44, CTLA4, GITR, KLRG1, CD127 (the IL-7 receptor), granzyme B, and Blimp1, a phenotype that is considered as prototypical for effector Treg (eTreg) cells (Fig. 2D, 2E, 2I) (25). Next, we characterized the expression of Bcl-2, Bim, Ki67, and Annexin V in Treg cells from Bach2 KO mice to investigate whether the diminished number of Treg cells resulted from defects in cell survival and/or proliferation. The balance between Bcl-2 and Bim is believed to be important for survival of Treg cells (26). We found that the levels of Bcl-2 in Bach2 KO Treg cells were dramatically lower than in WT Treg cells (Fig. 2F), and the resulting Bim: Bcl-2 ratios were substantially increased in Bach2 KO Treg cells (Fig. 2F). The mRNA levels for the anti-apoptotic molecule Mcl-1 were also lower in Bach2 KO Treg cells (Fig. 3A) (27). Additionally, higher percentages of Treg cells from Bach2 KO mice exhibited the apoptotic Annexin V+ phenotype (Fig. 2G). To examine the effect of Bach2 deficiency on the proliferation of Foxp3+ Treg cells, we compared the cell-cycle status of WT and Bach2 KO Treg cells by staining for the proliferative marker Ki67. Bach2 deficiency augmented the frequency of Ki67+ Treg cells in the thymus and spleen (Fig. 2H). In spleen, whereas only 20% of

FIGURE 1. Bach2 regulates peripheral T cell homeostasis. Splenocytes were collected from naive WT and Bach2 KO mice. (A–C) Activation of T cells was analyzed by staining with anti-CD44 and anti-CD62L. (A) Frequencies of naive versus activated CD4 and CD8 T cells. (B) Numbers of naive versus activated CD4 and CD8 T cells were calculated per spleen. (C) Median fluorescence intensity (MFI) of CD44 for each subset is plotted. (D) Splenocytes were stimulated for 5 h in vitro with PMA and ionomycin in the presence of brefeldin A, and cytokine production was measured by intracellular staining. (E) Expression of transcription factors such as T-bet and Gata3 was assessed by flow cytometry following intracellular staining. Data are representative of two independent experiments. *p , 0.05.


CD8 T cells with the activated/memory (CD44HI) phenotype occurred in spleens of Bach2 KO mice, as compared with those in WT mice (Fig. 1A, 1B). Notably, Bach2 deficiency increased the numbers of CD4 and CD8 T cells with the activated (CD44HI/ CD62LHI) and effector (CD44HI/CD62LLO) phenotypes. Interestingly, regardless of their activation status (naive/CD44LO versus activated/CD44HI), Bach2-deficient CD4 and CD8 T cells expressed higher levels of cell-surface CD44 per individual cell than their WT counterparts (Fig. 1C). Notably, a larger fraction of the Bach2 KO CD4 T cells produced readily detectable levels of Th1- (IFN-g) and Th2-type (IL-4 and IL-13) cytokines (Fig. 1D). The enhanced productions of Th1 and Th2 cytokines by Bach2 KO CD4 T cells were consistent with increased levels of the effector lineage-specific transcription factors, T-bet and Gata3, respectively (Fig. 1E). Collectively, data in Fig. 1 suggested that Bach2 promotes T cell quiescence by repressing the activation and differentiation of Th1/Th2 effector CD4 T cells under normal homeostatic conditions.

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WT Treg cells expressed Ki67, 50–60% of Treg cells were Ki67+ in the Bach2 KO mice. The increased Ki67+ cells among Bach2 KO Foxp3+ Treg cells suggested that Bach2 exerts antiproliferative effects. It is also possible that increased proliferation of Bach2 KO Tregs might reflect compensatory proliferation that occurs under conditions in which Treg cells’ maintenance is compromised (28). The intestinal tract is an anatomical location that is critical for the peripheral induction of Treg cells. To assess whether Bach2 regulates accumulation of Treg cells in the intestines, we quantified Foxp3+ Treg cells in the mesenteric lymph nodes (mLNs) and LP. Interestingly, there was a striking reduction in the frequencies of CD4 T cells in the LP (Fig. 2J) but not in the mLNs (not shown) of Bach2 KO mice. Consistent with other tissues examined, the percentages of Tregs in mLNs (p , 0.0004) and LP (p , 0.002) were significantly reduced in Bach2 KO mice (Fig. 2J). Taken together, data in Fig. 2 suggested that Bach2 plays a nonredundant role in Treg cell homeostasis in lymphoid and

nonlymphoid tissues by regulating the activation, generation, and maintenance of Treg cells. To determine whether the reduced Foxp3+ Treg cell number in Bach2 KO mice was associated with dysregulated Foxp3 expression, we compared the level of Foxp3 expressed per Treg cell in WT and Bach2 KO mice. Notably, the expression of Foxp3 protein was markedly reduced in Bach2 KO Treg cells in the spleen, but not in the thymus (Fig. 2I). This prominent feature was consistently documented in Bach2 KO mice that are .9 to 10 wk of age. A recent study has predicted Tcf1 and Blimp1 to be important transcription factors (other than Foxp3) that can potentially influence the expression of several genes constituting the Treg signature (12). Compared to WT Treg cells, Bach2 KO Treg cells displayed severely reduced Tcf1 expression, whereas Blimp1 levels were elevated (Fig. 2I). Based on these data, we inferred that Bach2 promotes the Treg cell transcriptional program by regulating the levels of Foxp3, Tcf1, and Blimp1.


FIGURE 2. Bach2 controls the homeostasis of Treg cells. Spleens and thymus were collected from naive WT and Bach2 KO mice (.9 wk old) and analyzed for Treg cells by flow cytometry. (A) Frequency and number of thymic Treg cells. (B) Phenotypic analysis of thymic Foxp3+ Treg cells. (C) Frequency and numbers of splenic Treg cells. (D) Phenotypic analysis of splenic Foxp3+ Treg cells. (E) Surface expression of KLRG1 and intracellular expression of granzyme B were analyzed for Foxp3+ Treg cells by flow cytometry. (F) Bcl-2 and Bim expression was assessed by intracellular staining in splenic Foxp3+ Treg cells and Foxp32 conventional CD4 T cells; numbers indicate median fluorescence intensity of Bcl-2. (G) Treg (CD4+CD25+GITR+) cell apoptosis was measured by Annexin V staining directly ex vivo. (H) Ki67 staining on thymic and splenic Foxp3+ Treg cells. (I) Expression levels of Foxp3, Blimp1, and Tcf1 on thymic (top panel) and splenic (bottom panel) Foxp3+ Treg cells were assessed by flow cytometry. (J) Treg cells from mLNs and small intestinal LP were quantified by flow cytometry. Data are representative of two independent experiments. *p , 0.05.


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Bach2 governs the balance of the effector and Treg transcription programs during Treg cell differentiation To understand the transcriptional basis for the altered Treg cell homeostasis in Bach2 KO mice, we compared the transcriptomes of CD4+CD25+GITR+ WT and Bach2 KO Treg cells. The scatter plot in Fig. 3A illustrates the altered expression of multiple genes that are known to regulate Treg cell development and/or homeostasis. First, the expressions of gata1 and lef1 that are members of the quintet of transcription factors that promote Treg differentiation were substantially reduced in Bach2 KO Treg cells (12). Likewise, Bach2 deficiency resulted in reduced levels of Foxo1 (29, 30), id3 (31), and rara (32, 33), which are known to play prominent roles in the development of Tregs. Signaling molecules tnfsf4 and tnfrsf4 (encodes OX40L and OX40, respectively) inhibit Foxp3 expression in Treg cells (34, 35), and interestingly, their expressions were elevated in Bach2 KO Treg cells. The elevated levels of prdm1 (encodes Blimp1) and irf4 along with itgae (CD103) and klrg1 are consistent with the activated or effector phenotype of Tregs in Bach2 KO mice (Fig. 2E, 2I) (25, 36). Additionally, genes like pde3b (10) and satb1 (37) that are typically expressed at low levels in Tregs were further decreased in Bach2 KO Treg cells. Bach2 deficiency was associated with diminished expression of prosurvival molecules bcl2 and mcl1, which might explain the enhanced apoptosis of Bach2 KO Treg cells (27). These data suggested that Bach2 might promote Treg cell homeostasis by activating genes that control their differentiation and survival. The relative dominance of the various competing effector transcription programs versus the Foxp3-centric Treg cell transcription

program is a key determinant factor in the differentiation and stability of the Treg cells. Therefore, we investigated whether Bach2 deficiency affected the competing Th1, Th2, or the Th17 transcriptional program in Treg cells (37). As illustrated in Fig. 3B and 3C, genes associated with Th1 were upregulated in Bach2 KO Treg cells and included the signature transcription factor tbx21 (encodes T-bet), ifng, and il12rb2. Much more impressive was the marked skewing of the transcriptional profile toward the Th2 lineage in Bach2 KO Treg cells; the expression of the Th2 lineage-defining transcription factor gata3 along with Th2-type cytokines genes including IL-4, IL-5, IL-6, and IL-13 were increased in Bach2 KO Treg cells. Although the expression of rorc (encodes Rorgt) was decreased (Fig. 3B, 3C), other genes associated with Th17 were largely unaltered in the absence of Bach2. On the basis of these data, we inferred that Bach2 might enable the differentiation of Treg cells by repression of the competing Th1 and Th2 effector transcriptional program. A cell-intrinsic role for Bach2 in regulating the homeostasis of conventional and Foxp3+ Treg cells To assess whether Bach2 regulated the development and/or survival of Foxp3 + Tregs by cell-intrinsic mechanism(s), we generated mixed BM chimeras by reconstituting lethally irradiated WT/Ly5.1 mice with a mixture of BMCs from WT/Ly5.1 and WT/Ly5.2 or Bach2 KO/Ly5.2 mice. Control chimeras (CCs) were reconstituted with a mixture of BMCs from WT/ Ly5.1 and WT/Ly5.2 mice, whereas the experimental chimeras (ECs) were derived by reconstitution of WT/Ly5.1 mice with BMCs from WT/Ly5.1 and Bach2 KO/Ly5.2 mice. Eight weeks


FIGURE 3. Bach2 regulates the balance of the effector and Treg transcription programs. Whole-genome transcriptional profiles from WT and Bach2 KO CD4+ CD25+GITR+ splenic Tregs were analyzed by DNA microarray. (A) Scatter plot represents comparison of normalized expression values in WT versus Bach2 KO. Expression of genes associated with Th1, Th2, Th17, or Treg functions in WT and Bach2 KO mice is presented as box whisker plots (B) and heat maps (C).

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FIGURE 4. Bach2 regulates the homeostasis of Treg cells by cell-intrinsic mechanisms. Mixed BM chimeras were generated by the transfer of a mixture of BMCs from WT/Ly5.1 and WT/Ly5.2 or Bach2 KO/Ly5.2 into lethally irradiated WT/Ly5.1 mice. Although CCs were reconstituted by a mixture of WT/Ly5.2 and WT/Ly5.1 BMCs, ECs were reconstituted by the mixture of Bach2 KO/Ly5.2 and WT/Ly5.1 BMCs. At least 8 wk after reconstitution, thymus and spleens were harvested and analyzed for Treg cells. (A) Spleens were harvested, and T cells were analyzed for their activation status. Cells were gated on WT/Ly5.2 (CC) and Bach2 KO/Ly5.2 (EC) for direct comparison. Frequencies and numbers of naive versus activated CD4 and CD8 T cells were assessed and calculated per spleen. (B) Frequencies of Foxp3+ Treg cells among the gated cells in both thymus and spleen were measured by flow cytometry. (B and C) Surface markers and Treg cell markers on WT/Ly5.2 (CC) and Bach2 KO/Ly5.2 (EC) Treg cells were measured by flow cytometry. (C) Frequency of Ly5.2+ Treg cells positive for CD44, CD69, and Ki67 and Blimp1 median fluorescence intensity (MFI). (D) MFIs of Foxp3 for Ly5.2+ WT and Bach2 KO Treg cells. Data are representative of two independent experiments. *p , 0.05.

were Ki67+ and expressed higher levels of CD44 and CD69, as compared with their WT/Ly5.2 or WT/Ly5.1 counterparts (Fig. 4C). Thus, the increased proliferation and the effector phenotype (CD44HI/CD69HI/Blimp1HI) of Bach2 KO Foxp3+ Treg cells are consequential to cell-intrinsic loss of Bach2. By extension, these data imply that Bach2 might repress the activation and proliferation of effector Foxp3+ Treg cells by cell-intrinsic mechanism(s). Also, loss of Bach2 might result in premature activation, proliferation, and perhaps apoptosis of effector Foxp3+ Treg cells. Another notable finding was that Bach2 KO Foxp3+ Treg cells expressed significantly lower levels of Foxp3 (p , 0.00003), in comparison with WT Treg cells (Fig. 4D). In summary, data in Fig. 4 provide compelling evidence that: 1) Bach2 promotes T cell quiescence by T cell–intrinsic mechanisms; 2) in the absence of Bach2, Treg cells fail to develop and/or persist in a competitive environment; 3) Bach2 restrains the activation and proliferation of


after reconstitution, the percentages of activated cells were greater among Bach2 KO CD4 and CD8 T cells, as compared with their WT counterparts (Fig. 4A). Additionally, in the thymus and spleen of CCs, the percentages of Foxp3+ Treg cells among WT/Ly5.1 CD4 T cells were slightly higher than among WT/Ly5.2 CD4 T cells (Fig. 4B). In striking contrast, the percentages of Foxp3+ Treg cells among Bach2 KO/Ly5.2 CD4 T cells in the thymus and spleen of ECs were 5- to 6-fold lower, as compared with the WT/ Ly5.1 CD4 T cells in the same mouse (Fig. 4B). Direct comparison between WT/Ly5.2 CD4 T cells in CCs with Bach2 KO/ Ly5.2 CD4 T cells in the ECs also showed 3–6-fold reduction in the percentages of Foxp3+ Treg cells among Bach2 KO CD4 T cells (Fig. 4B). Thus, Bach2 KO Foxp3+ Treg cells displayed loss of competitive fitness to develop and/or persist in mixed BM chimeras. The Bach2 KO Treg cells in ECs expressed more Blimp1, and a greater percentage of Bach2 KO Foxp3+ Treg cells


The Journal of Immunology Treg cells; and 4) Bach2 is required for full induction of Foxp3 in Treg cells by cell-intrinsic mechanism(s). Bach2 promotes the induction of Foxp3 and generation of Treg cells by repressing the effector transcription program Analyses of Bach2 KO mice and mixed BM chimeras clearly demonstrated a vital role for Bach2 in determining the competitive fitness of Foxp3 + Treg cells. It was of interest to investigate whether Bach2 regulated the development and/or persistence of subsets of Treg cells. The expression level of Helios, an Ikaros transcription factor, has been used to define subsets of Treg cells in the periphery (38). Intriguingly, we found that the percentages

991 and numbers of Helios+Foxp3+ Treg cells were not significantly different in spleens of WT and Bach2 KO mice (Fig. 5A). However, in Bach2 KO mice, the frequency (3-fold; p , 0.02) and numbers (5-fold; p , 0.03) of splenic Helios2Foxp3+ Treg cells were dramatically reduced, as compared with those in WT mice. These data suggested that that Bach2 deficiency selectively impaired the development and/or survival of the Helios2Foxp3+ Treg cells in the periphery (Fig. 5A). Next, we asked whether the in vitro induction of Foxp3 and generation/differentiation of Foxp3+ inducible Treg (iTreg) cells requires Bach2. TCR stimulation of naive conventional WT CD4 T cells in the presence of TGF-b readily induced Foxp3 expression


FIGURE 5. Bach2 is required for the induction of Foxp3 and differentiation of iTregs. (A) Splenocytes from WT and Bach2 KO mice were stained with anti-CD4, anti-Foxp3, and anti-Helios. Frequencies and numbers of Helios+ and Helios2 Tregs are displayed. (B–F) Conventional CD4 T cells (CD252 GITR2) from WT and Bach2 KO mice were purified by FACS and stimulated with anti-CD3 in the presence or absence of recombinant human TGF-b for 72 h. (B) Representative contour plots depict Foxp3 expression following in vitro Treg differentiation. Bar graph displays frequencies of Foxp3+ iTregs with or without anti-CD3 in the presence of indicated concentrations of TGF-b. (C) Median fluorescence intensities (MFIs) of Foxp3 for Foxp3+ iTregs from WT and Bach2 KO are plotted. (D and E) RNA from cells cultured for iTreg cell differentiation was reverse transcribed. Relative levels of Treg signature transcription factors (D), CD4 T cell lineage-specific transcription factors (E), and cytokines (F) were quantified by quantitative RT-PCR. Data are representative of two independent experiments. *p , 0.05.

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Bach2 KO mice develop a fatal lung disease Strikingly, we observed an exceptionally high incidence of eosinophilic crystalline pneumonia (ECP) and eosinophilic diseases in our colony of Bach2 KO mice. Through opportunistic postmortem sampling of lung tissue, the characteristic histologic lesions of ECP were first identifiable in some Bach2 KO mice at 8 wk of age. Beyond 8 wk, Bach2 KO mice exhibited a progressive age-dependent increase in morbidity and mortality. Some Bach2 KO mice exhibited moderate respiratory distress and hunched posture by 4 mo of age, and by 7 to 8 mo of age, nearly all Bach2 KO mice displayed severe respiratory distress necessitating humane euthanasia. Compared to normal histology of WT lungs (Fig. 6A), ECP lung lesions were present in only a few foci in younger subclinical mice, whereas the lesions were widespread in lungs of older clinically affected Bach2 KO mice (Fig. 6B). Histologically, there was disruption of the alveolar architecture by intra-alveolar aggregates of large macrophages that had hypereosinophilic acicular intracytoplasmic crystals (Fig. 6D), which are primarily composed of YM1 protein (also referred to as chitinase 3-like 3

FIGURE 6. Bach2 KO mice develop lung pathology. B6 WT and Bach2 KO mice were sacrificed and examined by H&E stain for lung immune pathology. (A) WT mouse, original magnification 315. Normal lung with large caliber bronchioles and pulmonary vessels located centrally (stars) and well-defined small-caliber and terminal airways and alveoli located peripherally (arrowheads). (B) Bach2 KO mouse, original magnification 315. Lung with ECP exhibiting regional loss of alveolar spaces resulting from densely cellular alveolar exudates and interstitial infiltrates (arrows). (C) Bach2 KO mouse, original magnification 3200. Lung with ECP: multiple alveolar spaces are filled with aggregates of hypereosinophilic inflammatory cells (stars). Infiltrates of eosinophils, histiocytes, and plasma cells expand the alveolar walls (arrows). Plasma cells and eosinophils encircle a pulmonary vein (arrowhead) (D) Bach2 KO mouse, original magnification 3600. Lung with ECP, affected alveolus: the densely cellular alveolar exudate is primarily composed of tightly packed variably sized macrophages containing intracytoplasmic hypereosinophilic acicular crystals (star). Eosinophils expand the adjacent interstitum (arrow). (E) Bach2 KO mouse, original magnification 3600. Lung with ECP: a medium-caliber pulmonary vein (arrow) is encircled by a cuff of plasma cells (arrowheads) admixed with fewer eosinophils and small lymphocytes. (F) Bach2 KO mouse, original magnification 3600. Densely cellular aggregates of eosinophils, plasma cells, and lymphocytes disrupt and fill an airway in a 7-mo-old mouse with severe concurrent multisystemic eosinophilic and plasmacytic inflammation. (G) mRNAs of indicated cytokines in WT and Bach2 KO lung lysates were quantitated by quantitative PCR.

crystals), a secretory product of pulmonary macrophages (40, 41). Notably, the genesis of YM1 protein-containing crystals in ECP and the Charcott-Leyden–like crystals (found in the lungs of human asthmatic patients) are both products of dysregulated Th2type immunity in the lungs (41, 42). Affected alveoli were lined by type II pneumocytes, and the interstitium was expanded by


in 26% of the stimulated WT conventional CD4 T cells (Fig. 5B). Remarkably, conventional CD4 T cells from Bach2 KO mice exhibited a profound impairment in Foxp3 expression and differentiation into Foxp3+ iTreg cells. Ag receptor stimulation and exposure to TGF-b induced Foxp3 only in 3% of the Bach2 KO conventional CD4 T cells (Fig. 5B). Moreover, iTreg cells that were derived from Bach2 KO CD4 T cells expressed low levels of Foxp3 on a per-cell basis (Fig. 5C). These data strongly suggested that Bach2 plays an essential role in the induction of Foxp3 and generation of iTreg cells by cell-intrinsic mechanism(s). Next, we sought to decipher the transcriptional basis for the impaired Foxp3 expression in Bach2 KO CD4 T cells in response to TGF-b. Specifically, we asked whether Bach2 deficiency dysregulated the expression of Foxp3 and/or the quintet of transcription factors that govern the differentiation of Treg cells (12). As illustrated in Fig. 5D, the expression of Foxp3 was reduced, but the levels of gata1 were particularly elevated in Bach2 KO CD4 T cells. Although the levels of ikzf4 (encodes Eos) and lef1 were mildly altered in Bach2 KO CD4 T cells, loss of Bach2 elevated the expression of ikzf2 (encodes Helios) and xbp1. We also interrogated whether loss of Bach2 resulted in aberrant induction of the Th cell effector program at the expense of the Treg cell differentiation. Notably, concomitant with impaired expression of Foxp3, the levels of Th2-type effector lineage-specification factor gata3 and prdm1 were strongly induced in Bach2 KO CD4 T cells (Fig. 5D, 5E). Strikingly, enhanced gata3 expression was associated with a dramatic induction (.1000-fold) of IL-4 in Bach2 KO CD4 T cells (Fig. 5F). Interestingly, in the absence of Bach2, the levels of IFN-g were also noticeably elevated with minimum alterations in the induction of tbx21. IL-10 expression by eTreg cells requires Blimp1 (25), and consistent with this report, enhanced Blimp1 levels were associated with markedly elevated levels of IL-10 mRNA in Bach2 KO CD4 T cells (Fig. 5F). The elevated expression of Blimp1 in the absence of Bach2 is likely a sequel to loss of Bach2-mediated repression of Blimp1. As compared with augmented expressions of IL-4, IFN-g, and IL-10, Bach2 deficiency resulted in less impressive alterations in the levels of IL-17. Taken together, data in Fig. 5 provide compelling evidence that Bach2 plays an essential role in the TGF-b–induced expression of Foxp3 and in vitro differentiation of Treg cells. Additionally, these data suggested that Bach2 represses the effector Th1/Th2 transcription program and promotes the commitment of CD4 T cells to the Treg program. This inference is consistent with a recent report that came out when this manuscript was in preparation (39).



Discussion It is abundantly clear that Bach2 plays a pivotal role as a transcriptional repressor in B cells by opposing the differentiation of plasma cells from activated B cells. Because Bach2 is considered primarily as a B cell–specific transcriptional factor, its role in T cells remains largely unexplored. In this manuscript, we ascribe crucial regulatory roles for Bach2 in the homeostasis of T cells. Specifically, we document that Bach2 is an integral member of the transcriptional network that enforces quiescence of mature T cells and regulates the competitive fitness, activation, and differentiation of Foxp3+ Treg cells. These findings have clinical implications because we also find that Bach2 deficiency results in fatal lung disease in mice, and Bach2 variants are implicated in the genesis of autoimmune diseases in humans (20–22). First, we show that in the global absence of Bach2, T cells display a spontaneously activated phenotype and produce inflammatory cytokines. Studies using mixed BM chimeras suggest that Bach2 promotes the quiescence of T cells, at least in part by cell-intrinsic mechanisms (Fig. 4A). The intrinsic mechanisms of Bach2mediated regulation of naive T cell quiescence include repression of transcription factors such as T-bet, Blimp1 (not shown), and Gata3 that drive the effector program in T cells. By repression of these transcription factors, Bach2 might mitigate the aberrant and premature activation of peripheral T cells in response to homeostatic cues. Prominently, activated/effector CD4 T cells in Bach2 KO mice display a highly skewed Th2-type polarity, which accompanied a modest elevation of the Th1-type T cells. The skewing toward the Th2 type in Bach2 KO mice is not restricted to conventional CD4 T cells because activation of CD4 T cells under Treg-inducing conditions (with TGF-b) also induces high levels of IL-4 and Gata3. Thus, Bach2-deficient T cells have an increased propensity to differentiate into Th2 effector cells, which is likely linked to the loss of Bach2dependent repression of Gata3 expression; when this manuscript was in preparation, it was reported that Bach2 might directly repress the induction of Gata3, at least in iTreg T cells (39). In addition to the maintenance of T cell quiescence, Bach2 plays a vital role in maintaining the size of the Treg cell niche in the thymus and periphery; loss of Bach2 led to a substantive reduction in the number of Foxp3+ Treg cells in the thymus and periphery. Our data suggest that regulation of Treg cell homeostasis by Bach2

is multifactorial and occurs by at least three mechanisms that are not mutually exclusive. First, Bach2 confers competitive fitness for Foxp3+ Treg cells in thymus and periphery by cell-intrinsic mechanisms that promote the differentiation and/or survival of Foxp3+ nTreg cells. One mechanism by which Bach2 might confer competitive fitness for Foxp3+ nTreg cells is by enhancing their survival because Bach2 KO Treg cells express diminished levels of the antiapoptotic Bcl-2 and Mcl-1, which are known to promote the viability of Treg cells (26, 27). The control of Treg cell survival by Bach2 deficiency might include potential repression of cellular Bcl-2 and Mcl-1 expressions, resulting from elevated levels of Blimp1 (25). Second, diminished levels of Foxp3 in Bach2 KO Treg cells might reduce the stability of the Treg cell transcription program and increase the plasticity of these cells in the periphery. Direct ex vivo analysis of the transcriptomes of peripheral Treg cells from Bach2 KO mice suggest that the balance of the Treg and the effector transcription programs is skewed in the absence of Bach2, in favor of the latter. In addition to the reduced expression of Treg cell– associated signature genes, there was a concomitant elevation in the levels of the genes linked to the Th1/Th2 effector programs in Bach2 KO Treg cells. These data suggest that Bach2 likely favors the Treg development by inducing the Treg transcriptional program and repression of the competing Th1/Th2 effector programs. This idea is consistent with a report from Wan and Flavell (9), which shows that attenuated Foxp3 expression in Treg cells results in lymphadenopathy and a fatal Th2-type immunopathology driven by aberrant differentiation of Treg cells into Th2 effector cells. Other groups also have demonstrated that proinflammatory cytokines such as IL-4, IL-21, and IFN-g produced by effector/ memory T cells strongly suppress Foxp3 induction, and the effect is reversed by retinoic acid (RA) signaling (32, 33). Interestingly, according to our transcriptome analysis, the expression of RA receptor a is much decreased in Bach2 KO Treg cells in addition to increased levels of IL-4, IL-21, and IFN-g. Therefore, the augmented Th1/Th2 immune environment in the Bach2 KO mice and the reduced expression of RA receptor might also dampen Foxp3 levels and impede the development and/or maintenance of Foxp3+ Treg cells in the periphery. In our transcriptome analysis, it should be noted that WT and Bach2 KO Treg cells are likely exposed to different inflammatory milieu before isolation. Hence, it is possible that some of the alterations in gene expression in Bach2 KO Treg cells are induced by the inflammatory environment, independent of Bach2 deficiency. This caveat can be addressed by isolating WT and Bach2 KO Tregs from BM chimeric mice, but a small number of Bach2 KO Treg cells in these mice precludes such analysis. Alternatively, we are developing an inducible system of Bach2 ablation in Foxp3+ cells that would largely mitigate the confounding effects of environment on gene expression in Treg cells. Third, it is possible that the in vivo development of Foxp3+ iTregs might occur less effectively without Bach2. Indeed, we find that Helios2Foxp3+ Treg cells are reduced in Bach2 KO mice, and in vitro induction of Foxp3 and Tregs from Bach2 KO CD4 T cells is severely impaired. Activation of Bach2 KO CD4 T cells with anti-CD3 and TGF-b results in aberrant induction of cells that express IL-4, IL-13, and Gata3, instead of Foxp3. These data again suggest that Bach2 promotes the dominance of the Treg transcriptional program over the competing Th1/Th2 effector transcriptional program during differentiation of iTreg cells by cell-intrinsic mechanisms. Additionally, the cell-extrinsic dysregulated immune environment in Bach2 KO mice might oppose the differentiation of Foxp3+ iTreg cells. Based on the data presented in this manuscript,


lymphoplasmacytic, eosinophilic, and histiocytic infiltrates (Fig. 6C). In the most severe cases, some airways were also filled with dense aggregates of eosinophils (Fig. 6F). Pulmonary veins in affected lung sections were often encircled by dense cuffs of plasma cells admixed with fewer eosinophils and lymphocytes Additionally, atypical plasma cells exhibiting marked cytomegaly and karyomegaly were a repeated finding in the perivascular cuffs of the most severely affected mice (Fig. 6E). Although ECP was the most common histologic lesion in the Bach2 KO mice, the most severely affected mice exhibited concurrent multisystemic plasmacytic and eosinophilic inflammation. Consistent with the pattern of lung lesions, the expressions of IL-4 and IL-13 in particular and IL-17A levels were markedly elevated in the lungs of Bach2 KO mice (Fig. 6G). Taken together, the nature of the lung pathology along with elevated levels of IL-4 and IL-13 in the affected lungs are highly reminiscent of Th2-type immunitydriven chronic pulmonary inflammation. It should be noted that identical Th2-type chronic lung inflammation and ECP occur in mice that are selectively deficient for iTreg cells (41). Therefore, the fatal lung inflammation in Bach2 KO mice is likely a sequel to perturbations in the development of iTreg cells that are tasked to suppress chronic Th2-type lung inflammation.

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Disclosures The authors have no financial conflicts of interest.

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we propose that Bach2 promotes the differentiation and/or survival of both thymus- and pTreg cells. Further, in Bach2 KO mice and BM chimeric mice, the residual Foxp3+ Treg cells uniformly displayed an activated phenotype that is reminiscent of eTreg cells (25). Thus, similar to its role in conventional T cells, Bach2 might promote quiescence of Foxp3+ Treg cells by opposing their activation and proliferation in the thymus and periphery. Blimp1 and IRF4 facilitate the maturation of eTreg cells (25), and hyperinduction of Blimp1 in Bach2 KO Treg cells might enhance the induction of eTreg cells. Importantly, altered immune homeostasis in Bach2 KO mice invariably resulted in fatal lung disease, characterized by ECP and eosinophilic inflammation. ECP is widely recognized as a progressive and fatal idiopathic disease syndrome in mice that occurs with increased frequency in the C57BL/6 from which the Bach2 KO mice are derived and frequencies up to 80–100% in certain strains such as moth-eaten and 129S4/SvJae (43, 44). The pulmonary crystals of ECP are predominantly composed of YM1 protein, a secretory product of pulmonary macrophages, which have been alternatively activated under the influence of Th2-associated cytokines IL-4 and IL-13 (40, 41). The pathologic role of these characteristic YM1 crystals in ECP remains unknown; however, it is likely that their production by alveolar macrophages, particularly to the degree observed in ECP, is a sentinel of pathologic Th2 immune dysregulation. The occurrence of systemic eosinophilic inflammation also is strongly indicative of aberrant Th2 immunity and chronic allergic inflammation in Bach2 KO mice. Indeed, the levels of IL-4 and IL13 are strikingly elevated in the lungs of Bach2 KO mice. What is the immunologic mechanism underlying the pathogenesis of lung disease in Bach2 KO mice? Intriguingly, identical chronic inflammatory lung disease characterized by ECP and obstructive airways also develops spontaneously in mice that are deficient for iTreg cells, but not for nTreg cells (41). As in the Bach2 KO mice, the primary immunologic lesion in the lungs of iTreg cell–deficient mice is the hyperinduction of Th2 cytokines. Therefore, it is highly likely that defective development of iTreg cells that mitigate the development of Th2-type inflammatory lung pathology underlies the fatal pulmonary disease in Bach2 KO mice. Thus, Bach2 plays a nonredundant role in the development of iTregs that protect against chronic inflammatory pulmonary disease, which has significant implications in the pathogenesis of diseases such as asthma in humans (40, 42). Although Bach2 KO mice die of pulmonary pathology, inflammatory disease is evident in other tissues including the uterus and the intestines. The lesions in the intestines of Bach2 KO mice are consistent with a diagnosis of multifocal lymphoplasmacytic and eosinophilic enteritis (not shown). It is worth noting that intestinal inflammation in Bach2 KO mice is associated with diminished numbers of Treg cells in the LP and mLNs. Studies by Roychoudhuri et al. (39) have shown that the development of intestinal inflammatory disease in Bach2 KO mice can be mitigated by providing WT Treg cells, which suggests that Bach2 protects against immune pathology by promoting dominant immune regulatory mechanisms, including the development and survival of Treg cells. In conclusion, our studies have identified a novel role for Bach2 in the transcriptional regulation of T cell quiescence, Treg cell differentiation, and homeostasis in vivo. These findings have provided crucial insights into the molecular regulation of immune homeostasis and suggest a molecular and cellular basis for the pathogenesis for Th2-type immune-mediated lung disease. Additionally, the findings in this manuscript have implications in understanding the pathogenesis of immune-mediated diseases of humans such as celiac disease, vitiligo, and type 1 diabetes, which have been linked to Bach2 gene variants.



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IL-21 contributes to fatal inflammatory disease in the absence of Foxp3+ T regulatory cells.

Unstable FoxP3+ T regulatory cells in NZW mice.

Foxp3+ regulatory T cells promote lung epithelial proliferation.

The Regulatory T-cell Transcription Factor Foxp3 Protects against Crescentic Glomerulonephritis.

Osteoprotegerin-Mediated Homeostasis of Rank+ Thymic Epithelial Cells Does Not Limit Foxp3+ Regulatory T Cell Development.

Advantages of Foxp3(+) regulatory T cell depletion using DEREG mice.

Functional dynamics of Foxp3⁺ regulatory T cells in mice and humans.

Donor CD4+ Foxp3+ regulatory T cells are necessary for posttransplantation cyclophosphamide-mediated protection against GVHD in mice.

Epigenetic and transcriptional control of Foxp3+ regulatory T cells.

Age-related changes in CD4+CD25+FOXP3+ regulatory T cells and their relationship with lung cancer.

HIV-1-mediated insertional activation of STAT5B and BACH2 trigger viral reservoir in T regulatory cells.

Lineage stability and phenotypic plasticity of Foxp3⁺ regulatory T cells.

Vitamin A supplementation leads to increases in regulatory CD4+Foxp3+LAP+ T cells in mice.

FOXP3+ regulatory T cells and their functional regulation.

Indirubin Increases CD4+CD25+Foxp3+ Regulatory T Cells to Prevent Immune Thrombocytopenia in Mice.

Differential control of immune cell homeostasis by Foxp3(+) regulatory T cells in murine peripheral lymph nodes and spleen.

Enhanced suppressor function of TIM-3+ FoxP3+ regulatory T cells.

Transcriptome profiling of human FoxP3+ regulatory T cells.

Foxp3+ regulatory T-cell homeostasis quantitatively differs in murine peripheral lymph nodes and spleen.

Development of thymic Foxp3(+) regulatory T cells: TGF-β matters.

IL-17+Foxp3+ T cells: an intermediate differentiation stage between Th17 cells and regulatory T cells.

Protection of Mice from Acute Graft-versus-Host Disease Requires CD28 Co-stimulation on Donor CD4+ Foxp3+ Regulatory T Cells.

Adipose tissue macrophages induce PPARγ-high FOXP3(+) regulatory T cells.

Homeostasis of thymus-derived Foxp3+ regulatory T cells is controlled by ultraviolet B exposure in the skin.