SATB1 Plays a Critical Role in Establishment of Immune Tolerance Motonari Kondo, Yuriko Tanaka, Taku Kuwabara, Taku Naito, Terumi Kohwi-Shigematsu and Akiko Watanabe This information is current as of September 24, 2021. J Immunol published online 14 December 2015 http://www.jimmunol.org/content/early/2015/12/11/jimmun ol.1501429 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published December 14, 2015, doi:10.4049/jimmunol.1501429 The Journal of Immunology

SATB1 Plays a Critical Role in Establishment of Immune Tolerance

Motonari Kondo,*,† Yuriko Tanaka,*,1 Taku Kuwabara,* Taku Naito,* Terumi Kohwi-Shigematsu,‡ and Akiko Watanabe†,1

Special AT-rich sequence binding protein 1 (SATB1) is a genome organizer that is expressed by T cells. T cell development is severely impaired in SATB1 null mice; however, because SATB1 null mice die by 3 wk of age, the roles of SATB1 in T cell development have not been well clarified. In this study, we generated and analyzed SATB1 conditional knockout (cKO) mice, in which the SATB1 gene was deleted from all hematopoietic cells. T cell numbers were reduced in these mice, mainly because of a deficiency in positive selection at the CD4+CD8+ double-positive stage during T cell development in the thymus. We also found that SATB1 cKO mice developed autoimmune diseases within 16 wk after birth. In SATB1 cKO mice, the numbers of Foxp3+ regulatory T (Treg) cells

were significantly reduced at 2 wk of age compared with wild-type littermates. Although the numbers gradually increased upon Downloaded from aging, Treg cells in SATB1 cKO mice were still less than those in wild-type littermates at adulthood. Suppressive functions of Treg cells, which play a major role in establishment of peripheral tolerance, were also affected in the absence of SATB1. In addition, negative selection during T cell development in the thymus was severely impaired in SATB1 deficient mice. These results suggest that SATB1 plays an essential role in establishment of immune tolerance. The Journal of Immunology, 2016, 196: 000–000.

dentity of T cells is established by T cell–specific gene ETPs, which initiates T cell differentiation and shuts off devel- http://www.jimmunol.org/ regulatory networks, which are composed of multiple tran- opmental potential to other lineages (7–9). T lineage commitment I scription factors (1). T cell development starts in the thymus is established during the DN2 stage, more specifically at the when multipotent progenitors or common lymphoid progenitors in transition from the DN2a to the DN2b stage (10, 11). Recombi- the bone marrow (BM) transit to the thymus through blood flow nation of the TCRb genes is completed in the DN3 stage. Only and are recognized as CD42CD82 double-negative (DN) thy- DN3 cells with productive TCRb-chains can transit to the CD4+ mocytes (2–4). The most immature thymocytes, called early T cell CD8+ double-positive (DP) stage after b-selection (12). DP cells progenitors (ETPs), in the DN thymocyte fraction have not only rearrange the TCRa-chain gene, and their fate is determined by T cell potential but also B cell and myeloid potential (5, 6). In the two subsequent selection events, positive and negative selection thymic microenvironment, Notch signaling is activated in these (2, 13). DP cells that successfully express TCRab-chains and that by guest on September 24, 2021 recognize MHC–peptide complexes with moderate affinity are positively selected and can advance to the next stage, either the *Department of Molecular Immunology, Toho University School of Medicine, Tokyo CD4+ or CD8+ single-positive (SP) stage (14–16). If the TCR on 143-8540, Japan; †Department of Immunology, Duke University Medical Center, Durham, NC 27710; and ‡Life Sciences Division, Lawrence Berkeley National Lab- the DP cells binds to MHC–peptide complexes with high affinity, oratory, University of California, Berkeley, CA 94720 these cells are autoreactive T cells and die by apoptosis (17–19). 1Y.T. and A.W. contributed equally to this work. This elimination process is termed negative selection and is in- ORCIDs: 0000-0002-1565-8804 (M.K.); 0000-0002-9278-7677 (Y.T.); 0000-0001- dispensable for establishment of central tolerance (13, 20, 21). 6440-5566 (T.N.); 0000-0002-5988-0669 (T.K.-S.); 0000-0003-4741-4705 (A.W.). Protection of DP cells from apoptosis after positive selection, Received for publication June 23, 2015. Accepted for publication November 13, which is supported at least in part by the IL-7/IL-7R system, is 2015. important in the transition from DP to SP cells (22). Because This work was supported in part by Japan Society for the Promotion of Science intrathymic T cell development is a multiple-step and complicated Grants-in-Aid for Scientific Research (24390121 and 26670240), the Uehara Memo- rial Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical process, elucidation of gene expression patterns and their regu- Research, a Strategic Research Foundation Grant-aided Project for Private Schools at lation by a network of multiple transcription factors is necessary Heisei 23rd (S1101016) and Heisei 26th (S1411015) from the Ministry of Education, to fully understand how T cell development and immune tolerance Culture, Sports, Science and Technology, a Research Promotion Grant from Toho University Graduate School of Medicine (11-01 and 14-02), a Bridging Grant from is regulated at the molecular level. Duke University Medical Center (to M.K.), Project Research Grants from Toho Transcription factors bind to cis-elements in the promoters of University School of Medicine (to Y.T.), and by National Institutes of Health Grant target genes and activate transcription of these genes (1). Because (R37CA39681 to T.K.-S.). M.K. was a scholar of the Leukemia & Lymphoma Society. not only individual transcription factors but also a set of tran- Address correspondence and reprint requests to Dr. Motonari Kondo and Dr. Yuriko scription factors that form transcriptional networks govern the Tanaka, Toho University School of Medicine, 5-21-16 Omori-Nishi, Ota-ku, Tokyo identity of developing T cells at the various maturational stages in 143-8540, Japan. E-mail addresses: [email protected] (M.K.) and the thymus, it is important to understand how global epigenetic [email protected] (Y.T.) regulation of genes is ensured (1, 23). Several nuclear proteins The online version of this article contains supplemental material. are known to possess functions that regulate chromatin structure. Abbreviations used in this article: BM, bone marrow; cKO, conditional knockout; DN, double-negative; DP, double-positive; ETP, early T cell progenitor; HPC, hema- One such protein family is the SWI/SNF–chromatin remodeling topoietic progenitor cell; HSC, hematopoietic stem cell; SATB1, special AT-rich complex, which moves or removes nucleosomes and regulates sequence binding protein-1; SP, single-positive; Tconv, conventional T; TG, trans- nucleosome positioning and density at the proper genomic location genic; Treg, regulatory T; WT, wild-type. (24). When the functions of SWI/SNF complexes are inhibited, the Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$30.00 expression of CD4 and CD8 molecules in developing T cells in the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501429 2 LOSS OF SATB1 LEADS TO AUTOIMMUNE DISORDERS thymus are misregulated (25), which demonstrates that the regu- Real-time quantitative RT-PCR lation of chromatin structures by SWI/SNF complexes plays a role Total RNA from various cell populations was extracted using the TRIzol in proper gene expression in T cells. However, the action of SWI/ reagent (Invitrogen). The High-Capacity cDNA Reverse Transcription Kit SNF complexes is necessary for a broad range of cell types (Applied Biosystems) was used for first-strand synthesis. Quantitative RT- including pluripotent stem cells (26). Therefore, the existence PCR was performed according to the protocol of TaqMan gene expression of another factor that regulates epigenetic gene expression in a assay kits (Applied Biosystems) with an ABI 7500 Fast system (Applied Biosystems) using the following primers: Hprt, Mm00446968_m1, and T-lineage–specific manner has been postulated. SATB1, Mmoo485920_m1. Results were normalized to the expression of Special AT-rich binding protein 1 (SATB1) is an NF that binds Hprt mRNA. AT-rich sequences (also known as base unpairing regions) and can Flow cytometry function as a genome organizer (27, 28). SATB1 forms complexes with SWI/SNF factors and positively and negatively regulates Abs used for cell-surface and intracellular staining were as follows: anti– the expression of a vast number of genes (28–30). SATB1 ex- CD4-PE–Cy7 (GK1.5), anti-CD5–FITC (53-7.3), anti-CD8–Pacific Blue pression is observed in hematopoietic stem cells (HSCs), and its (53-6.7), anti-CD24–PE (M1/69), anti-CD25–PE (PC61), anti-CD44–PE (IM7), anti-CD45.2–allophycocyanin (104), anti-CD62L–FITC (MEL-14), anti- expression is abundant in thymocytes (31, 32). Accordingly, TCRb–APC (H57-597), anti-Va2–PE (B20.1), anti-Vb5.1 5.2–FITC (PE- SATB1 and its target genes are involved in various cellular MR9-4), and anti-B220–FITC (RA3-6B2) were obtained from BioLegend. functions in different types of blood cells. For example, it has Anti-CD45.1–FITC (A20), anti-CD69–FITC (H1.2F3), anti-HY–APC been demonstrated that SATB1 is necessary for the self-renewal (T3.70), anti–c-Kit–APC (2B8), anti-Thy1.2–PE (53-2.1), and anti-Foxp3– FITC (FJK-16a) were purchased from eBioscience. Anti-TCRb–APC Abs of HSC (32). SATB1 also plays a role in lymphoid lineage (H57-597) were obtained from Tonbo Biosciences. specification and/or commitment (31). In SATB1 null mice, For staining, erythrocytes were depleted from a single-cell suspension Downloaded from however, a dramatic change in cell phenotype is observed in the derived from the various organs specified in the figure legends. After thymus, where T cell development is mostly stagnated at the DP the cells were washed with FACS buffer (2% FBS in 13 PBS with stage (30). In addition, it has been reported that downregulation 0.05% NaN3), they were incubated with a mixture of fluorescence- conjugated Abs on ice for 20 min and then washed twice with FACS of SATB1 is necessary for exhibition of proper inhibitory ac- buffer. A Cytofix/Cytoperm kit (BD Biosciences) was used for intracellular tions by regulatory T (Treg) cells, which negatively regulate staining by following the instructions of the kit. Flow cytometric analysis

immune responses and are major players in establishment of pe- was performed using FACSCanto II (BD Biosciences) and FACSAria III http://www.jimmunol.org/ ripheral tolerance (33). However, because SATB1 is expressed not (BD Biosciences). Cell sorting was done using FACSAria III (BD Bio- sciences). Dead cells were excluded as 7-aminoactinomycin D–positive only by hematopoietic cells but also by other types of cells such as (BD Biosciences) cells. Data were analyzed using FlowJo software neuronal cells, and because SATB1 null mice die by 3 wk after (Tree Star). birth, it is difficult to investigate the precise role of SATB1 in T cell development and function (30). BM chimeras In this study, we demonstrated that T cell development is BM cells isolated from SATB1cKO (CD45.2) mice, their WT littermates severely impaired in the absence of SATB1 by using a mouse (CD45.2), and congenic C57BL/6 (CD45.1) mice were stained with anti- B220–FITC, anti-Thy1.2–PE, and anti–c-Kit–APC Abs. Subsequently, model in which the SATB1 gene was conditionally deleted in 2 2 c-Kit+Thy1.2 /loB220 hematopoietic progenitor cells (HPCs) with HSCs by guest on September 24, 2021 hematopoietic cells. These SATB1 conditional knockout (cKO) were purified using cell sorting on an FACSAria III (BD Biosciences). mice were created by crossing SATB1 floxed mice with Vav-Cre HPCs (1 3 106 cells) from SATB1cKO mice or a mixture of HPCs from transgenic (TG) mice (34, 35). We showed that the T cell de- SATB1cKO mice and congenic C57BL/6 (CD45.1) mice at a ratio of 1:1 velopmental defect in SATB1cKO mice is mainly due to dys- were i.v. injected into lethal dose–irradiated (900 rad) C57BL/6 (CD45.1) mice. Splenocytes from these mice were analyzed on FACS machines at functional positive selection, resulting in stagnation at the 8 wk after injection of the HPCs. transition stage from the DP to the CD4+ or CD8+ SP stage. Notably, the mortality rate of SATB1cKO mice was higher than Histopathology and immunohistochemistry that of wild-type (WT) littermates. One cause of death of Mouse tissues were fixed in 10% formalin solution (Wako) and embedded SATB1cKO mice may be autoimmune manifestation because the in paraffin. Sections (6 mm) were stained with H&E and observed using concentration of autoantibodies in the serum was increased in a BX61 microscope (Olympus). SATB1cKO mice after 16 wk of age. In addition, inflammatory Autoantibody detection lesions or lymphocyte infiltrates were observed in multiple or- gans in SATB1cKO mice. We also found that negative selection Anti-dsDNA Abs in the serum were measured using a Mouse Anti-dsDNA and a regulatory function of Treg cells were impaired in ELISA kit (Shibayagi). SATB1cKO mice, suggesting that SATB1 plays an important In vitro Treg cell suppression assay role in regulation of the gene expression that is critical for es- 2 CD25 CD4+ conventional T (Tconv) cells and CD25+CD4+ Treg cells tablishment of immune tolerance. from spleen and peripheral lymph nodes were purified by cell sorting. Responder Tconv cells (CD45.1, 2 3 105 cells) were labeled with CFSE Materials and Methods according to the manufacturer’s instructions (Invitrogen) and cultured for Mice 4 d with irradiated splenocytes (1 3 105 cells; CD45.2) and anti-CD3ε Ab m + fl/fl (5 g/ml; 145-2C11; eBioscience) in the presence or absence of CD25 SATB1 mice were generated as described (34). Vav-Cre mice (35), OT-I CD4+ Treg cells (CD45.2) either from SATB1cKO mice or their WT lit- and OT-II TCR TG mice (36, 37), and RIPmOVA mice (38) were pur- termates. CFSE levels on Tconv cells were then measured by gating on chased from The Jackson Laboratory. Lck-Cre mice (39) were obtained CD45.1+ cells in FACS analysis. from the Laboratory Animal Resource Bank at Nibiohn. HY-TCR TG mice were provided by W. Zhang at Duke University. C57BL/6 mice were In vivo Treg cell suppression assay (T cell–mediated colitis 2/2 purchased from CLEA Japan. RAG2 and C57BL/6 (CD45.1) mice model) were maintained at the animal facility in Toho University. All mice were maintained on a C57BL/6 background and under specific pathogen-free Rag22/2 mice were injected i.v. with CD252CD4+CD45RBhi T cells (2 3 conditions at the Toho University School of Medicine animal facility. All 105) in the presence or absence of CD25+CD4+ Treg cells (2 3 105). The experiments using mice received approval from the Toho University Ad- mice were weighed weekly, assessed for clinical signs of colitis for 5 wk, ministrative Panel for Animal Care (15-55-262) and Recombinant DNA and then killed to determine the presence of colitis in colon sections. (15-52-260). Mice were used at 5–10 wk of age unless otherwise specified Colons were fixed in 10% formalin solution and embedded in paraffin. in the text. Sections (6-mm thick) were stained with H&E. The Journal of Immunology 3

In vivo injection of anti-CD3 Abs under the control of the Lck proximal promoter (Lck-Cre), which m ε are active in thymocytes after the DN2 stage (41), although the WT and SATB1 cKO mice were i.p. injected with 25 g anti-CD3 Abs in fl/fl 100 ml PBS. After 48 h, thymocytes were isolated, and the expression of number of ETPs in Lck-Cre-SATB1 mice was lower than that in CD4 and CD8 was analyzed using FACS. WT mice (Supplemental Fig. 2A). Statistical testing Peripheral T cells are activated in the absence of SATB1 Statistical analysis was performed with the mean difference hypothesis of We next analyzed peripheral T cells in SATB1cKO mice and their Student t test or the Mann–Whitney U test with assumption of different WT littermates. Reflecting the observed reduction in the number variances and a confidence level of 95%. of SP thymocytes, both CD4+ and CD8+ T cells were significantly decreased in the spleen of SATB1cKO mice (Fig. 2A). As in the Results case of SATB1 null mice (30), CD4+CD8+ DP cells were observed The effects of SATB1 deficiency on thymocyte development in the spleen of SATB1cKO mice (Fig. 2A), suggesting that SATB1 expression is observed in hematopoietic cells as early as SATB1 is involved in the downregulation of CD4 and/or CD8 the HSC stage (32). In this study, real-time quantitative RT-PCR molecules in peripheral T cells. In contrast to the T cell defect in analysis of thymus tissue indicated that, although all thymocytes SATB1cKO mice, the B cell number was not significantly dif- of WT mice expressed SATB1 mRNA, the expression level was ferent between SATB1cKO and WT littermates (Fig. 2B). the highest at the DP stage and decreased thereafter along with T lymphopenia may lead to activation of T cells as the result of maturation (Fig. 1A). This result implies that SATB1 expression is cell expansion by homeostatic proliferation (42). This also appears important at the DP stage during intrathymic T cell development. to be the case for SATB1cKO mice because the number of Downloaded from To investigate the effect of SATB1 deficiency on T cell devel- CD62LhighCD442/lo naive T cells was drastically reduced and that opment, we generated SATB1cKO mice by crossing mice with an of CD62L2CD44high effector/memory T cells was significantly SATB1 floxed allele (SATB1fl/fl mice) with Vav-Cre TG mice (34, increased in both CD4+ and CD8+ populations in the spleen of 35, 40). As in the case of SATB1 null mice (30), T cell devel- SATB1cKO mice (Fig. 2C, 2D). The mechanism that underlies opment was severely impaired in the SATB1cKO mice (Fig. 1B, this phenomenon is unclear; however, whereas naive T cells in 6 3 8

1C). Thymocyte numbers in SATB1cKO mice (0.55 0.18 10 ; WT mice showed from low to negative expression of CD44, naive http://www.jimmunol.org/ p , 0.05) were ∼40% of those in WT littermates (1.3 6 0.14 3 108). T cells in the SATB1cKO mice were mostly negative for CD44 Reduction in the number of thymocytes in SATB1cKO mice was (Fig. 2C). Activated T cells in SATB1cKO mice seem to enhance significant in the SP population (Fig. 1B, 1C), suggesting that humoral immunity because germinal center B cells, which are transition from the DP to the SP stage, in which positive selec- positive for GL7, were detected in the spleen and lymph nodes of tion is required, is impaired (13). SATB1cKO mice (data not shown). The decrease in the number of thymocytes in SATB1cKO mice To address whether T cell activation in SATB1cKO mice was observed even for the most immature population of thymo- was truly due to homeostatic proliferation under a lymphopenic cytes, the ETPs (Fig. 1D). A significant reduction was observed condition, we generated chimeric mice by injecting BM cells from in the number of common lymphoid progenitor cells but not in SATB1cKO (CD45.2) and WT C57BL/6 (CD45.1) mice into lethally by guest on September 24, 2021 HSCs in BM of SATB1cKO mice compared with WT mice irradiated WT mice (CD45.1). At 6 wk after BM reconstitution in the (Supplemental Fig. 1A). These findings are consistent with our mice injected with SATB1cKO BM alone, ∼10% of the CD4+ cells previous report that SATB1 deficiency might affect specification derived from SATB1cKO BM were naive cells (Fig. 3A, left panel, and/or commitment of the lymphoid lineage (32). Indeed, no Fig. 3B). When SATB1cKO BM cells were injected with WT BM significant difference was observed in the number of ETPs be- cells, the percentage of naive CD4+ T cells was increased to ∼20% tween WT mice and SATB1 floxed mice with Cre transgenes (Fig. 3A, right panel), which is comparable to the number of CD4+

FIGURE 1. Defective thymocyte development in SATB1 cKO mice. (A) Expression of SATB1 mRNA in thymocytes and splenic T cells derived from WT mice as analyzed using quantitative RT-PCR. The level of SATB1 mRNA was normalized relative to that of hprt expression (n = 3). (B) CD4 and CD8 expression on thymocytes in WT and SATB1cKO mice. Representa- tive FACS plots from more than five independent ex- periments are shown. Numbers of cells in various thymocyte populations (C) and subfractions of the DN population (D) in WT (open bars, n = 5) and SATB1 cKO mice (closed bars, n = 5). Student t tests were performed for statistical analysis. *p , 0.05. 4 LOSS OF SATB1 LEADS TO AUTOIMMUNE DISORDERS

FIGURE 2. T cells in the periphery are activated in SATB1 cKO mice. (A) CD4 and CD8 expression on splenocytes from WT and SATB1cKO mice. Repre- sentative FACS plots from more than five independent analyses are shown. (B) Cell numbers of the indicated populations in the spleen derived from WT (open bars, n = 5) and SATB1 cKO mice (closed bars, n = 5). (C) Representative FACS analysis of the expression of CD62L and CD44 on CD4+ (top panel) or CD8+ (bottom panel) splenic T cells from WT (left panel)or SATB1 cKO mice (right panel). (D) The percentage of CD62LhiCD44lo (naive) and CD62LloCD44hi (effector/ memory) subfractions of CD4+ (top panel) or CD8+ Downloaded from (bottom panel) T cells from WT (open circles) and SATB1 cKO (closed circles) mice. Each symbol rep- resents an individual mouse, and horizontal lines indi- cate the mean value. Student t tests were used for statistical analyses. *p , 0.05. http://www.jimmunol.org/

naive T cells from WT BM (Fig. 3B). This result demonstrates selection in the TCR TG mice (Fig. 4A, 4B, left panels). However, by guest on September 24, 2021 that the activated phenotype of T cells in SATB1cKO mice is a OT-I+ DP thymocytes in a SATB1cKO background did not dif- result of homeostatic proliferation in a T lymphopenic condition. ferentiate into CD8+ SP cells (Fig. 4A, middle and right panels). Similarly, fewer OT-II+CD4+ SP cells were observed in the SATB1 is required for positive selection SATB1cKO background than in the SATB1 sufficient OT-II+ mice We observed a marked decrease in the number of CD4+ and (Fig. 4B), although the developmental block from the DP to the SP CD8+ SP cells in the thymus of SATB1cKO mice (Fig. 1B). To stage was less severe in OT-II+ SATB1cKO mice than in OT-I+ determine if this decrease in the number of SP thymocytes was a SATB1cKO mice. These results clearly demonstrate that positive result of a defect in positive selection, we examined the devel- selection is severely impaired in the absence of SATB1. opmental fate of thymocytes expressing fixed TCRs. We crossed In accordance with this defect in positive selection, upregula- SATB1cKO mice with OT-I (36) and OT-II (37) TCR TG mice. tion of CD69 and TCRb, which normally occurs after successful These TCRs recognize OVA peptides presented by MHC class I positive selection at the DP stage, was impaired in SATB1cKO and II, respectively, and therefore, OT-I+ and OT-II+ DP cells give mice (Fig. 4C). Finally, we analyzed the CD5 expression level on rise to CD8+ and CD4+ T cells, respectively, upon positive thymocytes from SATB1cKO mice and their WT littermates. The

FIGURE 3. T cells in SATB1 cKO mice were activated due to homeostatic proliferation under the T lymphopenic condition. (A) c-Kit+ BM cells from SATB1 cKO mice (CD45.2) with (right panel) or without (left panel) c-Kit+ BM cells from WT mice (CD45.1) were i.v. injected into lethally irradiated congenic mice (CD45.1). At 8 wk after BM reconstitution, CD4+ T cells from SATB1 cKO mice were analyzed by FACS for expression of CD62L and CD44. (B) The frequency of CD62LhiCD44lo naive CD4+ T cells from the indicated donors in mice injected with SATB1cKO c-Kit+ BM cells alone or with both SATB1cKO and WT c-Kit+ BM cells (n = 3). Data are representative of two independent experiments. *p , 0.05. The Journal of Immunology 5

FIGURE 4. Defect of positive se- lection in SATB1 cKO mice. (A) FACS analysis of thymocytes from WT (left panel) or SATB1 cKO mice (middle panel) expressing OT-I TCR transgenes (Va2+). The results shown are pregated on a Va2+ population. The ratio of CD8+ SP cells to DP cells is shown in the right panel.(B) FACS analysis of thymocytes from WT (left panel) or SATB1 cKO mice (middle panel) expressing OT-II TCR transgenes (Va2+). The ratio of CD4+ SP cells to DP cells is shown (right panel). (C) FACS analysis of expres- sion of the TCRb-chain and CD69 on thymocytes in WT (left panel)and Downloaded from SATB1 cKO mice (right panel). (D) FACS analysis of CD5 expression on DP and CD4+CD8int thymocytes from WT (solid line) or SATB1 cKO mice (dotted line). A representative result of more than three independent experiments is shown. *p , 0.05. http://www.jimmunol.org/

expression of CD5 was obviously impaired in SATB1cKO mice, of age, the frequency of Treg cells was higher than that in WT mice especially in DP thymocytes (Fig. 4D). This decrease in CD5 (Fig. 6A); however, the absolute number of splenic Treg cells in expression persisted at the CD4+CD8int stage in SATB1cKO mice SATB1cKO mice was less than that of WT mice (Fig. 6B). A re- (Fig. 4D). The CD5 expression level on thymocytes, especially on duction in the number of Treg cells was also observed in inguinal by guest on September 24, 2021 CD4+CD8int cells, is known to reflect the avidity of the interaction and mesenteric lymph nodes of SATB1cKO mice compared with of the TCR with self-peptide/MHC complexes (43). Therefore, WT mice (Fig. 6B). Similarly, slightly fewer Treg cells were pre- impairment of positive selection might be due, at least in part, to sent in older (24-wk-old) SATB1cKO mice than in WT mice insufficient TCR engagement of DP thymocytes in SATB1cKO mice. (Supplemental Fig. 1B). Also, no significant difference in the number of Treg cells was observed between healthy and sick SATB1cKO mice develop autoimmune disease SATB1cKO mice (data not shown). However, Treg cell numbers During the breeding and maintenance of SATB1cKO mice and their were significantly different between SATB1cKO and WT mice at littermates, we noticed that the SATB1cKO mice died earlier than 2 wk after birth (Supplemental Fig. 1B), suggesting that the loss of their WT littermates (Fig. 5A). We found that levels of autoanti- SATB1 may affect Treg cell development. bodies, such as anti-dsDNA Abs, in the serum of SATB1cKO mice We next analyzed the suppressive ability of SATB1-deficient were higher than those of WT mice (Fig. 5B), suggesting that Treg cells in vitro. To determine whether SATB1-deficient Treg SATB1cKO mice are prone to autoimmunity. In addition to the cells can suppress the proliferation of CD4+CD252 Tconv cells, presence of autoantibodies, apparent inflammatory cell infiltration we cultured activated Tconv cells from WT congenic mice was observed in various organs of the SATB1cKO mice (Fig. 5C). (CD45.1) with or without CD4+CD25+ Treg cells, all of which It seems that this autoimmune disorder is caused by T cells in the expressed Foxp3, from either WT or SATB1cKO mice (CD45.2) absence of SATB1 because autoimmune phenotypes were ob- for 4 d. After culture, we analyzed the proliferation of the Tconv served in Lck-Cre-SATB1fl/fl mice, in which SATB1 is deleted cells by CFSE dilution on a FACS machine and found that the only in T cells (Supplemental Fig. 2B, 2C). suppressive ability of SATB1-deficient Treg cells was slightly Immune tolerance is the key for blocking autoimmune mani- reduced compared with that of WT Treg cells (Fig. 6C, 6D). We festation in healthy conditions. Once immune tolerance is dis- also examined suppressive abilities of Treg cells derived from turbed, the bodies’ immune system may attack itself and various SATB1cKO mice in in vivo settings by using a T cell–induced organs may get damaged, resulting in autoimmune diseases (44). colitis model. Upon transfer of naive CD4+CD45RBhigh cells into Treg cells, which are CD4+ T cells that express the transcription RAG22/2 mice, the mice became wasted and developed colitis, factor Foxp3, play a central role in establishment of peripheral whereas cotransfer of either WT or SATB1-deficient CD4+CD25+ tolerance and immune homeostasis (45). Treg cell deficiency and Treg cells prevented development of T cell–induced colitis dysfunction may cause a T cell–mediated autoimmune disorder (Fig. 6E, 6F). These results suggest that although the suppressive (45). Therefore, to determine the involvement of Tregs in the au- ability of SATB1-deficient Treg cells was not clearly observed in toimmunity of the SATB1cKO mice, we first compared the fre- in vivo assays, Treg function is impaired in SATB1cKO mice in quency and the cell number of splenic Treg cells in SATB1cKO terms of reduction in the number and impairment of in vitro mice with those of WT littermates. In SATB1cKO mice at 5–10 wk suppressive ability of Treg cells in SATB1cKO mice. 6 LOSS OF SATB1 LEADS TO AUTOIMMUNE DISORDERS

FIGURE 5. SATB1cKO mice develop autoimmune diseases. (A) Survival rates of SATB1 cKO mice (solid line, n = 17) and WT littermates (dotted line, n = 12) at the indicated periods. (B) Concentration of anti-dsDNA Abs in the serum of WT or SATB1 cKO mice at dif- ferent ages. Each symbol represents an individual mouse, and horizontal lines indicate the mean. (C) H&E staining of sections of various organs from WT and SATB1 cKO mice. Scale bars, 200 nm. Log-rank tests (A) and the Mann–Whitney U test (B) were used for statistical analyses. *p , 0.05. Downloaded from http://www.jimmunol.org/

Negative selection is impaired in the absence of SATB1 protein, AIRE. Large-scale deletion of OT-II TCR+ thymocytes is Because SATB1cKO mice displayed a modest defect in a function seen when both the OT-II TCR transgene and RIP-mOVA trans- gene are present (46). Indeed, we observed significant but partial of Treg cells, we next examined whether negative selection, which + is required for establishment of central tolerance, is defective in deletion of CD4 SP cells in the OT-II TG mice in the presence of RIP-mOVA (from 87.6 to 54%; Fig. 7C). As shown above, fewer these mice. For this purpose, we crossed SATB1cKO mice with + HY-TCR TG mice (14). Because female mice do not have HYAgs, CD4 SP cells were observed in OT-II TG-SATB1cKO mice than HY-TCR+ DP cells on the WT background were positively se- in OT-II TG mice on the WT background due to a defect in

+ positive selection (Fig. 7C). In the presence of RIP-mOVA, by guest on September 24, 2021 lected and gave rise to CD8 T cells (Supplemental Fig. 3, left + panel). Because positive selection is perturbed in the absence of however, the number of CD4 SP cells was increased in the OT- SATB1, fewer CD8+ SP cells were observed in the HY-TCR+ II SATB1cKO mice (Fig. 7D). These results demonstrate that SATB1cKO female mice versus the HY-TCR+ WT mice SATB1 is necessary for establishment of central tolerance. Al- (Supplemental Fig. 3, right panel). In the male mice, most of though self-reactive T cells should be in the periphery of the HY-TCR+ thymocytes were deleted because of the presence of SATB1cKO mice, we could not reproduce autoimmune phenotype HY Ags, resulting in very few CD8+ SP cells in the thymus (Fig. in the naive mice by transferring SATB1-deficient T cells, maybe 7A, left panel). On a SATB1cKO background, however, more because of low activation potential of T cells in SATB1cKO mice HY-TCR+ CD8+ SP cells were observed in the thymus than on the due to weak TCR signal strength as shown in Supplemental Fig. 4. WT background (Fig. 7A, 7B). These results suggested that neg- Because T cells in patients with systemic lupus erythematosus ative selection was impaired in SATB1cKO mice. It should be have some abnormalities, such as T cells’ reduced ability to produce noted that there were two different CD4/CD8 staining patterns in cytokines (47), T cells in SATB1cKO mice might share some char- HY-TCR+ SATB1cKO mice; one pattern was similar to that of the acteristics with T cells in human autoimmune patients. HY-TCR+ WT mice (Fig. 7A, pattern 1), and in the other pattern, Finally, we examined the sensitivity of the DP thymocytes in the CD8+ SP population was more prominent (pattern 2). The SATB1cKO mice to clonal deletion induced by strong TCR signals. cause of these different thymocyte staining patterns in HY-TCR+ We i.p. injected anti-CD3 Abs into SATB1cKO and WT littermates SATB1cKO mice is unclear; we did not observe a significant and analyzed thymocytes from each mouse 4 d later. Although difference in the peripheral phenotype of these mice compared almost all DP thymocytes were depleted in WT mice by this with WT mice (data not shown). Whatever the cause of these treatment, DP cells in SATB1cKO mice were resistant to apoptosis different staining patterns, the results shown in Fig. 7A and 7B induced by anti-CD3 Ab treatment (Fig. 8). Therefore, dysregu- suggest that negative selection is impaired in the absence of lation of negative selection as well as reduction in the suppressive SATB1 and that more HY-TCR+ thymocytes have escaped from function of Treg cells in the absence of SATB1 might be causes of clonal deletion in male mice. autoimmune disorder in the SATB1cKO mice. Overall, the results We also examined whether negative selection of CD4+ T cells in this study demonstrate that SATB1 plays a role in the estab- was impaired in SATB1cKO mice. For this purpose, we crossed lishment of immune tolerance in T cells. SATB1cKO mice to OT-II 3 RIP-mOVA double TG mice ac- cording to the method used by Anderson et al. (46). RIP-mOVA Discussion TG mice express a membrane-bound form of OVA under the In this study, we demonstrated that SATB1 is required for both control of the insulin promoter (38). In addition to expression in positive and negative selection events during intrathymic T cell the pancreatic islet, this transgene is also expressed by medullary development. As previously reported by Alvarez et al. (30) for thymic epithelial cells in the thymus under the control of a nuclear SATB1 null mice, T cell development was severely impaired at The Journal of Immunology 7

FIGURE 6. Generation and function of Treg cells in SATB1cKO mice. (A) Foxp3 expression in the CD4+ spleen T cell population from WT (left panel) and SATB1 cKO mice (right panel). The FACS plots shown were pregated on the CD4+ fraction. (B) Absolute numbers of Treg (Foxp3+CD4+) cells in the spleen, inguinal lymph node, and mesenteric lymph node of WT (open bars) and SATB1 cKO (closed bars) mice. (C and D) In vitro suppression assay with CFSE-labeled Tconv cells (CD4+CD252) as responders and Treg cells de- rived from WT (open circles) or SATB1 cKO (closed circles) mice as suppressors (D). Representative results without Treg cells (left panel), or with WT Treg (middle panel) or SATB1 cKO Treg (right panel) cells at a 1:4 Tconv/Treg ratio are Downloaded from shown in (C). (E) CD4+CD45RBhi T cells were i.v. injected into Rag22/2 mice with or without WT or SATB1 cKO Treg cells. The body weight of each mouse was measured at the indicated period after cell injection. Three mice were used in each experimental group. The mean value + SD is indicated. (F) H&E staining of colon sections from the mice from each group in (E) at 5 wk after T cell transfer (original magnifi- http://www.jimmunol.org/ cation 3100). by guest on September 24, 2021 the DP stage in the absence of SATB1. Although no obvious T cell of Lck between MHC class I– and class II–restricted positive developmental defect at the DN stage is seen in SATB1 null mice, selection events might affect the cell fate of DP thymocytes in the we showed that, in SATB1cKO mice, T cell development was absence of SATB1. Expression patterns of various cytokine re- obstructed even at the stage of the earliest thymocyte population, ceptor subunits in thymocytes are changed in SATB1 null mice the ETP population (Fig. 1D). SATB1 plays a role in the main- compared with WT mice (30). Because intrathymic cytokines, tenance of self-renewal potential in HSC (32) and in specification/ such as IL-7, affect the specification of CD4 and CD8 lineage commitment of HSC/multipotent progenitors to the lymphoid choice at the DP stage (50), it is also possible that this dysregu- lineage as we previously reported (31), T cell development might lation of cytokine receptor expression might result in different be affected at a time point as early as the HSC stage in severity in the phenotype of CD4+ T and CD8+ T cells in SATB1cKO mice. However, we did not observe a significant SATB1cKO mice. Further investigation is necessary for identifi- difference in the number of HSCs in the BM between SATB1cKO cation of the reason why SATB1 is required for the process of mice and their WT littermates, which may not be consistent with a proper positive selection events. previous report by Will et al. (32). Because SATB1 null mice die A significant finding of this study was that SATB1 plays a role in by 3 wk after birth, HPCs from fetal liver or BM in infants were establishment of immune tolerance. SATB1cKO mice died earlier used in the experiments in the previous report, whereas we ana- than WT mice (Fig. 5A) and displayed multiple characteristics of lyzed adult mice in this study. Therefore, the difference between autoimmune prone mice, such as increased levels of autoanti- the previous studies and our present report might stem from the bodies and infiltration of immune cells into various organs (51, ages of the mice used in the investigations. More detailed analysis 52). Thus, impairment of negative selection might be one of the is necessary for clarification of the precise requirement of SATB1 causative reasons of development of autoimmune disorders in the at the various maturational stages of hematopoietic cells. absence of SATB1. Hwang et al. (53) have demonstrated that a Because T cell development of the SATB1 null mice is severely weak TCR signal in mice with a mutated ITAM in the TCRz blocked at the DP stage in the thymus, it was hypothesized that chains leads to impairment of both positive and negative selection. SATB1 deficiency might lead to impairment of positive selection Therefore, it is possible that dysregulation of both positive and (48). In this study, we demonstrate that positive selection is indeed negative selection in SATB1cKO mice is also caused by weak dysfunctional in the absence of SATB1 (Fig. 4). It should be noted TCR signal strength at the DP stage. However, it has been reported that this developmental arrest is more severe for CD8+ T cells than that symptoms of autoimmune diseases are not observed in TCRz for CD4+ T cells. Signal cascades via the TCR are triggered by a mutant mice because of increased numbers of Foxp3+ Treg cells cytoplasmic tyrosine kinase, Lck, which associates more effi- (53). SATB1cKO mice have impairment of Treg functions com- ciently with CD4 than with CD8 (49). Therefore, different usage pared with WT mice at adulthood (Fig. 6B). Therefore, Treg cell 8 LOSS OF SATB1 LEADS TO AUTOIMMUNE DISORDERS

FIGURE 7. Negative selection is impaired in SATB1 cKO mice. (A) FACS analysis of male HY-TCR+ thymocytes on a WT or SATB1 cKO background. The results shown were pregated on the T3.70+ fraction. (B) Numbers of HY-TCR+ CD8+ SP cells in WT (open circles) or SATB1 cKO mice (closed circles). (C) Ex- pression of CD4 and CD8 in thy- mocytes from OT-II TCR+ mice on a WT or SATB1 cKO background in the presence or absence of RIP- Downloaded from mOVA transgenes. (D) Numbers of OT-II+ (Va2+)CD4+ SP cells in each line of mice shown in (C). *p , 0.05. http://www.jimmunol.org/

numbers and functions might be critical for prevention of auto- resting Treg cells (55–57). It is therefore not clear why the sup- immune diseases, when autoreactive T cells are increased in the pressive function of SATB1-deficient Treg cells is impaired. periphery. Further investigation is necessary for clarification of the roles of by guest on September 24, 2021 Reduction in the number of Treg cells was significantly different SATB1 in development and in the functions of Treg cells. from WT in SATB1cKO mice at 2 wk after birth (Supplemental We demonstrated in this study that SATB1 deficiency in he- Fig. 1B). Because it has been recently demonstrated that there are matopoietic cells results not only in T cell deficiency but also in distinct Treg populations that are generated in an age-dependent autoimmune manifestations. It has been shown that homeostatic manner and have different TCR repertoires (54), it might be proliferation of T cells is an important event for development possible that SATB1-dependent and SATB1-independent Treg of autoimmune diseases due to an increase in the number of cells are present. In addition to the decreased number, the sup- autoreactive T cells and the activation of such T cells (58, 59). pressive functions of Treg cells were slightly impaired in the ab- Therefore, the T lymphopenia that was observed in the SATB1cKO sence of SATB1 (Fig. 6C, 6D). However, our preliminary results mice might accelerate the onset of autoimmune disorder even as suggest that Treg cells in SATB1cKO mice express higher levels early as 16 wk of age in these mice under the condition in which of GITR than the Treg cells of WT mice. GITR is observed in Treg cell development is also affected in the absence of SATB1. preactivated Treg cells that have higher suppressive functions than Because SATB1 globally regulates the expression of numerous

FIGURE 8. DP thymocytes in SATB1 cKO mice are resistant to TCR-mediated deletion. (A) WT and SATB1 cKO mice were i.p. injected with anti-CD3 Abs. At 48 h after injection, the thymocytes were har- vested, stained for CD4 and CD8, and analyzed using FACS. (B) The percentage of DP thymocytes in WT and SATB1 cKO mice before (open bars) or after (filled bars) injection of anti-CD3 Abs. *p , 0.05. The Journal of Immunology 9 genes by modification of chromosomal structure (28, 29), clarifi- 26. Savarese, F., A. Da´vila, R. Nechanitzky, I. De La Rosa-Velazquez, C. F. Pereira, R. Engelke, K. Takahashi, T. Jenuwein, T. Kohwi-Shigematsu, A. G. Fisher, and cation of the gene regulatory network modulated by SATB1 in R. Grosschedl. 2009. Satb1 and Satb2 regulate embryonic stem cell differenti- developing and mature T cells should provide new insights into ation and Nanog expression. Genes Dev. 23: 2625–2638. understanding the establishment of immune tolerance, which may 27. Dickinson, L. A., T. Joh, Y. Kohwi, and T. Kohwi-Shigematsu. 1992. A tissue- specific MAR/SAR DNA-binding protein with unusual binding site recognition. lead to the development of a new treatment of autoimmune diseases. Cell 70: 631–645. 28. Cai, S., C. C. Lee, and T. Kohwi-Shigematsu. 2006. SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine Acknowledgments genes. Nat. Genet. 38: 1278–1288. We thank Dr. Weiguo Zhang (Duke University Medical Center) for HY- 29. Yasui, D., M. Miyano, S. Cai, P. Varga-Weisz, and T. Kohwi-Shigematsu. 2002. TCR TG mice. SATB1 targets chromatin remodelling to regulate genes over long distances. Nature 419: 641–645. 30.Alvarez,J.D.,D.H.Yasui,H.Niida,T.Joh,D.Y.Loh,andT.Kohwi- Disclosures Shigematsu. 2000. The MAR-binding protein SATB1 orchestrates temporal Genes The authors have no financial conflicts of interest. and spatial expression of multiple genes during T-cell development. Dev. 14: 521–535. 31. Satoh, Y., T. Yokota, T. Sudo, M. Kondo, A. Lai, P. W. Kincade, T. Kouro, R. Iida, K. Kokame, T. Miyata, et al. 2013. The Satb1 protein directs hemato- References poietic stem cell differentiation toward lymphoid lineages. Immunity 38: 1105– 1. Rothenberg, E. V. 2014. Transcriptional control of early T and B cell develop- 1115. mental choices. Annu. Rev. Immunol. 32: 283–321. 32. Will, B., T. O. Vogler, B. Bartholdy, F. Garrett-Bakelman, J. Mayer, L. Barreyro, 2. Germain, R. N. 2002. T-cell development and the CD4-CD8 lineage decision. A. Pandolfi, T. I. Todorova, U. C. Okoye-Okafor, R. F. Stanley, et al. 2013. Satb1 Nat. Rev. Immunol. 2: 309–322. regulates the self-renewal of hematopoietic stem cells by promoting quiescence and repressing differentiation commitment. Nat. Immunol. 14: 437–445.

3. Lai, A. Y., and M. Kondo. 2007. Identification of a bone marrow precursor of the Downloaded from earliest thymocytes in adult mouse. Proc. Natl. Acad. Sci. USA 104: 6311–6316. 33. Beyer, M., Y. Thabet, R. U. Muller,€ T. Sadlon, S. Classen, K. Lahl, S. Basu, 4. Serwold, T., L. I. Ehrlich, and I. L. Weissman. 2009. Reductive isolation from X. Zhou, S. L. Bailey-Bucktrout, W. Krebs, et al. 2011. Repression of the ge- bone marrow and blood implicates common lymphoid progenitors as the major nome organizer SATB1 in regulatory T cells is required for suppressive function source of thymopoiesis. Blood 113: 807–815. and inhibition of effector differentiation. Nat. Immunol. 12: 898–907. 5. Bell, J. J., and A. Bhandoola. 2008. The earliest thymic progenitors for T cells 34. Skowronska-Krawczyk, D., Q. Ma, M. Schwartz, K. Scully, W. Li, Z. Liu, possess myeloid lineage potential. Nature 452: 764–767. H. Taylor, J. Tollkuhn, K. A. Ohgi, D. Notani, et al. 2014. Required enhancer- 6. Wada, H., K. Masuda, R. Satoh, K. Kakugawa, T. Ikawa, Y. Katsura, and matrin-3 network interactions for a homeodomain transcription program. Nature H. Kawamoto. 2008. Adult T-cell progenitors retain myeloid potential. Nature 514: 257–261. 452: 768–772. 35. de Boer, J., A. Williams, G. Skavdis, N. Harker, M. Coles, M. Tolaini, T. Norton, http://www.jimmunol.org/ 7. Pui, J. C., D. Allman, L. Xu, S. DeRocco, F. G. Karnell, S. Bakkour, J. Y. Lee, K. Williams, K. Roderick, A. J. Potocnik, and D. Kioussis. 2003. Transgenic T. Kadesch, R. R. Hardy, J. C. Aster, and W. S. Pear. 1999. Notch1 expression in mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. early lymphopoiesis influences B versus T lineage determination. Immunity 11: Immunol. 33: 314–325. 299–308. 36. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and 8. Radtke, F., A. Wilson, G. Stark, M. Bauer, J. van Meerwijk, H. R. MacDonald, F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selec- and M. Aguet. 1999. Deficient T cell fate specification in mice with an induced tion. Cell 76: 17–27. inactivation of Notch1. Immunity 10: 547–558. 37. Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCR 9. Sambandam, A., I. Maillard, V. P. Zediak, L. Xu, R. M. Gerstein, J. C. Aster, expression in transgenic mice constructed using cDNA-based alpha- and beta- W. S. Pear, and A. Bhandoola. 2005. Notch signaling controls the generation and chain genes under the control of heterologous regulatory elements. Immunol. differentiation of early T lineage progenitors. Nat. Immunol. 6: 663–670. Cell Biol. 76: 34–40. 10. Yui, M. A., N. Feng, and E. V. Rothenberg. 2010. Fine-scale staging of T cell 38. Kurts, C., W. R. Heath, F. R. Carbone, J. Allison, J. F. Miller, and H. Kosaka. lineage commitment in adult mouse thymus. J. Immunol. 185: 284–293. 1996. Constitutive class I-restricted exogenous presentation of self antigens by guest on September 24, 2021 11. Masuda, K., K. Kakugawa, T. Nakayama, N. Minato, Y. Katsura, and in vivo. J. Exp. Med. 184: 923–930. H. Kawamoto. 2007. T cell lineage determination precedes the initiation of TCR 39. Takahama, Y., K. Ohishi, Y. Tokoro, T. Sugawara, Y. Yoshimura, M. Okabe, beta gene rearrangement. J. Immunol. 179: 3699–3706. T. Kinoshita, and J. Takeda. 1998. Functional competence of T cells in the ab- 12. Krangel, M. S. 2009. Mechanics of T cell receptor gene rearrangement. Curr. sence of glycosylphosphatidylinositol-anchored proteins caused by T cell- Opin. Immunol. 21: 133–139. specific disruption of the Pig-a gene. Eur. J. Immunol. 28: 2159–2166. 13. Starr, T. K., S. C. Jameson, and K. A. Hogquist. 2003. Positive and negative 40. Hao, B., A. K. Naik, A. Watanabe, H. Tanaka, L. Chen, H. W. Richards, selection of T cells. Annu. Rev. Immunol. 21: 139–176. M. Kondo, I. Taniuchi, Y. Kohwi, T. Kohwi-Shigematsu, and M. S. Krangel. 14. Teh, H. S., P. Kisielow, B. Scott, H. Kishi, Y. Uematsu, H. Bluthmann,€ and 2015. An anti-silencer- and SATB1-dependent chromatin hub regulates Rag1 H. von Boehmer. 1988. Thymic major histocompatibility complex antigens and and Rag2 gene expression during thymocyte development. J. Exp. Med. 212: the alpha beta T-cell receptor determine the CD4/CD8 phenotype of T cells. 809–824. Nature 335: 229–233. 41. Shimizu, C., H. Kawamoto, M. Yamashita, M. Kimura, E. Kondou, Y. Kaneko, 15. Marusic´-Galesic´, S., D. L. Longo, and A. M. Kruisbeek. 1989. Preferential S. Okada, T. Tokuhisa, M. Yokoyama, M. Taniguchi, et al. 2001. Progression of differentiation of T cell receptor specificities based on the MHC glycoproteins T cell lineage restriction in the earliest subpopulation of murine adult thymus encountered during development. Evidence for positive selection. J. Exp. Med. visualized by the expression of lck proximal promoter activity. Int. Immunol. 13: 169: 1619–1630. 105–117. 16. Kaye, J., M. L. Hsu, M. E. Sauron, S. C. Jameson, N. R. Gascoigne, and 42. Boyman, O., S. Le´tourneau, C. Krieg, and J. Sprent. 2009. Homeostatic prolif- S. M. Hedrick. 1989. Selective development of CD4+ T cells in transgenic mice eration and survival of naı¨ve and memory T cells. Eur. J. Immunol. 39: 2088– expressing a class II MHC-restricted antigen receptor. Nature 341: 746–749. 2094. 17. Murphy, K. M., A. B. Heimberger, and D. Y. Loh. 1990. Induction by antigen of 43. Azzam, H. S., A. Grinberg, K. Lui, H. Shen, E. W. Shores, and P. E. Love. 1998. intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250: CD5 expression is developmentally regulated by T cell receptor (TCR) signals 1720–1723. and TCR avidity. J. Exp. Med. 188: 2301–2311. 18. Vasquez, N. J., J. Kaye, and S. M. Hedrick. 1992. In vivo and in vitro clonal 44. Mathis, D., and C. Benoist. 2007. A decade of AIRE. Nat. Rev. Immunol. 7: 645– deletion of double-positive thymocytes. J. Exp. Med. 175: 1307–1316. 650. 19. Swat, W., L. Ignatowicz, H. von Boehmer, and P. Kisielow. 1991. Clonal deletion 45. Sakaguchi, S., K. Wing, and T. Yamaguchi. 2009. Dynamics of peripheral tol- of immature CD4+8+ thymocytes in suspension culture by extrathymic antigen- erance and immune regulation mediated by Treg. Eur. J. Immunol. 39: 2331– presenting cells. Nature 351: 150–153. 2336. 20. Kappler, J. W., N. Roehm, and P. Marrack. 1987. T cell tolerance by clonal 46. Anderson, M. S., E. S. Venanzi, Z. Chen, S. P. Berzins, C. Benoist, and elimination in the thymus. Cell 49: 273–280. D. Mathis. 2005. The cellular mechanism of Aire control of T cell tolerance. 21. Palmer, E. 2003. Negative selection–clearing out the bad apples from the T-cell Immunity 23: 227–239. repertoire. Nat. Rev. Immunol. 3: 383–391. 47. Moulton, V. R., and G. C. Tsokos. 2011. Abnormalities of T cell signaling in 22. Akashi, K., M. Kondo, U. von Freeden-Jeffry, R. Murray, and I. L. Weissman. systemic lupus erythematosus. Arthritis Res. Ther. 13: 207. 1997. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-deficient mice. 48. Murre, C. 2013. Boosting lymphocyte production. Immunity 38: 1081–1083. Cell 89: 1033–1041. 49. Wiest, D. L., L. Yuan, J. Jefferson, P. Benveniste, M. Tsokos, R. D. Klausner, 23. Zhang, J. A., A. Mortazavi, B. A. Williams, B. J. Wold, and E. V. Rothenberg. L. H. Glimcher, L. E. Samelson, and A. Singer. 1993. Regulation of T cell re- 2012. Dynamic transformations of genome-wide epigenetic marking and tran- ceptor expression in immature CD4+CD8+ thymocytes by p56lck tyrosine ki- scriptional control establish T cell identity. Cell 149: 467–482. nase: basis for differential signaling by CD4 and CD8 in immature thymocytes 24. Wilson, B. G., and C. W. Roberts. 2011. SWI/SNF nucleosome remodellers and expressing both coreceptor molecules. J. Exp. Med. 178: 1701–1712. cancer. Nat. Rev. Cancer 11: 481–492. 50. Park, J. H., S. Adoro, T. Guinter, B. Erman, A. S. Alag, M. Catalfamo, 25. Chi, T. H., M. Wan, K. Zhao, I. Taniuchi, L. Chen, D. R. Littman, and M. Y. Kimura, Y. Cui, P. J. Lucas, R. E. Gress, et al. 2010. Signaling by intrathymic G. R. Crabtree. 2002. Reciprocal regulation of CD4/CD8 expression by SWI/ cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes SNF-like BAF complexes. Nature 418: 195–199. the differentiation of cytotoxic-lineage T cells. Nat. Immunol. 11: 257–264. 10 LOSS OF SATB1 LEADS TO AUTOIMMUNE DISORDERS

51. Sakaguchi, S., T. Yamaguchi, T. Nomura, and M. Ono. 2008. Regulatory T cells 55. Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, and A. Rudensky. 2002. Homeo- and immune tolerance. Cell 133: 775–787. stasis and anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat. Immunol. 3: 33–41. 52. Lee, B. H., A. E. Gauna, K. M. Pauley, Y. J. Park, and S. Cha. 2012. Animal 56. Thornton, A. M., E. E. Donovan, C. A. Piccirillo, and E. M. Shevach. 2004. models in autoimmune diseases: lessons learned from mouse models for Sjo¨gren’s Cutting edge: IL-2 is critically required for the in vitro activation of CD4+CD25+ syndrome. Clin. Rev. Allergy Immunol. 42: 35–44. T cell suppressor function. J. Immunol. 172: 6519–6523. 53. Hwang, S., K. D. Song, R. Lesourne, J. Lee, J. Pinkhasov, L. Li, D. El-Khoury, 57. Shimizu, J., S. Yamazaki, T. Takahashi, Y. Ishida, and S. Sakaguchi. 2002. and P. E. Love. 2012. Reduced TCR signaling potential impairs negative Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks im- selection but does not result in autoimmune disease. J. Exp. Med. 209: munological self-tolerance. Nat. Immunol. 3: 135–142. 1781–1795. 58. Baccala, R., and A. N. Theofilopoulos. 2005. The new paradigm of T-cell ho- 54. Yang, S., N. Fujikado, D. Kolodin, C. Benoist, and D. Mathis. 2015. Immune meostatic proliferation-induced autoimmunity. Trends Immunol. 26: 5–8. tolerance. Regulatory T cells generated early in life play a distinct role in 59. King, C., A. Ilic, K. Koelsch, and N. Sarvetnick. 2004. Homeostatic expansion of maintaining self-tolerance. Science 348: 589–594. T cells during immune insufficiency generates autoimmunity. Cell 117: 265–277. Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 The Journal of Immunology

Corrections

Kondo, M., Y. Tanaka, T. Kuwabara, T. Naito, T. Kohwi-Shigematsu, and A. Watanabe. 2016. SATB1 plays a critical role in establishment of immune tolerance. J. Immunol. 196: 563–572.

A funding source was omitted in the footnotes for this article. The funding information footnote should read:

This work was supported in part by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (24390121 and 26670240), the Uehara Memorial Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, a Strategic Research Foundation Grant-aided Project for Private Schools at Heisei 23rd (S1101016) and Heisei 26th (S1411015) from the Ministry of Education, Culture, Sports, Science and Technology, a Research Promotion Grant from Toho University Graduate School of Medicine (11-01 and 14-02), a Bridging Grant from Duke University Medical Center (to M.K.), Project Research Grants from Toho University School of Medicine (to Y.T.), National Institutes of Health Grant (R37CA39681 to T.K.-S.), and by the Public Foundation of the Vaccination Research Center. M.K. was a scholar of the Leukemia & Lymphoma Society. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600293

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