Malt1 Protease Is Critical in Maintaining Function of Regulatory T Cells and May Be a Therapeutic Target for Antitumor Immunity

This information is current as Liqing Cheng, Nan Deng, Naixue Yang, Xueqiang Zhao and of October 1, 2021. Xin Lin J Immunol published online 12 April 2019 http://www.jimmunol.org/content/early/2019/04/11/jimmun ol.1801614 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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 12, 2019, doi:10.4049/jimmunol.1801614 The Journal of Immunology

Malt1 Protease Is Critical in Maintaining Function of Regulatory T Cells and May Be a Therapeutic Target for Antitumor Immunity

Liqing Cheng,*,† Nan Deng,*,† Naixue Yang,‡ Xueqiang Zhao,*,† and Xin Lin*,†

The Malt1 is a key molecule in mediating Ag receptor–induced NF-kB activation in , but the role of Malt1 in the function of regulatory T (Treg) cells is still unclear. In this article, we reported that specific deletion of Malt1 in Treg cells would lead to Scurfy-like lethal autoimmune disease, which was caused by Treg cell dysfunction but not number loss. Interestingly, Foxp3CreMalt1fl/C472A mice, in which Malt1 protease was specifically inactivated in Treg cells, also displayed spontaneous inflam- matory disorders, with severe hair loss and skin hyperplasia. Consistently, Foxp3CreMalt1fl/C472A mice showed enhanced antitumor response because of their decreased function and infiltration of Treg cells, as well as reduced CD8+ exhaustion.

expression profiling analysis revealed dysregulated expression pattern of Treg effector upon Malt1 deletion or its protease Downloaded from inactivation. Together, our data unraveled a critical role of Malt1, especially its protease activity, in maintaining homeostasis and function of Treg cells. The Journal of Immunology, 2019, 202: 000–000.

egulatory T (Treg) cells, a subpopulation of CD4+ TCR-induced NF-kB activation, such as PKCu, Carma1, Bcl10, T cells, are responsible for maintaining immune toler- Malt1, or TAK1, showed impaired Treg cells development, but the R ance and preventing autoimmune disorders by inhibiting detailed mechanism of this process is not fully elucidated (14–18). http://www.jimmunol.org/ activation of effector T cells and immune response (1, 2). Foxp3, In contrast to conventional T cells (Tconv), which show naive located on the X , is a Treg-specific transcription phenotype at steady-state, Treg cells display an effector-like factor that can orchestrate the development, maintenance, and phenotype with higher expression level of GITR, CD44, and suppressive function of Treg cells (3, 4). Mice with Foxp3 mutant KLRG1 (19, 20). Previous studies have revealed the requirement can develop spontaneous lethal , similar to what is of continuous TCR signaling for maintaining Treg cells homeo- seen in humans, and are known as Scurfy-mice (5, 6). Foxp3 can stasis, especially for effector phenotype (21, 22). Consistently, the cooperate with other transcription factors, such as NF-AT, NF-kB, transcription factor NF-kB, downstream of TCR signaling, can or IRF4, to regulate transcriptional program of Treg cells (7–9). control lineage-specific transcription landscapes and maintain the by guest on October 1, 2021 Treg cells develop in the thymus and require a relatively higher identity and function of Treg cells (2, 19). However, the role of affinity of TCR engagement with self- MHC class II in the intermediate molecules of TCR signaling pathway in Treg cells is presence of CD28 costimulatory signals and IL-2 (1, 10, 11). TCR poorly understood. engagement can induce the intracellular activation of transcription Malt1 is well known to form complex with Carma1 and Bcl10 to factors including NF-kB. c-Rel, one of its subunits, can bind to mediate Ag receptor–induced activation (23, 24). It CNS2 and CNS3 elements of Foxp3 gene promotor region, which was originally identified as a translocation fused with facilitates chromatin remodeling and transcription initiation cIAP2 in mucosa-associated lymphoid tissue B cell lymphomas, (7, 12, 13). Indeed, mice with perturbation of genes that mediate leading to constitutive activation of NF-kB, which is critical for lymphoma cells survival and proliferation (25–28). Given the fact *Institute for Immunology, Tsinghua University School of Medicine, Beijing 100084, that Malt1 contains a caspase-like domain, which shares homol- China; †Tsinghua University–Peking University Center for Life Sciences, Beijing, ogy with caspase, it is also named as paracaspase (29). Generation ‡ 100084, China; and Tsinghua University School of Medicine, Beijing 100084, China of Malt1-deficient mice provided initial evidence of the function ORCIDs: 0000-0003-0906-1806 (L.C.); 0000-0002-8187-5583 (X.Z.); 0000-0003- of Malt1 in adaptive immune response (23, 24). Upon TCR en- 0956-3654 (X.L.). gagement, Malt1 and Bcl10 will associate with Carma1, and Received for publication December 11, 2018. Accepted for publication March 14, 2019. Malt1 can function as a scaffolding protein to recruit E3 ligase This work was supported in part by National Nature Science Foundation of China TRAF6 to Carma1-Bcl10-Malt1 complex (30–32). The ubiq- Grants 81630058, 81570211, and 91542107 (to X.L.) and 31670904 (to X.Z.) and a uitination modification of Bcl10 and Malt1 will activate IkB start-up fund from Tsinghua University–Peking University Center for Life Sciences. kinase, which induces IkBa phosphorylation, degradation, and The sequencing result presented in this article has been submitted to the Gene release of canonical NF-kB (33). The released NF-kB can trans- Expression Omnibus under accession number GSE124089. locate into the nucleus to initiate transcription programs (34). Address correspondence and reprint requests to Dr. Xin Lin, School of Medicine, Besides its scaffolding function, Malt1 protease activity can also Tsinghua University, 30 Shuangqing Road, Haidian Qu, Beijing 100084, China. E-mail address: [email protected] be activated upon TCR engagement. It can cleave deubiquitinases The online version of this article contains supplemental material. A20 and CYLD, which remove K63 polyubiquitin chains and are Abbreviations used in this article: BM, bone marrow; cKI, conditional KI; cKO, negative regulators of NF-kB or JNK activation, to amplify signal conditional knockout; iTreg, induced Treg; KI, knock-in; KO, knockout; RNA-seq, (35, 36). RelB, a noncanonical NF-kB member that can inhibit RNA sequencing; Tconv, conventional T cell; Tfh, follicular helper T cell; TIL, canonical NF-kB activation, is also a substrate of Malt1 (37). In tumor infiltrating lymphocyte; Treg, regulatory T; WT, wild type. addition, Malt1 has been reported to cleave mRNA-binding pro- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 teins Roquin and Regnase1 to regulate mRNA stability (38, 39).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801614 2 Malt1 CONTROLS THE FUNCTION OF Treg CELLS

The substrates mentioned above can be recognized as negative FITC anti-Foxp3 (clone FJK-16s), eFluor 450 anti-CD44 (clone IM7), regulators of T cell activation, whereas HOIL-1, a member of allophycocyanin-eFluor 780 anti-CD62L (clone MEL-14), allophycocyanin- LUBAC (linear ubiquitin chain assembly complex), was reported eFluor 780 anti-CD45.2 (clone 104), eFluor 450 anti-PD-1 (clone RMPI-30), eFluor 450 anti-IgD (clone 11-26c), eFluor 660 anti-GL7 (clone GL7) were to be a substrate of Malt1 recently, the cleavage of which would purchased from eBioscience; allophycocyanin anti-IFN-g (clone XMG1.2) dampen TCR signaling and prevented T cells from overt activation was purchased from BioLegend. For CXCR5 staining, cells were incubated (40, 41). The physiological role of Malt1 protease activity has not with biotin-labeled anti-CXCR5 (clone 2G8; BD Biosciences) for 1 h on ice, been defined until the generation of Malt1 protease-dead mutant followed by staining with second Ab PE-CF594 Streptavidin (BD Biosci- ences). Foxp3 was stained according to the manufacturer’s protocol from mice. This strain of mice displayed spontaneous autoimmune and Foxp3 staining kit (eBioscience). For cytokine staining, cells were stimulated multiorgan inflammation with defective differentiation of Treg, with 50 ng/ml PMA (Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma- marginal zone B, and B1 B cells (31, 42–44). Although Malt1 and Aldrich) in the presence of GolgiStop (BD Biosciences) for 5 h at 37˚C its protease activity has been well studied in Tconv, their role in and stained for cell surface markers followed by fixation/permeabilization Treg cells has not been fully appreciated. and intracellular cytokine staining (BD Biosciences). Samples were analyzed by LSR Fortessa cytometers (BD Biosciences), and data were analyzed by In this article, by using genetic modified mouse model, we reported FlowJo software. For cell sorting, CD4+ T cells were enriched by using that Treg-specific deletion of Malt1 could lead to early-onset lethal mouse CD4+ microbeads (Miltenyi Biotec), and then cells were sorted by autoimmune disease, which was not caused by decreasing the number FACSAria (BD Biosciences). but impaired suppressive function of Treg cells. Interestingly, we Generation of mixed bone marrow chimera mice found that mice with Malt1 protease inhibition specific in Treg cells 3 6 developed chronic but severe inflammatory disorders with obvious A total of 5 10 bone marrow (BM) cells from femurs and tibias of CD45.1 WT mice were mixed at a ratio of 1:1 with BM cells from CD45.2 hair loss and skin hyperplasia, and this strain of mice displayed 2/2 C472A/C472A

WT, Malt1 ,orMalt1 mice, and cells were i.v. transferred Downloaded from enhanced antitumor effect. profiling revealed some into lethally irradiated (5.5Gy 3 2) recipient mice. Two months later, Treg Treg signature genes could be regulated by Malt1 and its protease cell development was analyzed by FACS. activity. In summary, our studies revealed an unappreciated role of Histopathology and immunohistochemistry Malt1, especially its protease activity, in the function of Treg cells, further underscoring the importance of Malt1-mediated signal For histopathology analysis, mouse lung, liver, dorsal skin, and colon were transduction in maintaining the homeostasis of Treg cells. fixed in 4% formaldehyde overnight. The fixed samples were embedded in paraffin, followed by slicing and H&E staining in Servicebio. http://www.jimmunol.org/ Materials and Methods Induced Treg differentiation in vitro 2 2 2 2 Mice CD4+CD25 CD44 CD62L+ naive T cells were sorted from WT, Malt1 / , or Malt1C472A/C472A mice. Cells were cultured in complete RPMI 1640 me- C472A/C472A C57BL/6 (wild type; WT) mice were bred in house. Malt1 and dium (Life Technologies) and stimulated by 5 mg/ml plate-coated anti-CD3/28 fl/fl Malt1 mice were generated by CRISPR/Cas9 method as described below. alone or in the presence of human IL-2 (100 IU/ml) and TGF-b (10 ng/ml). 2/2 2/2 Malt1 mice have been described previously (24). Rag1 ,CD45.1, Three days later, cells were collected and stained for analysis. CD4Cre,andFoxp3Cre-YFP tool mice were kindly provided by Dr. C. Dong at Tsinghua University. All the mice were bred in the specific pathogen-free In vivo colitis model animal facility at Tsinghua University. Animal protocol was approved by + 2 2 + Institutional Animal Care and Use Committees at Tsinghua University. CD4 CD25 CD44 CD62L naive T cells were sorted from CD45.1 WT by guest on October 1, 2021 mice, and YFP+ Treg cells were sorted from Foxp3 Cre (WT), Foxp3 CreMalt1fl/fl Generation of Malt1C472A/C472A and Malt1fl/fl mice (conditional knockout [cKO]), or Foxp3 CreMalt1fl/C472A (conditional KI [cKI]) mice. 5 3 105 naive T cells alone or mixed with 1 3 105 WT,cKO,orcKI C472A/C472A fl/fl Malt1 and Malt1 mice were generated on C57BL/6 back- Treg cells were i.v. injected into recipient Rag12/2 mice. Weight change was ground by CRISPR/Cas9 method. The single guide RNA (sgRNA) tar- monitored weekly to indicate colitis progression. Colon, spleen, and mesenteric geting sequence (Supplemental Table I) was designed using the tool online lymph node were collected for H&E staining or FACS analysis. (http://crispr.mit.edu/), which was then synthesized by using Streptococcus pyogenes Cas9–guide RNA efficiency of target detection kit (Viewsolid In vitro Treg suppression assay Biotech). Then, sgRNA and Cas9 mRNA were transcribed in vitro using 4 + MEGAshortscript T7 Transcription Kit and mMESSAGE mMACHINE T7 A total of 2.5 3 10 CFSE-labeled CD45.1 naive CD4 T cells were ULTRA Transcription Kit (Life Technologies) according to the manufac- cocultured with different ratio of WT, cKO, or cKI Treg cells in the 5 ture’s protocol, respectively. Both sgRNA and Cas9 mRNA were purified presence of 1 3 10 irradiated splenocytes as APC and 2 mg/ml anti-CD3 using MEGAclear Kit (Life Technologies) and dissolved in RNase-free in a 96-well U-bottom plate. Three days later, CFSE dilution was detected, water. Donor DNA, single-strand oligos containing point mutation or which indicated that Treg cells suppression ability. loxp sequence, was synthesized in Sangon Biotech (Shanghai, China). For Malt1C472A/C472A generation, to increase the knock-in (KI) efficiency and Tumor model prevent the sgRNA-guided Cas9 from cleaving mutant Malt1 genomic A total of 2 3 105 E.G7 tumor cells were inoculated s.c. into the shaved flank , we also designed five synonymous mutations within the donor. of each mice. Tumor growth was monitored every other day by using caliper, A total 20 ml of mixture containing sgRNA (20 ng/ml), Cas9 mRNA and tumor size was calculated with the following formula: (length / 2) 3 (40 ng/ml), and donor oligo (60 ng/ml) was injected into zygotes. The (width / 2) 3 p. For isolation of tumor infiltrating lymphocytes (TIL), tu- microinjection was accomplished at Laboratory Animal Research Center, fl/fl mors were digested with 1 mg/ml type 2 collagenase (Worthington) in the Tsinghua University. Notably, for Malt1 generation, two sgRNA (10 ng/ml presence of 10 U/ml DNase I for 1 h at 37˚C prior to centrifuge on a 40 and for each) and two donor oligoes (30 ng/ml for each), which target two dis- 70% discontinuous Percoll gradient (GE Healthcare). tinct loxp-inserting sites at Malt1 genome locus, were contained in the mixture for injection. The founder mice were genotyped by using manually Real-time quantitative PCR designed specific primers (Supplemental Table I), confirmed by TA cloning (TransGen Biotech) and sequencing. Total RNA was extracted by TRIzol (Invitrogen), and cDNA was synthe- sized by using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Flow cytometry and cell sorting Scientific). Real-time quantitative PCR using 23 SYBR Green PCR Master Mix (Genestar) was performed at ABI 7500 Real-Time PCR sys- Single-cell suspension was prepared from peripheral spleen or lymph node. tem (Applied Biosystems). Results were carried out using the 22OOCT Cells were incubated with Abs as follows: allophycocyanin anti-CD4 (clone method. Primers were shown in Supplemental Table II. GK1.5), V500 anti-CD4 (clone RM4-5), allophycocyanin-Cy7 anti-CD19 (clone 1D3), PE anti-CD45.1 (clone A20), PE-Cy7 anti-CD95 (clone Jo2), Western blot PE anti-IL-17A (clone TC11-18H10) were purchased from BD Biosciences; eFluor 450 anti-CD4 (clone GK1.5), FITC anti-CD4 (clone GK1.5), PE anti- Pan T cells were isolated from spleen using EasySep Mouse CD90.2 CD8 (clone 53-6.7), allophycocyanin-eFluor 780 anti-CD8 (clone 53-6.7), Positive Selection Kit II (Stemcell Technologies). Sorted YFP+ Treg cells The Journal of Immunology 3 were stimulated by PMA and ionomycin for the indicated time. Cells were Malt1 loss in Treg cells leads to early-onset lethal lysed in lysis buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 1 mM EDTA, autoimmune disease 1% Nonidet P-40, and protease inhibitors), and total lysates were subjected to SDS-PAGE, followed by blotting with indicated Abs, IkBa (Santa Cruz To evaluate the role of Malt1 in the homeostasis and function of Biotechnology), Malt1 (Santa Cruz Biotechnology), RelB (Cell Signaling Treg cells, we crossed Malt1fl/fl with Foxp3Cre-YFP mice to spe- Technology), GAPDH (Easy Bio), Tubulin (Santa Cruz Biotechnology), cifically delete Malt1 in Treg cells but not Tconv. We observed and second Ab (Easy Bio). lethal systemic inflammation with lymphadenopathy and spleno- RNA sequencing analysis megaly in Foxp3CreMalt1fl/fl mice (Fig. 2A), which died within CD4+YFP+ Treg cells were sorted from relative mouse strains, and RNA 40 d after birth with a Scurfy-like phenotype (Fig. 2B). Histo- was prepared by using TRIzol (Invitrogen). Qualification of RNA samples, logical results showed a significant infiltration of lymphocytes in cDNA library construction (KAPA mRNA HyperPrep Kit), and sequencing the lung and liver of Foxp3CreMalt1fl/fl mice compared with control (Illumina HiSeq X Ten) were done in Genomics and Synthetic Biology mice (Fig. 2C). The percentage of activated effector memory Core Facility in Tsinghua University. The reads were aligned with HISAT2 (CD44+CD62L2) and central memory (CD44+CD62L+) conven- (45), and transcriptomes from RNA sequencing (RNA-seq) data were as- + + sembled with Cufflinks (46). Differential expression was done with tional CD4 and CD8 T cells was dramatically increased in Cuffdiff (46). The sequencing result was uploaded to the Gene Expression Foxp3CreMalt1fl/fl mice in both spleen and lymph nodes (Fig. 2D, Omnibus under accession GSE124089, and the URL is https://www.ncbi. 2E). We also observed increased proportion of IFN-g and IL-17A– nlm.nih.gov/geo/query/acc.cgi?acc=GSE124089. producing CD4+ T cells in Foxp3CreMalt1fl/fl mice (Fig. 2F, 2G). Statistical analysis Because these mice showed autoimmune disease phenotype, we detected follicular helper T cell (Tfh) and germinal center B cell Data were analyzed in Prism 7 software, and statistical analysis was per- formed by using unpaired Student t test. The statistical significance was differentiation and found a spontaneous formation of a germinal Downloaded from Cre fl/fl shown as *p , 0.05, **p , 0.01, ***p , 0.001, and ****p , 0.0001 center in Foxp3 Malt1 mice with increased percentage of Tfh (“ns” indicates no significance). and germinal center B cells in spleen (Fig. 2H, 2I). However, Malt1 loss led to decreased percentage of Treg cells in spleen but Results not in lymph nodes, and Treg number was unaffected in spleen but Malt1 and its protease activity is required for the development actually increased in lymph nodes (Fig. 2J, 2K), implying that Malt1 was crucial for maintaining Treg homeostasis. of Treg cells http://www.jimmunol.org/ To delineate the role of Malt1 in Treg cells, we generated Malt1 Mice with Treg-specific inhibition of Malt1 protease activity protease-dead mutant (Malt1C472A/C472A)andloxp-flanked Malt1 al- develop spontaneous inflammatory disorders fl/fl leles (Malt1 )micebyusingCRISPR/Cas9method(Supplemental To specifically assess the role of Malt1 protease activity in Treg Fig. 1A, 1B). First, we explored its role in natural Treg cell devel- cells, we crossed Malt1fl/fl with Malt1C472A/C472A and Foxp3Cre-YFP Cre fl/fl C472A/C472A opment by crossing CD4 mice with Malt1 and Malt1 to get Foxp3CreMalt1fl/C472A mice, in which Malt1 protease was Cre fl/fl Cre fl/C472A to get CD4 Malt1 or CD4 Malt1 mice (Supplemental specifically inactivated in Treg cells. Unlike Foxp3CreMalt1fl/fl Fig. 1C). We observed a significant decrease in both proportion mice, which developed lethal autoimmune phenotype at early time and number of Foxp3+ Treg cells upon Malt1 loss or its protease Cre fl/C472A after birth, Foxp3 Malt1 mice showed a delay of illness by guest on October 1, 2021 inactivation in the thymus (Fig. 1A, 1B). A similar tendency was also onset after 6–8 wk with spontaneous and severe hair loss, skin observed in peripheral organs, spleen, and lymph nodes (Fig. 1C, inflammation, lymphadenopathy, and splenomegaly with de- Cre fl/C472A 1D), although the decrease was mild in CD4 Malt1 mice creased body weight (Fig. 3A, 3B). Histological staining showed Cre fl/fl compared with CD4 Malt1 mice, in which Treg cells were al- a significant infiltration of lymphocytes in the dorsal skin and most diminished. These data suggested that Malt1 or its protease lung of Foxp3CreMalt1fl/C472A mice compared with control mice activity was required for the development of Treg cells. Interestingly, (Fig. 3C). Moreover, conventional CD4+ and CD8+ T cells showed Cre fl/C472A CD4 Malt1 mice also showed spontaneous body weight loss a dramatically increased effector phenotype (Fig. 3D, 3E). How- (Supplemental Fig. 1D), a similar phenotype as observed in germline ever, no difference was observed for the percentage of CD4+ Malt1 protease-dead mutant mice (31, 42–44). T cells producing IFN-g and IL-17A (Fig. 3F, 3G), but there was To assess whether Malt1 played an intrinsic or extrinsic role in an increased frequency of CD8+ T cells secreting IFN-g (Fig. 3H, directing the development of Treg cells, we generated mixed BM 3I). Despite the severe disease, the portion and number of Treg + + chimera mice by cotransferring CD45.1 WT BM with CD45.2 WT, cells were actually increased in peripheral lymphoid tissues 2/2 C472A/C472A or BM cells from Malt1 (knockout; KO) or Malt1 (KI) (Fig. 3J, 3K). Taken together, these data indicated that Malt1 2/2 mice into irradiated Treg-deficient TCRbd mice. BM cells from protease activity was also required for maintaining the homeo- + + either CD45.1 or CD45.2 WT mice could differentiate into Treg stasis of Treg cells and preventing overt inflammation-induced cells normally, whereas Malt1-deficient or Malt1 protease-dead tissue damage. mutant BM cells could not, implicating Malt1 played an intrinsic role in controlling the development of Treg cells (Fig. 1E, 1F). Malt1 and its protease activity is required for the suppressive Finally, we investigated a potential role of Malt1 in induced Treg function of Treg cells (iTreg) differentiation in vitro. CD4+ naive T cells were isolated Although Foxp3 CreMalt1fl/fl and Foxp3 CreMalt1fl/C472A mice exhibited from WT, Malt12/2 (KO), or Malt1C472A/C472A (KI) mice, stimu- obvious illness, the amount of Treg cells actually increased in these lated by anti-CD3/28 alone or in the presence of IL-2 and TGF-b. mice. To investigate the effect of Malt1 deficiency on the suppressive Under the situation of anti-CD3/28 stimulation alone, a small function of Treg cells, we performed an adoptive T cell transfer– portion of WT cells could express Foxp3, whereas KO or KI cells induced colitis model in vivo, in which CD45.1+ naive T cells were not (Fig. 1G, 1H). In contrast to a dramatic decrease of Treg transferred to Rag12/2 recipients alone or with WT or Malt1- cells in Malt12/2 and Malt1C472A/C472A mice compared with WT deficient Treg cells. In contrast to WT Treg cells, Malt1-deficient mice in vivo (31), the difference was pretty modest for Foxp3 (cKO) Treg cells could not prevent naive T cell–induced body induction in vitro (Fig. 1G, 1H), suggesting that Malt1 or its weight loss in recipient mice (Fig. 4A). Histological examination of protease activity was partially required for iTreg differentiation large intestine showed exacerbated damage in mice transferred with in vitro. Malt1-deficient Treg cells or CD45.1+ naive T cells only (Fig. 4B). 4 Malt1 CONTROLS THE FUNCTION OF Treg CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Malt1 and its protease activity is required for the development of Treg cells. (A and B) Thymus from 6- to 8-wk-old CD4CreMalt1fl/wt, CD4CreMalt1fl/fl, and CD4CreMalt1fl/C472A mice were analyzed by FACS (n = 3–5 mice per group). (A) Representative dot plots of CD4+Foxp3+ Treg cells gated on CD4+CD82 cells in the thymus. (B) Statistical analysis of CD4+Foxp3+ cell percentage (left) and absolute number (right) in the thymus. (C and D) FACS analysis of peripheral lymphoid tissues of mice described as in (A). (C) Representative of Foxp3+ Treg cells gated on CD4+ in the spleen. (D) Percentage (left) and absolute number (right) of CD4+Foxp3+ Treg cells in both peripheral spleen (SP) and lymph node (LN). (E) CD45.1+ WT BM cells were cotransferred with CD45.2+ WT, Malt12/2 (KO), or Malt1C472A/C472A (KI) BM cells into lethally irradiated TCRbd2/2 recipient mice. Treg de- velopment was analyzed 2 mo later. (F) Statistical analysis of the percentage of Treg cells derived from relative donors (n = 3–5 mice per group). (G and H) CD4+CD252CD442CD62L+ naive T cells sorted from pooled spleen and lymph nodes of WT, Matl12/2 (KO), or Malt1C472A/C472A (KI) mice were stimulated by plate-bound anti-CD3/28 (5 mg/ml) alone (Th0) or in the presence of human IL-2 (100 IU/ml) and TGF-b (10 ng/ml) (iTreg). (G) Foxp3 induction in viable cells was detected by FACS 3 d later gated on CD4+ and a summary of Foxp3+ percentage was shown in (H). In (A)–(H), One rep- resentative of two independent experiments was shown. Values were shown as mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, nonsignificant. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Malt1 loss in Treg cells leads to early-onset lethal autoimmune disease. (A) Images of 21-d-old mice and thymus, mesenteric lymph node, cervical lymph node, and spleen of Foxp3CreMalt1fl/wt and Foxp3CreMalt1fl/fl littermate mice. (B) Survival curves of Foxp3CreMalt1fl/wt and Foxp3CreMalt1fl/fl littermate mice. (C) H&E staining of lung and liver sections from 21-d-old mice. Scale bars, 100 mm. (D–K) FACS analysis of spleen or lymph node of 21-d-old mice. (D) Representative of CD44 and CD62L expression in spleen CD4+Foxp32 and CD8+ T cells. (E) Percentage of naive (CD442CD62L+), central memory T cells (TCM; CD44+CD62L+), and effector memory T cells (TEM; CD44+CD62L2) spleen CD4+Foxp32 or CD8+ Tcells.(F) Representative of IFN-g or IL-17A–producing CD4+ Foxp32 T cells in spleen. (G) Statistical results of percentage of IFN-g or IL-17A–producing CD4+Foxp32 Tcellsinboth spleen and lymph node. (H) Tfh (left) and germinal center B cell (right) differentiation in spleen. Tfh were gated on CD4+, and germinal center B cells were gated on CD19+IgDlow.(I) Statistical results of percentage of Tfh and germinal center B cells in spleen. (J) Representative of Foxp3 expression in spleen CD4+ Tcells.(K) Cumulative percentage (left) and number (right) of Treg cells in both spleen and lymph node. In (E)–(K), values shown were mean 6 SEM. One representative of two independent experiments was shown (n = 4 mice per group). *p , 0.05, **p , 0.01, ***p , 0.001. ns, nonsignificant. 6 Malt1 CONTROLS THE FUNCTION OF Treg CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. Mice with Treg-specific inhibition of Malt1 protease activity develop spontaneous inflammatory disorders. (A) Images of 8-wk-old mice and thymus, inguinal lymph node, cervical lymph node, mesenteric lymph node, and spleen of Foxp3CreMalt1fl/wt and Foxp3CreMalt1fl/C472A littermate mice. (B) Body weight curve of Foxp3CreMalt1fl/wt and Foxp3CreMalt1fl/C472A littermate mice. (C) H&E staining of skin and lung sections from 8-wk-old mice. Scale bars, 50 mm. (D–K) FACS analysis of spleen or lymph node of 8 wk-old mice. (D) Representative of CD44 and CD62L expression in spleen CD4+Foxp32 and CD8+ T cells. (E) Percentage of naive (CD442CD62L+), TCM (CD44+CD62L+) and TEM (CD44+CD62L2) spleen CD4+Foxp32 or CD8+ T cells. (F) Representative of IL-17A or IFN-g–producing CD4+Foxp32 T cells in spleen. (G) Statistical results of percentage of IFN-g or IL-17A–producing CD4+Foxp32 T cells in both spleen and lymph node. (H) Representative of IFN-g–producing CD8+ T cells in spleen. (I) Statistical results of percentage of IFN-g+CD8+ T cells in spleen and lymph node. (J) Representative of Foxp3 expression in spleen CD4+ T cells. (K) Cumulative percentage (left) and number (right) of Treg cells in both spleen and lymph node. In (E)–(K), values shown were mean 6 SEM. One representative of two independent experiments was shown (n = 3 mice per group). *p , 0.05, **p , 0.01, ***p , 0.001. ns, nonsignificant. The Journal of Immunology 7

We also observed significant splenomegaly and mesenteric lymph with previous study, suggested a critical role of Malt1, especially nodes enlargement, and there were more IL-17A and IFN-g– its protease activity, in maintaining the identity of Treg cells. producing cells in Malt1-deficient Treg cell–transferred mice Malt1 protease activity in Treg cells could be a potential target (Fig. 4C). Besides, Malt1-deficient Treg cells lost Foxp3 expression for enhancing antitumor response after transfer (Fig. 4D). We also performed in vitro suppression assay by coculturing WT or Malt1-deficient Treg cells with CFSE-labeled The observation that Malt1 protease-dead mutant could impair Treg naive T cells, and the results indicated Malt1 loss could impair the function led us to speculate a potential role of Malt1 protease suppressive function of Treg cells (Fig. 4E). The same experiments activity in antitumor response. To test this hypothesis, we inocu- C472A/C472A were performed to determine the suppressive function of Treg cells lated E.G7 lymphoma cells in Malt1 mice and found from Foxp3CreMalt1fl/C472A (cKI) mice. Similarly, cKI Treg cells that tumor development was partially inhibited in these mice could not prevent recipient mice from T cell–induced body weight (Fig. 6A), which was consistent with a previous report (51). To loss and tissue damage (Fig. 4F, 4G), whereas there was no differ- specifically assess Malt1 protease activity in Treg cells, we chal- Cre fl/C472A ence in the suppression assay in vitro (Fig. 4H). Together, these data lenged Foxp3 Malt1 mice with tumor and found that tu- Cre fl/C472A indicated that Malt1 was required for the suppressive function of mor development was delayed in Foxp3 Malt1 mice with Treg cells both in vivo and in vitro. decreased tumor weight at the end stage (Fig. 6B). However, there was no significant difference of CD8+ T or NK cell infiltration in Malt1 and its protease activity is essential in maintaining Treg the tumor microenvironment (Fig. 6C) or IFN-g and granzyme B– cell identity producing CD8+ T cells (Fig. 6D). But, we observed decreased + To comprehensively and unbiased analyze the mechanism under- Treg cell infiltration and PD-1 expression level on CD8 T cells in

Cre fl/C472A Downloaded from lying defective Treg cell function upon Malt1 deletion or its protease Foxp3 Malt1 mice (Fig. 6E–G), indicating less exhausted + activity inhibition, we performed RNA-seq using CD4+YFP+ Treg CD8 T cells and enduring killing activity. Taken together, cells sorted from Foxp3CreMalt1fl/wt (WT), Foxp3 CreMalt1fl/fl (cKO), inhibiting Malt1 protease activity in Treg cells could be a potential and Foxp3 CreMalt1fl/C472A (cKI) mice. The results showed an ob- strategy for enhancing antitumor response. vious downregulation of Treg signature genes in cKO and cKI cells, whereas it was relatively moderate in Malt1 protease-dead mutant Discussion Treg cells (Fig. 5A, Supplemental Fig. 2A). We also did real-time Tconv and Treg cells develop from the same progenitor and share http://www.jimmunol.org/ PCR to confirm some of the putative genes based on RNA-seq similar TCR signaling pathways, but they have opposite functions data, and Treg-associated suppressive genes, such as CTLA4, in immune response. TCR signaling has been well studied in Tconv, IRF4, IL-10, and TGF-b, showed decreased expression level in whereas its function in Treg cells remains to be fully elucidated. In Foxp3 CreMalt1fl/fl cells (Fig. 5B). Among these genes, KLRG1, which the current study, we showed that Malt1, a TCR intermediate was reported to be an effector gene of Treg cells, was downregulated signaling molecule, was required for the development of natural significantly in both Foxp3 CreMalt1fl/fl and Foxp3 CreMalt1fl/C472A Treg Treg cells. However, in contrast to a previous study showing that cells (Fig. 5C). KLRG1 expression could be regulated by IL-2– naive T cells lacking both NF-kB subunits could not differentiate STAT5 signaling pathway (47), but we did not observe difference for to iTreg cells (2), we found Malt1 or its protease activity was only by guest on October 1, 2021 STAT5 phosphorylation upon IL-2 stimulation (Supplemental partially required for iTreg differentiation in vitro, suggesting Fig. 2B). Another effector gene, CD44, was also downregu- other signaling pathways that can activate NF-kB might also be lated (Supplemental Fig. 2C). Malt1 was well known to mediate involved in this process. TCR-induced NF-kB activation, so we used PMA and ionomycin To specifically assess the role of Malt1 in the function of Treg to stimulate the cells, and the results showed that IkBa degra- cells, we generated mice with Treg-specific deletion of Malt1 or dation was severely dampened in Foxp3 CreMalt1fl/fl but moder- Treg-specific Malt1 protease-dead mutant. We observed lethal ately in Foxp3 CreMalt1fl/C472A Treg cells (Fig. 5D). Besides, RelB autoimmune disease upon specific deletion of Malt1 in Treg cells, cleavage was blocked in both cKO and cKI Treg cells compared and Malt1 protease activity inhibition in Treg cells also led to with WT (Fig. 5E), which showed a similar tendency as in Tconv chronic but severe inflammatory disorders, both of which were (31). Besides, we also used iTreg cells differentiated from WT, caused by dysfunction of Treg cells but not number loss. The Mal12/2 (KO), or Malt1C472A/C472A (KI) CD4+ naive T cells in vitro different phenotype observed between Foxp3CreMalt1fl/fl and and found IkBa degradation was inhibited in Malt1 deficient iTreg Foxp3CreMalt1fl/C472A mice suggested both scaffolding and prote- cells, whereas RelB cleavage was blocked in both KO and KI ase activity were necessary for Malt1’s function. Besides, the iTreg cells (Fig. 5F, 5G). These data suggested Malt1 was critical number of Treg cells was increased in Foxp3CreMalt1fl/fl and in mediating TCR-induced NF-kB activation, and its protease Foxp3CreMalt1fl/C472A mice, which was in contrast to the almost activity can regulate this process via cleaving NF-kB negative complete loss of Treg cells in Malt12/2 or Malt1C472A/C472A mice. modulators, such as RelB or other molecules. Previous study has This supported the notion that Malt1 was required for Foxp3 ex- reported the transcription factor NF-kB, downstream of Malt1, pression in Treg cells at the initial stage, whereas Foxp3 were essential in maintaining identity and function of Treg cells expression-induced Malt1 deletion or protease inactivation would (2). So, we compared our RNA-seq data with c-Rel and RelA not dampen the proliferation of Treg cells in vivo. The increased double KO Treg cell sequencing data and observed an obvious number of Treg cells might be due to inflammation caused by the overlap of either upregulated or downregulated genes among these dysfunction of Treg cells, because overt inflammation would genotypes (Supplemental Fig. 2D), implying defective NF-kB trigger the proliferation of Treg cells but impaired their suppres- signaling in both Malt1-deficient or protease-dead mutant Treg sive function by regulating metabolism (52), whereas the detailed cells. According to a previous report that Treg cells showed rel- mechanism remains to be further investigated. atively higher constitutive signaling, known as tonic signaling, in It is interesting to notice the different phenotypes among the the basal state (19). Nr4a1, which was reported to be associated mice with Malt1 deletion or its downstream transcription factor with TCR signal strength and indicative of tonic signaling in Treg NF-kB deletion specifically in Treg cells (2, 19). RelA deletion cells (48–50), was also significantly decreased in both cKO and could lead to profound autoimmune disease and the mice would cKI Treg cells (Supplemental Fig. 2E). This analysis, combining die within 5–15 wk after birth, which was not as severe as 8 Malt1 CONTROLS THE FUNCTION OF Treg CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. Malt1 and its protease activity are required for the suppressive function of Treg cells. (A–D) In vivo colitis model by transferring CD45.1+ naive T cells alone or with CD45.2+YFP+ Treg cells sorted from Foxp3Cre (WT Treg) or Foxp3CreMalt1fl/fl (cKO Treg) mice into recipient Rag12/2 mice. One representative of two independent experiments was shown (n = 3–5 mice per group). (A) Body weight changes of recipient mice were shown as percentage of starting weight. (B) H&E staining of colon 7 wk after transfer. Scale bars, 100 mm. (C) Cellularity of IL-17A and IFN-g–producing CD45.1+ T cells in spleen (left) and mesenteric lymph node (right). (D) Foxp3 expression in both spleen and mesenteric lymph node gated on CD4+CD45.2+, shown as mean fluorescence intensity (MFI). (E) In vitro Treg suppression assay, shown as Treg inhibition of responder T cells (Tresp) proliferation by detecting CFSE dilution after 72 h. One representative of three experiments was shown. (F and G) In vivo colitis model for detecting the suppressive function of cKI Treg cells by monitoring recipient mice body weight (F) and histopathological change (G). One representative of two independent experiments was shown. (H) In vitro cKI Treg cells suppression assay. This experiment was repeated for three times. (A, C–F,andH)*p , 0.05, **p , 0.01, ****p , 0.0001. ns, nonsignificant.

Foxp3CreMalt1fl/fl mice, and c-Rel deletion alone led to mild in- transcription factor NF-kB, we also observed differences of au- flammatory phenotype. Only deletion of both NF-kB subunits toimmune disease severity among Foxp3CreMalt1fl/fl and other c-Rel and RelA in Treg cells could cause a similar phenotype as mice with Treg-specific deletion of genes such as TAK1 or Ubc13, we observed in Foxp3CreMalt1fl/fl mice; for example, the mice which were also molecules mediating NF-kB activation down- would die around 1 mo after birth. Aside from the downstream stream of Malt1 (53, 54). But these mice did not display The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Malt1 and its protease activity are essential in maintaining Treg cell identity. (A) RNA-seq was performed by using CD4+YFP+ Treg cells sorted from Foxp3CreMalt1fl/wt (WT), Foxp3CreMalt1fl/fl (cKO), or Foxp3CreMalt1fl/C472A (cKI) mice. Heatmap of Treg genes downregulated in cKO or cKI Treg cells compared with WT Treg cells. (B) Real-time quantitative PCR results of several Treg signature genes expression in Foxp3CreMalt1fl/wt (WT), Foxp3CreMalt1fl/fl (cKO), and Foxp3 CreMalt1fl/C472A (cKI) Treg cells. (C) Representative of KLRG1 expression gated on CD4+Foxp3+.(D and E) Western blot results of Foxp3 CreMalt1fl/wt (WT), Foxp3CreMalt1fl/fl (cKO), and Foxp3CreMalt1fl/C472A (cKI) Treg cells stimulated by PMA and ionomycin (P/I) for 15 min (D). For detecting RelB cleavage, the Treg cells were pretreated with MG132 for 15 min and followed by P/I stimulation for 30 min (E). (F and G) CD4+CD252CD442CD62L+ naive T cells sorted from WT, Matl12/2 (KO), or Malt1C472A/C472A (KI) mice were stimulated by plate-bound anti-CD3/28 (5 mg/ml) in the presence of human IL-2 (100 IU/ml) and TGF-b (10 ng/ml) to be differentiated into iTreg cells in vitro, then IkBa degradation (F) and RelB cleavage (G)was detected upon P/I stimulation by Western blot. (B)–(D) were repeated for two times. (E)–(G) were shown as one time result. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. ns, nonsignificant. 10 Malt1 CONTROLS THE FUNCTION OF Treg CELLS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 6. Malt1 protease activity in Treg cells could be a potential target for enhancing antitumor response. (A and B) E.G7 tumor cells were in- oculated s.c. in different cohorts. Tumor growth was measured by caliper at indicated time points and tumor weight was calculated at end stage. (A) Six- to eight-week-old WT or Malt1C472A/C472A mice were used. (B) Six- to eight-week-old Foxp3CreMalt1fl/wt and Foxp3CreMalt1fl/C472A mice were challenged with tumor. (C) CD8+ T and NK cell percentage in the TIL gated on CD45.2+.(D) Percentage of IFN-g and Granzyme B–producing CD8+ T cells gated on CD8+.(E) Treg percentage in the TIL gated on CD45.2+.(F and G) FACS plot of PD-1–expressing CD8+ T cells gated on CD8+ (F), and statistical result of PD-1+CD8+ percentage and mean fluorescence intensity (MFI) of PD-1 expression on CD8+ T cells (G). (A and B) One representative of three independent experiments was shown. (C–G) One representative of two independent experiments was shown. WT, Foxp3CreMalt1fl/wt; cKI, Foxp3CreMalt1fl/C472A. *p , 0.05, **p , 0.01, ***p , 0.001. ns, nonsignificant. autoimmune pathology as severe as Foxp3CreMalt1fl/fl mice, sug- activity could regulate c-Rel expression via cleaving the mRNA- gesting that Malt1 played an essential role in regulating the binding protein Regnase-1, which can regulate c-Rel mRNA stabil- function of Treg cells. ity, we did not observe very significant decrease of c-Rel mRNA in The generation of Foxp3 CreMalt1fl/fl and Foxp3 CreMalt1fl/C472A mice Treg cells expressing the Malt1 protease-dead mutant, suggesting helped to bypass the impediment caused by loss of Malt1 or its there were other factors that could be regulated by Malt1 protease protease activity on the development of Treg cells, which enabled us activity besides of c-Rel. Foxp3 was a key transcription factor for to sort out Treg cells for profiling gene expression. RNA-seq results Treg cells, but we actually did not observe the change of Foxp3 revealed Malt1 or its protease activity was critical for maintaining the expression upon Malt1 loss or its protease inhibition, supporting the identity of Treg cells, especially the expression profile of effector genes, notion that Foxp3 alone was not enough to maintain the homeostasis such as CTLA4 and KLRG1, as we observed impaired suppressive of Treg cells and it needs to cooperate with other transcription factors functionofTregcellsinFoxp3 CreMalt1fl/fl and Foxp3 CreMalt1fl/C472A to coregulate the expression of Treg genes. mice. According to previous reports, there was more NF-kB locating Although Treg cells are critical in maintaining peripheral tol- in the nucleus of Treg cells under the normal situation, and contin- erance, they may also be detrimental in the tumor microenviron- uous TCR signaling was required for the suppressive function of Treg ment. Because Malt1 has protease activity, it could be a potential cells (19, 21, 22), and our data showed defective NF-kB signaling in therapeutic target for drug development. When inoculating tumor Malt1-deficient Treg cells. Consistently, we also found that there were into mice with different genotypes of Malt1, we found that Malt1 sets of genes commonly downregulated or upregulated in Treg cells protease inactivation specific in Treg cells could potentiate anti- from Foxp3 CreMalt1fl/fl and Foxp3 CreMalt1fl/C472A mice compared tumor effect. A similar phenotype was previously observed in with Foxp3 CreRelfl/flRelAfl/fl mice (2). Although Malt1 protease Foxp3CreRelfl/fl but not in Foxp3CreRelAfl/fl mice (55), which also The Journal of Immunology 11 showed profound autoimmune disease, suggesting autoimmunity 11. Josefowicz, S. Z., and A. Rudensky. 2009. Control of regulatory T cell lineage could not always have antitumor effect. Interestingly, we did not commitment and maintenance. 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Supplementary Figures

Figure S1. Generation of Malt1C472A/C472A and Malt1fl/fl mice, related to Fig 1. (A)-(B) Generation of Malt1C472A/C472A and Malt1fl/fl mice by using CRISPR/Cas9 method. The detailed was described in Materials and Methods. (A) Schematic graph of Malt1 protease dead mutant mice generation (left) and genotyping result (right). (B) Two loxp element were inserted into Intron 2 and 10 respectively (left) and genotyping result was shown in right. (C) Deletion efficiency was measured by detecting Malt1 expression via Western Blot in WT Pan T, CD4CreMalt1fl/fl (cKO) Pan T and non-Pan T cells. (D) Body weight curve of CD4CreMalt1fl/C472A and control mice. * p<0.05.

Figure S2. Malt1 and its protease activity is essential in maintaining Treg cell identity, related to Fig 5. (A) Treg signature genes (22) expression change in Foxp3CreMalt1fl/fl (cKO) or Foxp3CreMalt1fl/C472A (cKI) group was calculated in a pie. (B) Sorted Foxp3CreMalt1fl/wt (WT) or Foxp3CreMalt1fl/fl (cKO) CD4+YFP+ Treg cells were stimulated by hIL-2 (25IU/ml) for 20min, and STAT5 phosphorylation was detected by FACS. (C) Representative of CD44 expression in Foxp3CreMalt1fl/wt, Foxp3CreMalt1fl/fl and Foxp3CreMalt1fl/C472A Treg cells. (D) Overlapped genes of commonly upregulated (left) or downregulated (right) among Foxp3CreRelAfl/flRelfl/fl (DKO), Foxp3CreMalt1fl/fl (cKO) and Foxp3CreMalt1fl/C472A (cKI) Treg cells were shown in the Venn diagram. (E) Nr4a1 mRNA level in Foxp3CreMalt1fl/wt (WT), Foxp3CreMalt1fl/fl (cKO) and Foxp3CreMalt1fl/C472A (cKI) Treg cells was determined by qPCR. (B)-(C) and (E) were repeated for two times. ns, non-significant.

Supplementary Table 1. Oligo sequences for mouse generation

Oligo name Sequences (5’-3’)

C472A sgRNA TTTTCCGACACATATCCAAC

C472A donor catactgaaattaatgcaagaaaaggagactggcctgaatgtgttTctCCtggaCatgG CGcgCaaaaggtaaaatgtctcatctctctatcaagtagcaaccttgacaaagtctatgta

1st loxp sgRNA TAGATGCCTAAGTAGTGCAC

1st loxp donor tctcaggtctttagttcatgtctagttgaaaaaaacaacaactagcctATAACTTCGT ATAATGTATGCTATACGAAGTTATgtgcactacttaggcatctaactc agctgaagtgctggccatatgagt

2nd loxp sgRNA GTGACATGCTCTAGATCCAC

2nd loxp donor gactagcagtcactgcagattctctccagacattaacaaaccacacctATAACTTC GTATAATGTATGCTATACGAAGTTATgtggatctagagcatgtcac caggctgtcctgtcttacaagtgggaag

C472A-WT-F (genotyping) TGTTCCTGTTGGATATGTGTCGG

C472A-KI-F (genotyping) TGTTTCTCCTGGACATGGCGCGC

C472A-R (genotyping) GGGTCTCACTATGCATCCCTGCTG

1st-loxp-F (genotyping) GCTTCTAGTCAGTCACCAGCTCAGTT

1st-loxp-R (genotyping) CCAGGCAGACATCATATTACAGTTCTC

2nd-loxp-F (genotyping) CTCTCTCTAGAGTCCATGTCCATGT

2nd-loxp-R (genotyping) TCACTACTTCTGACCCTTGGGGAATG

Supplementary Table 2. The primer sequences for RT-PCR

Gene name Forward (5’-3’) Reverse (5’-3’)

IRF4 TCCTCTGGATGGCTCCAGATG CACCAAAGCACAGAGTCACCT

IL-10 ATAACTGCACCCACTTCCCAGTC CCCAAGTAACCCTTAAAGTCCTGC

CTLA4 GCTTCCTAGATTACCCCTTCTGC CGGGCATGGTTCTGGATCA

TGFb CCACCTGCAAGACCATCGAC CTGGCGAGCCTTAGTTTGGAC

Actin TGGAATCCTGTGGCATCCATGAAAC TAAAACGCAGCTCAGTAACAGTCCG

KLRG1 GGACGAGGAATGGTAGCCAC GTAAGGAGATGGTAGCCAC

c-Rel CAACTGGAGAAGGAAGATTCA TGGAACTCCTGAAGACCTG

Nr4a1 ATGCCTCCCCTACCAATCTTC CACCAGTTCCTGGAACTTGGA

HPRT GGACTAATTATGGACAGGACTG GCTCTTCAGTCTGATAAAATCTAC