Bach2 Controls T Follicular Helper Cells by Direct Repression of Bcl-6 Annette Lahmann, Julia Kuhrau, Franziska Fuhrmann, Frederik Heinrich, Laura Bauer, Pawel Durek, Mir-Farzin This information is current as Mashreghi and Andreas Hutloff of September 30, 2021. J Immunol published online 4 March 2019 http://www.jimmunol.org/content/early/2019/03/01/jimmun ol.1801400 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 March 4, 2019, doi:10.4049/jimmunol.1801400 The Journal of Immunology

Bach2 Controls T Follicular Helper Cells by Direct Repression of Bcl-6

Annette Lahmann,* Julia Kuhrau,* Franziska Fuhrmann,* Frederik Heinrich,† Laura Bauer,* Pawel Durek,† Mir-Farzin Mashreghi,† and Andreas Hutloff*

T follicular helper (Tfh) cells are a specialized T cell subset that regulates the long-lived production of highly specific Abs by B cells during the germinal center (GC) reaction. However, the transcriptional network sustaining the Tfh cell phenotype and function is still incompletely understood. In this study, we identify the Bach2 as a central negative regulator of Tfh cells. Ectopic overexpression of Bach2 in murine Tfh cells resulted in a rapid loss of their phenotype and subsequent breakdown of the GC response. Low Bach2 expression levels are required to maintain high expression of the signature cytokine IL-21, the coinhibitory TIGITand the transcriptional repressor Bcl-6. In stark contrast to the regulatory network in GC B cells, Bach2 in Tfh cells

is not coexpressed with Bcl-6 at high levels to inhibit the antagonizing factor Blimp-1, but suppresses Bcl-6 by direct binding to the Downloaded from promoter. These data reveal that by replacing an activating complex of Batf and Irf-4 at the Bcl-6 promoter, Bach2 regulates the transcriptional network of Tfh cells in a different way, as in GC B cells. The Journal of Immunology, 2019, 202: 000–000.

n effective humoral immune response requires cognate The specific signals maintaining the Tfh cell phenotype in later interaction of Ag-specific B and T cells (1). In the ger- phases of the GC reaction are poorly understood. Furthermore, A minal center (GC) reaction, B cells proliferate and un- little is known how the Tfh cell master transcription factor Bcl-6 http://www.jimmunol.org/ dergo affinity maturation under the control of a specialized T cell itself is regulated. subset of T follicular helper (Tfh) cells (2). Consequently, Tfh cells Much about the molecular regulation of Tfh cells can be learned are the prerequisite for the generation of high affinity memory from B cells, because the transcriptional networks defining the GC B cells and long-lived plasma cells. Therefore, manipulation of the B cell and Tfh cell phenotype share many similarities (5). The prime Tfh response is of particular clinical interest to either promote the example is Bcl-6, which has a key role for both the development of generation of protective Abs during vaccination or to eliminate GC B cells and Tfh cells. Bcl-6 inhibits the expression of Blimp-1, harmful Abs in autoimmune diseases or allergy (3). which is the transcription factor driving alternative fate differenti- Tfh cells provide B cell help by expression of CD40L and IL-21. ation of B and T cells to plasma cells and non-Tfh effector cells, They are characterized by high expression of the chemokine re- respectively (5). Another important transcription factor to maintain by guest on September 30, 2021 ceptor CXCR5, the coinhibitory receptor PD-1, and the lineage- the phenotype of GC B cells is Bach2 (6). Bach2 is highly defining transcription factor Bcl-6. The development of Tfh cells expressed in GC B cells and cooperates with Bcl-6 to suppress from naive CD4+ T cells is a multistep process that requires inter- expression of Blimp-1, thereby preventing terminal differentiation action with different cell types and only takes place in the complex of GC B cells into plasma cells (7–9). For many years, Bach2 was anatomical structure of secondary lymphoid organs (4). Although considered to be a B cell–specific transcription factor (10). Only our knowledge about molecules regulating Tfh cells has sub- recently, Bach2 was also demonstrated to be expressed in T cells to stantially expanded in past years, many details are still ill defined. promote the development of regulatory T cells and prevent differ- entiation of Th2 CD4+ T cells (11–13). However, whether Bach2 has a function also for Tfh cells is still unknown. *Chronic Immune Reactions, German Rheumatism Research Center, 10117 Berlin, In this study, we show that Bach2 is highly differentially Germany; and †Therapeutic Regulation, German Rheumatism Research Center, expressed in Tfh versus non-Tfh effector cells. However, in striking 10117 Berlin, Germany contrast to GC B cells, Tfh cells are characterized by low ex- ORCIDs: 0000-0001-7521-7472 (A.L.); 0000-0001-9567-7273 (F.F.); 0000-0001- pression of Bach2, indicating that the function of this transcription 6097-5422 (F.H.); 0000-0002-1435-8121 (L.B.); 0000-0002-6179-0670 (P.D.); 0000-0002-8015-6907 (M.-F.M.); 0000-0002-0572-8151 (A.H.). factor in T cells is completely different. Ectopic overexpression of Received for publication October 16, 2018. Accepted for publication January 27, Bach2 in already-differentiated Tfh cells results in rapid loss of 2019. their phenotype, identifying Bach2 as a key molecule to maintain This work was supported by Deutsche Forschungsgemeinschaft Grants HU 1294/7-1 Tfh cells in later phases of the GC reaction. Bach2 overexpression and TRR 130 P23 (to A.H.) and the European Regional Development Fund (Programme in Tfh cells inhibits a specific set of , including IL-21, TIGIT, 2014–2020, Europa¨ische Fonds fu¨r Regionale Entwicklung 1.8/11, Deutsches Rheuma- Forschungszentrum) to F.H. and M.-F.M. and Bcl-6. Furthermore, we can demonstrate that Bach2 directly The original transcriptome data presented in this article have been submitted to the represses Bcl-6 by binding in the promotor region and that inhi- Omnibus (www.ncbi.nlm.nih.gov/projects/geo/) under accession bition of Bcl-6 promoter activity resulted from displacing an ac- numbers GSE118821 and GSE118822. tivating complex of Irf-4 and Batf. Address correspondence and reprint requests to Dr. Andreas Hutloff, German Rheumatism Research Center, Charite´platz 1, 10117 Berlin, Germany. E-mail address: [email protected] Materials and Methods The online version of this article contains supplemental material. Mice Abbreviations used in this article: ChIP, chromatin immunoprecipitation; GC, ger- minal center; KO, knockout; NP, nitrophenol; Tfh, T follicular helper. TCR-transgenic OT-II (14) and Smarta (15) mice were additionally crossed to B6PL mice (Jax stock 000406; Thy-1.1+) to track cells in adoptive Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 transfer experiments. For the tamoxifen-inducible retroviral overexpression

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801400 2 Bach2 IN T FOLLICULAR HELPER CELL REGULATION system, both strains were crossed to CreERT2 mice (16). For CRISPR/ for biotinylated Abs. Fc-receptors were blocked with 2.4G2 (anti-CD16/ Cas9-mediated knockout (KO), Smarta mice were crossed to Cas9-GFP 32). Abs were bought from commercial suppliers or were purified from transgenic mice (17). For analysis of Il21 expression, we used Smarta hybridoma supernatants and coupled to fluorophores by standard proce- CreERT2 mice crossed to Il21-IRES-FP635 reporter mice (18, 19). dures. To detect early apoptotic cells, a FITC-conjugated inhibitor of Nitrophenol (NP)–specific BCR knock-in B1-8i mice (20) were crossed to caspase-3 (CaspGLOW; eBioscience) was used according to the manu- k-L chain KO mice (21) to ensure NP-specificity of all B cells and Ly-5.1 facturer’s instructions. Intracellular staining for transcription factors and mice (Jax stock 002014; CD45.1+) for tracking of transferred cells. As apoptotic was performed using the Foxp3 Staining Buffer Set recipient mice for adoptive transfer experiments, C57BL/6 or CD28 KO from eBioscience. DNA-incorporated BrdU was labeled with the APC mice (22) were used. All mice were bred under specific pathogen-free BrdU Flow Kit (BD Biosciences) according to the manufacturer’s in- conditions in the animal facility of the Federal Institute for Risk Assess- structions. To discriminate dead cells, either DAPI was added to live cells ment, Berlin. Female mice were used for experiments at an age of 8–16 immediately before analysis or cells were stained on ice for 25 min with wk. Animal handling and experiments were conducted according to the 84 nM Alexa 700 succinimidyl ester (Thermo Fisher Scientific) prior to German animal protection laws and approved by the responsible govern- fixation. Cells were analyzed on a BD LSRFortessa (BD Biosciences). mental authority (Landesamt fu¨r Gesundheit und Soziales). Analysis gates were set on live cells defined by scatter characteristics and exclusion of DAPI- or Alexa 700-positive cells. Data were analyzed with Retroviral overexpression and CRISPR/Cas9-mediated KO FlowJo Software (Tree Star). For constitutive overexpression, cDNAs for Bach2, Tigit, Irf4, or Bcl6, followed by an IRES site and either eGFP or mAmetrine were cloned into Quantitative RT-PCR and next-generation sequencing the retroviral expression vector pMSCV (Takara Bio). For inducible RNA was isolated using the RNeasy Micro Kit (Qiagen) or Roche High overexpression, Bach2 was cloned behind a loxP-flanked dsRed stop cassette Pure RNA Isolation Kit, and quality was checked on an Agilent 2100 (23). For KO of genes, guide RNAs were selected using CrispRGold ver- Bioanalyzer. For quantitative RT-PCR, cDNA was synthesized with the sion 1.1 (https://crisprgold.mdc-berlin.de). Corresponding oligonucleotides Applied Biosystems High Capacity cDNA Kit. Gene expression was analyzed Downloaded from (Supplemental Table I) were cloned into the BbsI site of a pMSCV-based on an Applied Biosystems 7500 Real Time PCR System using TaqMan gene guide RNA vector (17). Viral particles were generated in HEK293T cells by expression assays for murine Bach2 (Mm00464379_m1), human Bach2 calcium phosphate transfection using the packaging plasmids pECO and (Hs00222364_m1), and Hprt for standardization (Mm01545399_m1 and pCpG. In vitro stimulated OT-II or Smarta T cells (OVA323–339 or LCMV Hs02800695_m1). GP61–80 peptide for 24 h) were infected with retroviral supernatants by For gene expression analysis by next-generation sequencing, either the 3 centrifugation for 90 min at 400 g,32˚C,and8mg/ml polybrene. Cells TruSeq Stranded Total RNA Library Prep Kit (Illumina) or the SMART-Seq were cultured for additional 40 h and sorted for fluorescent – v4 Ultra Low Input RNA Kit (Clontech Laboratories) and Nextera XT expressing cells on a FACSAria II flow sorter (BD Biosciences). Library Preparation Kit (Illumina) were used according to the manufacturer’s http://www.jimmunol.org/ To determine the efficiency of CRISPR/Cas9-mediated KO, some cells instructions. Paired-end sequencing with 2 3 75 nt was performed on a were cultured for additional 24 h and sorted for Cas9-GFP and guide RNA– NextSeq500 (Illumina), and raw sequence reads were mapped to the mouse expressing T cells to prepare genomic DNA. The genomic region around GRCm38/mm10 genome with TopHat2 (25) in very-sensitive settings for the guide RNA binding site was amplified by PCR using the primers listed Bowtie2 (26). Gene expression was quantified either by HTSeq (27) for in Supplemental Table II. PCR products were cloned into pJet1.2 (Thermo total RNA or featureCounts (28) for mRNA and analyzed using DESeq2 Fisher Scientific) and subjected to Sanger sequencing. Sequences from at (29). Differential expression between TFH and non-TFH or empty control least 11 individual clones were aligned to the original DNA sequence. vector and Bach2 was regarded as significant when the adjusted p value was #0.05 and the fold change was $1.3. Adoptive transfer experiments

5 Luciferase reporter assay A total of 2.5 3 10 retrovirally infected transgenic T cells were adoptively by guest on September 30, 2021 transferred by i.v. injection into C57BL/6 mice. For some experiments, EL4 T cells (American Type Culture Collection, TIB-39; tested for my- 1 3 106 B1-8i B cells were cotransferred. Recipient mice were injected s.c. coplasma) were transfected by nucleofection (Amaxa Cell Line Nucleofector at the tailbase with cognate Ag (50 mg NP-conjugated OVA for OT-II Kit L; Lonza Biosciences) with the pGL3 basic luciferase plasmid containing T cells or 20 mg NP and GP61–80 coupled to mouse serum albumin for the Bcl-6 promotor (bp +51 to 2947), internal control pRL-TK Renilla Smarta T cells) in complete Freund’s adjuvant (Sigma-Aldrich). To induce plasmid (both from Promega) and the Bach2, Batf, and Irf4 coding sequence nuclear translocation of CreERT2 recombinase, mice were injected i.p. with in pcDNA3.1 (Thermo Fisher Scientific). Luciferase activity was measured 1.5 mg tamoxifen (Sigma-Aldrich) dissolved in sunflower oil. For deter- on a SpectraMax i33 microplate reader (Molecular Devices) after 24 h using mination of cell proliferation, 1 mg bromodeoxyuridine (BrdU; Sigma- the dual luciferase assay system (Promega). Data were normalized to the Aldrich) was injected i.p. 12 and 24 h after tamoxifen administration. To activity of Renilla luciferase. block lymphocyte egress, 30 mg FTY720 (Sigma-Aldrich) was administered i.p. Draining inguinal lymph nodes were isolated at indicated time points and Chromatin immunoprecipitation mashed through 70-mm sieves to prepare single-cell suspensions for flow Smarta T cells were stimulated with GP61–80 peptide under Tfh-polarizing cytometric analysis. Cells were counted with a Guava EasyCyte capillary flow conditions in the presence of IL-21, IL-6 (BioLegend), a–IL-12 (17.8), cytometer and ViaCount solution (Merck Millipore). Transgenic T cells were 2 2 a–IL-4 (11B11), a–IFN-g (AN18.14), and a–TGF-b (1D11) as described identified in flow cytometry as CD4+B220 CD8 Thy-1.1+. Transgenic B cells 2 2 2 (30) and retrovirally infected with either FLAG-tagged or control Irf-4 in were defined as CD19+CD3 CD8 CD45.2 CD45.1+. combination with a Bach2 expression or empty control vector. Chromatin Sorting of Ag-specific Tfh cells immunoprecipitation (ChIP) was performed with sorted double-transduced cells as described previously (23). Briefly, 1 3 106 cells were cross-linked Sorting of transgenic murine Tfh and non-Tfh cells was performed as with 1% formaldehyde, followed by chromatin shearing by sonication and described (24). In brief, transgenic T cells from inguinal lymph nodes were immunoprecipitation with an anti-FLAG Ab (clone M2; Sigma-Aldrich). first enriched by magnetic sorting using Thy-1.1 MicroBeads (Miltenyi Enrichment at the Bcl-6 promoter was measured by SYBR Green–based Biotec), followed by sorting on an BD FACSAria II flow sorter (BD RT-PCR using the primer pair 59-GAGGGAGAGTGCGCTTTGC-39 and Biosciences) for live B2202CD82CD4+Thy-1.1+CD44high and either 59-GGAGCCGAGTTTATGGG-TAGGA-39. Binding was determined by CXCR5/PD-1 double-positive or negative cells. Human tonsils from pa- calculating the percent of input relative to the nonflagged control. tients undergoing routine tonsillectomy were obtained after informed consent. Mononuclear cells were prepared by mechanical disruption of Statistical analysis tissue and Ficoll density centrifugation. Tfh and non-Tfh cells were sorted + + + 2 Experimental groups consisted of four to seven individual mice per as live CD3 CD4 CD45RO CD45RA and ICOS/CXCR5 double positive group (randomly assigned). Sample size was predetermined using G*Power or double negative, respectively. 3 (31). Data are presented either showing the mean and results from single Flow cytometry animals or the mean with error bars (SEM). Flow cytometry plots are al- ways shown as concatenated data from all animals in the group. No data Single-cell suspensions from lymph nodes were stained with different exclusion criteria were used. Data were analyzed using GraphPad Prism 7. combinations of mAbs (Supplemental Table III) conjugated to Biotin, For samples with normal distribution according to Shapiro–Wilk, differ- FITC, PE, PerCP, PE-Cy7, Alexa Fluor 647, Alexa Fluor 700, APC-Cy7, ences between groups were calculated using a two-tailed Student t test or Pacific Blue, Brilliant Violet 421, Brilliant Violet 605, Brilliant Violet 711, one-way ANOVA with Bonferroni multiple comparison test. Otherwise, or Brilliant Violet 785. Streptavidin/PE-Cy7 was used as secondary reagent data were analyzed by Mann–Whitney U test. The Journal of Immunology 3

Results or follicular regulatory T cells in our system. However, intracel- Tfh cells are characterized by low expression of Bach2 lular staining for Foxp3 in transgenic T cells transfected with the Bach2 overexpression vector did not reveal any differences com- To identify novel factors that might be important to maintain the pared with the control group (Supplemental Fig. 1). phenotype of already-differentiated Tfh cells, we analyzed the To confirm the Bach2-mediated regulation of Tfh cells by a transcriptomes of Ag-specific Tfh and non-Tfh effector cells. To reciprocal approach, we tested whether the KO of Bach2 would be this end, TCR-transgenic cells expressing the congenic marker able to increase the numbers of Tfh cells. We used T cells from Thy-1.1 were transferred into syngenic Thy-1.2+ mice, which were Cas9 and Smarta TCR–transgenic mice and retroviral delivery of a immunized s.c. with cognate Ag. On day 8, the maximum of the GC Bach2-specific guide RNA (Fig. 2D). This system was demon- response in this system, TCR-transgenic, OVA-specific, Thy-1.1+ strated to be highly efficient, with KO frequencies up to 90% (17). OT-II T cells were reisolated from draining lymph nodes and sorted Draining lymph nodes from recipients of transgenic T cells were into Tfh and non-Tfh cells according to expression of CXCR5 and analyzed by flow cytometry on day 6. In line with the reduced Tfh PD-1. Next-generation sequencing identified more than 980 genes frequencies upon Bach2 overexpression, CRISPR/Cas9-mediated with a highly significant (p , 0.01) and more than 1.3-fold dif- KO of Bach2 conversely resulted in increased frequencies of transgenic ferential expression between CXCR5/PD-1 double-positive versus Tfh cells in vivo (Fig. 2E). double-negative cells. Because of their key function for cell subset differentiation, we Bach2 does not regulate Tfh cell proliferation, survival, focused our analysis on the gene family of transcription factors or migration

(Fig. 1A). Among the 29 differentially expressed transcription Downloaded from There are several possible explanations for the reduction of Tfh factors, Bach2 was of special interest because of its already- cells upon Bach2 overexpression: 1) decreased survival of Tfh reported major role for B cells during the GC reaction. Bach2 cells, 2) decreased proliferation, 3) migration out of the draining reads were almost four times lower in Tfh compared with non-Tfh lymph node, or 4) loss of the Tfh cell phenotype. To address the effector cells (Fig. 1B). Similar results were obtained for Tfh cells effect of Bach2 overexpression on apoptosis, we determined the isolated from human tonsil; Bach2 expression in CXCR5/ICOS frequency of Smarta TCR–transgenic T cells positive for active double-positive Tfh cells was even 12 times lower than in double- caspase-3, a central protease involved in the apoptosis pathway. http://www.jimmunol.org/ negative cells (Fig. 1C). It has been previously reported that the No difference was observed between the Bach2 overexpression KO of Bach2 resulted in a preferential differentiation of CD4+ and control group 36 h after induction of Bach2 overexpression T cells toward a Th2 phenotype (13). Therefore, we analyzed (Fig. 3A). In particular, the frequencies of active Caspase-3– Bach2 expression in different T helper subsets. Whereas Bach2 positive Tfh cells were almost identical. As a second readout for expression in Th1 and Th17 cells was more than two times higher cell survival, the expression ratio of the antiapoptotic factor Bcl-2 than in naive T cells, a downregulation by more than 85% was and the proapoptotic factor Bim (Bcl2L11) was determined by observed in Th2 and Tfh cells, indicating that Th subsets differ- flow cytometry. Again, no differences were observed between the entially rely on Bach2 and that Bach2 expression seems to be two groups, neither for total transgenic T cells nor for Tfh cells critical for Th2 and Tfh cells (Fig. 1D). (Fig. 3B). Therefore, the reduced number of Tfh cells is not the by guest on September 30, 2021 This low expression of Bach2 in Tfh cells was a highly unex- result of enhanced apoptosis. pected finding, because in B cells, Bach2 is known to directly The proliferation of Ag-specific T cells was assessed by incor- repress the transcription factor Blimp-1, which itself antagonizes poration of the thymidine-analogue BrdU. BrdU application was Bcl-6 (5). Consequently, high Bach2 expression levels in GC started together with tamoxifen-induced Bach2 overexpression, and B cells prevent Blimp-1 expression and ensure Bcl-6 expression mice were analyzed 36 h later by flow cytometry. BrdU incorporation (32). In contrast, Tfh cells seem to downregulate Blimp-1 inde- was the same between all transgenic T cells or Tfh cells overexpressing pendent of Bach2, indicating that Bach2 in Tfh cells functions in a Bach2 and control cells (Fig. 3C), ruling out any proliferative effects way completely different from the regulatory network in B cells. as explanation for the reduced numbers of Tfh cells. Overexpression of Bach2 results in loss of the Tfh cell To test whether Bach2 overexpression directs Tfh cells to se- phenotype and collapse of the GC reaction lectively migrate out of the draining lymph node, we treated re- cipient mice with the sphingosine-1-phosphate receptor antagonist To functionally analyze the role of Bach2 for Tfh cells, we ectopically + FTY720 parallel to tamoxifen-induced Bach2 overexpression. This overexpressedBach2inalready-differentiated Tfh cells. Thy-1.1 compound effectively blocks lymphocyte egress from lymph OT-II T cells expressing tamoxifen-inducible Cre recombinase nodes, which was confirmed by the presence of only very low were transfected with a retroviral vector containing Bach2 cDNA numbers of transgenic T cells in the peripheral blood and their behind a loxP-flanked dsRed stop cassette. T cells were trans- accumulation in the draining lymph node. At the same time, the ferred into recipient mice, and tamoxifen was applied 4 d after frequency of transgenic T cells present in the lymph node remained immunization when Tfh cells had already developed in vivo the same after Bach2 overexpression compared with the control + + (Fig. 2A). Analysis of Ag-specific CXCR5 PD-1 Tfh cells in group (Fig. 3D). Importantly, FTY720 treatment was not able to draining lymph node revealed that their frequency and absolute prevent the Bach2 overexpression-induced loss of CXCR5+/PD-1+ Tfh number were decreased by almost 70% only 3 d after tamoxifen- cells (Fig. 3E). induced Bach2 overexpression (Fig. 2B). The functional conse- Taken together, these experiments show that neither increased quences of the Bach2-mediated loss of Tfh cells for the GC re- apoptosis, reduced proliferation, nor egress out of the lymph node action were analyzed by cotransferring B cells specific for NP and cause the loss of Tfh cells upon Bach2 overexpression. Instead, it immunization of recipient mice with cognate Ag. Within 6 d of can be concluded that low levels of Bach2 are required to prevent Bach2 overexpression in Ag-specific T cells, the number of Ag- dedifferentiation of Tfh cells into non-Tfh effector cells. specific PNA+GL7+ GC B cells was reduced by 80% (Fig. 2C). Because Bach2 has been previously described to be important for Bach2 represses a specific set of Tfh cell–related genes the homeostasis of regulatory T cells (11, 12), we tested whether To identify downstream targets of Bach2, which directly interfere Bach2 overexpression might increase the frequency of regulatory with the stability of the Tfh cell phenotype, we performed global 4 Bach2 IN T FOLLICULAR HELPER CELL REGULATION

transcriptome analysis. Based on expression of CXCR5 and PD-1, Smarta TCR–transgenic Tfh and non-Tfh cells were sorted ex vivo 18 h after tamoxifen-induced Bach2 overexpression. At that time, transgenic Bach2 protein levels could be expected to be already high. However, the Tfh phenotype, as defined by expression of CXCR5 and PD-1, had not changed yet, enabling us to define early events of the Tfh phenotype loss and to find genes directly regulated by Bach2. The transcriptome of Tfh cells transduced with the Bach2 overexpression vector was compared with cells transfected with control vector by RNA sequencing. Because Bach2 is known as a transcriptional repressor, we focused on genes that were downregulated in Tfh cells by Bach2 overexpression. To further narrow down the number of 110 genes meeting this first criterion, we compared them with genes upregulated in Tfh versus non-Tfh cells, meaning they are likely to promote the Tfh cell phenotype. This finally resulted in a list of only 16 genes that were both Tfh signature genes and downregulated by Bach2 (Fig. 4A). The top three hits of this list were Bcl-6, the coinhibitory cell

surface receptor TIGIT, and IL-21, the main effector cytokine Downloaded from of Tfh cells. Further genes suppressed by Bach2 included heme oxygenase 1 (Hmox1), which is able to degrade heme. As heme can inactivate Bach2 (33), the Bach2-mediated inhibition of Hmox1 appears to create a negative feedback loop. Expression of the three top hits in the list (Bcl-6, TIGIT, and

IL-21) was further analyzed by flow cytometry (Fig. 4B). This http://www.jimmunol.org/ analysis did not only confirm the highly differential expression of all three molecules between Tfh and non-Tfh cells, but also demonstrated their suppression by Bach2. Bcl-6 is the major target of Bach2 in Tfh cells To functionally test whether suppression of one of our candidate genes would result in a similar loss of the Tfh cell phenotype as observed with Bach2 overexpression, we performed CRISPR/

Cas-mediated KO of the top genes from the list shown in by guest on September 30, 2021 Fig. 4A. T cells from Smarta Cas9 transgenic mice were retrovirally infected with appropriate guide RNAs (see Fig. 2D). Successful deletion of genes was verified by sequencing of the targeted region (Supplemental Table II). Transduced cells were transferred into recipient mice, and the frequency of Ag-specific Tfh cells was determined on day 6 after immunization. Only the KO of Bcl-6 resulted in a strong reduction in Tfh cells, whereas KO of the remaining genes did not have any effect (Fig. 5A). To confirm this result, we additionally tested whether overexpression of Bcl-6 together with Bach2 could rescue the Bach-induced loss of the Tfh cell phenotype. In this test, we also included TIGIT, which was among the most strongly Bach2-downregulated targets and is a mol- ecule with a long-known differential expression but still undefined function for Tfh cells (34, 35). The Bach2 overexpression plasmid was cotransfected with either Bcl-6 or TIGIT overexpression plasmids into Smarta T cells. Both plasmids contained different fluorescence reporters (GFP and mAmetrine, respectively), so that single- and double-transfected cells could be easily discriminated. Transduced

populations was analyzed by RNA sequencing. (A) The heatmap indicates relative gene expression of differentially expressed transcription factors. (B) The number of normalized reads of Bach2 in Tfh and non-Tfh cells. (C) Bach2 expression in Tfh and non-Tfh cells from human tonsil was ana- lyzed by quantitative RT-PCR. (D) OT-II T cells were stimulated in vitro under different Th subset–polarizing conditions (rIL-12 and anti–IL-4 for FIGURE 1. Tfh cells are characterized by low Bach2 expression. (A and Th1, rIL-4 and anti–IL-12/IFN-g for Th2, rTGF-b1, IL-6, IL-23, and anti– B) Tfh and non-Tfh cells from an adoptive transfer of OT-II TCR-transgenic IL-4/IFN-g for Th17). Tfh cells were isolated ex vivo as described above. cells were sorted from draining lymph nodes (day 8 after immunization) Bach2 expression was analyzed by quantitative RT-PCR. ****p , 0.0001, according to CXCR5 and PD-1 expression. Gene expression in both **p , 0.01. ns, nonsignificant. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 2. Tfh cells require low Bach2 expression to maintain their phenotype.(A–C) OT-II CreERT2 T cells (Thy-1.1+) were retrovirally infected with an inducible Bach2 overexpression vector or empty control vector (EV) and transduced cells sorted for transfer into recipient mice (Thy-1.2+) that were immunized s.c. with NP-OVA. Bach2 overexpression was induced by tamoxifen on day 4. (B) Ag-specific T cells were analyzed by flow cytometry for a CXCR5+/PD-1+ Tfh phenotype on day 7. (C) NP-specific B1-8i B cells were cotransferred with transduced OT-II CreERT2 T cells into CD28 KO recipient mice, which cannot generate endogenous Tfh cells. Ag-specific B1-8i B cells were analyzed for a GL7+/PNA+ GC phenotype on day 10. Data are rep- resentative for at least two independent experiments with six to seven animals per group. (D and E) Smarta Cas9-GFP T cells were retrovirally transduced with a Bach2-specific guide RNA or an EVand transferred into C57BL/6 recipients. Ag-specific T cells were analyzed for a Tfh phenotype on day 6. Pooled data from two independent experiments together with nine animals per group. **p , 0.01, *p , 0.05. cells were transferred into recipient mice, which were analyzed 6 d phenotype (Fig. 5B). This clearly shows that TIGIT is not the Bach2 after immunization for Ag-specific Tfh cells. As expected, single downstream target responsible for the observed loss of the Tfh cell overexpression of Bach2 resulted in an almost complete loss of Tfh phenotype. However, this does not rule out that the Bach2-induced cells (Fig. 5B). With the constitutive overexpression vector, this effect downregulation of TIGIT still might have a role for specific effector was even more pronounced than with the inducible vector (compare functions of Tfh cells. Fig. 2B). As shown before (see Fig. 4B), Bach2 overexpression also For overexpression of Bcl-6, the results of the rescue assay were resulted in a substantial downregulation of TIGIT on the cell surface very different. As expected, overexpression of Bcl-6 alone resulted (Fig. 5B). Simultaneous overexpression of TIGIT restored the ex- in a substantial increase in Tfh cells, with almost 70% of all pression almost to the same level observed in the empty vector control. transgenic Smarta T cells displaying a CXCR5+PD-1+ Tfh cell However, TIGIT overexpression was not able to rescue the Tfh cell phenotype. Vice versa, Bach2 overexpression reduced Tfh cell 6 Bach2 IN T FOLLICULAR HELPER CELL REGULATION Downloaded from http://www.jimmunol.org/

FIGURE 3. Loss of Tfh cells is not the result of enhanced apoptosis, reduced proliferation, or migration out of the lymph node. Smarta CreERT2 T cells were retrovirally transduced with an inducible Bach2 overexpression or empty control vector, and dsRed+ cells were sorted for adoptive transfer into C57BL/6 recipients. Four and a half days after s.c. immunization, Bach2 overexpression was induced by tamoxifen application. (A and B) Apoptosis of Ag- specific total (Thy-1.1+) T cells or Tfh cells (Thy-1.1+CXCR5+PD-1+) was assessed by flow cytometry on day 6 by staining for (A) active caspase-3 and (B) intracellular staining for Bcl-2 and Bim. (C) To determine proliferation, BrdU was given on day 5 and 5.5, and BrdU incorporation of Ag-specific T cells by guest on September 30, 2021 and Tfh cell was analyzed on day 6. (D and E) To block lymphocyte egress from the draining lymph node, recipient mice were treated with FTY720 on day 5 and analyzed by flow cytometry on day 6 for d) the frequencies of Ag-specific T cells in blood and lymph node and (E) frequencies of CXCR5+PD-1+ Tfh cells. Data are representative for two independent experiments with five to six animals per group. **p , 0.01, *p , 0.05. ns, nonsignificant. frequencies almost 10-fold compared with the empty vector con- of gene expression. Interestingly, it has been shown that Batf binds trol. Importantly, simultaneous Bach2 and Bcl-6 overexpression to the Bcl6 gene in the same 1-kb region upstream of transcription resulted in a highly significant rescue of the Tfh cell population start as Bach2 to regulate gene expression (38). At the same time, (Fig. 5C). The partial recovery of Tfh cell frequencies goes in line it has been demonstrated that Irf-4 promotes Bcl-6 expression and with the incomplete recovery of Bcl-6 expression levels in the the Tfh cell differentiation program (39, 40). Therefore, compet- Bach2/Bcl-6 double-overexpression group. The rescue of the Tfh itive gene regulation similar to as demonstrated for the Il4 gene cell population by additional Bcl-6 overexpression shows that is conceivable for the suppression of Bcl-6 by Bach2. Indeed, suppression of Bcl-6 by Bach2 is the mechanism for how Bach2 analysis of ChIP sequencing data (https://www.ncbi.nlm.nih.gov/ regulates Tfh cells. geo/query/acc.cgi?acc=GSE39756) revealed that both Batf and Irf4 bind to the same region in the Bcl-6 promotor (Fig. 6C). To Bach2 directly binds to the Bcl-6 promotor investigate whether Bach2 expression resulted in a replacement of To determine whether Bach2 directly binds in the Bcl-6 promotor, Irf-4 at this site, Irf-4–FLAG-expressing T cells were additionally we used publicly available Bach2 ChIP sequencing data from transduced with a Bach2 expression vector and cultured under Tfh in vitro–stimulated, wild-type, and Bach2-deficient CD4+ Tcells cell–promoting conditions; in the presence of IL-21, IL-6 and Abs (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65084). against IL-12, IL-4, TGF-b, and IFN-g as previously described These data showed a strong enrichment for Bach2 binding within (30). Analysis by ChIP and RT-PCR confirmed that Irf-4 binding a region of 1 kb immediately in front of the transcriptional start to the Batf/Bach2 binding site in the Bcl-6 gene was abrogated by (Fig. 6A). Bach2 is known to bind to AP-1 motifs (10), and bioinformatic Bach2 overexpression, indicating that Bach2 replaces Irf-4 to form analysis revealed that this region contains several potential Bach2 binding an inhibiting complex with Batf instead (Fig. 6D). sites (Fig. 6B). To finally demonstrate that Bach2 not only binds to, but also Bach transcription factors typically form heterodimers with functionally suppresses Bcl-6 transcription, a luciferase assay members of the transcription factor family (MafF, with the Bcl-6 promotor was performed in EL4 T cells. Cells MafG, and MafK) (36). However, recently, it has been shown that were transfected with expression plasmids for Batf and Irf4 and Bach2 can also form a dimer with another AP-1 family tran- either an empty control vector or a Bach2 expression vector to scription factor, Batf, which binds to palindromic AP-1 motives in test whether Bach2 can replace Irf4 in the activating Batf/Irf-4 the IL-4 promotor (37). The Bach2/Batf dimer displaced an acti- complex. Indeed, Bach2 cotransfection resulted in a signifi- vating complex of Batf, Irf4, and JunD, which resulted in a shutdown cant reduction of luciferase activity (Fig. 6E). Importantly, this The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 4. Bach2 regulates a specific set of Tfh cell–associated genes. Smarta CreERT2 T cells were retrovirally transduced with an inducible Bach2 overexpression or empty control vector, and sorted dsRed+ cells adoptively transferred into C57BL/6 recipients. Five days after s.c. immunization, Bach2 overexpression was induced by tamoxifen application. Eighteen hours later, Ag-specific Tfh cells (pooled lymph nodes from 15 mice) were sorted and analyzed by RNA sequencing. (A) The Venn diagram reveals 16 genes that are higher expressed in Tfh versus non-Tfh cells and, at the same time, suppressed by Bach2. The 16 genes are listed below in a heat map that shows normalized reads of the indicated samples. (B) Smarta CreERT2 IL-21/FP635 T cells were retrovirally transduced with an inducible Bach2 overexpression or empty control vector and adoptively transferred into C57BL/6 recipients. Tamoxifen was given on day 4.5 after immunization, and transgenic T cells from draining lymph nodes were analyzed by flow cytometry for Bcl-6, TIGIT, and IL-21 expression on day 6. Data are shown from a representative experiment out of two with six mice per group. ****p , 0.0001, ***p , 0.001. displacement effect was only visible when EL4 T cells were ad- Bach2 KO mice had substantially reduced Foxp3+ regulatory ditionally transfected with Batf and Irf4. These data suggest that T cells, which resulted in an autoinflammatory phenotype (11, 12). Bach2 functions as a suppressor of Bcl-6 expression by displacing Moreover, a second study showed increased IL-4 production by an activating Batf/Irf4 complex in the Bcl-6 promotor. effector T cells, indicating a specific function of Bach2 for Th2 cells (13). However, other Th subsets have not been analyzed so far. Discussion Our data now indicate that Bach2 has much more complex and Bach2 has been known as an important transcriptional regulator of specific roles for different Th cell subsets. The exceptionally high B cells for more than 20 y. However, only recently it was discovered expression of Bach2 in Th1 and Th17 cells indicates that these two that Bach2 is also expressed in T cells (11–13). These studies Th subsets rely on high Bach2 expression similarly to regulatory demonstrated that Bach2 has a critical role for regulatory T cells. T cells. To the contrary, Tfh cells show very low expression levels 8 Bach2 IN T FOLLICULAR HELPER CELL REGULATION Downloaded from http://www.jimmunol.org/ by guest on September 30, 2021

FIGURE 5. Bcl-6 is the critical downstream target of Bach2 for maintenance of the Tfh cell phenotype. (A) Smarta Cas9-GFP T cells were retrovirally transduced with guide RNAs for the indicated genes and sorted and transferred into C57BL/6 recipients. Ag-specific T cells were analyzed for a Tfh cell phenotype on day 6 after immunization. Pooled data from two independent experiments together with nine mice per group. (B and C) Smarta T cells were retrovirally transduced with different combinations of GFP or mAmetrine-tagged vectors expressing Bach2 and either TIGIT or Bcl-6. Infected cells were either directly transferred (B) or sorted (C) before transfer into C57BL/6 recipients. Ag-specific GFP+ mAmetrine+ T cells from draining lymph nodes were analyzed for a Tfh cell phenotype and expression of (B) TIGIT and (C) Bcl-6 on day 6 after immunization. Representative data from one out of two experiments. ****p , 0.0001, ***p , 0.001, **p , 0.01, *p , 0.05. ns, nonsignificant. The Journal of Immunology 9

FIGURE 6. Bach2 directly binds to the Bcl-6 promo- tor. (A) Analysis of ChIP sequencing data (https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65084) for Bach2 binding in the Bcl-6 promotor. Normalized reads of in vitro stimulated and anti-Bach2 precipitated wild-type (WT; black) and Bach2 KO (gray) CD4+ T cell samples were mapped to the reference genomic sequence of Bcl-6 using Integrative Genomics Viewer (IGV) browser (56). Increased numbers of reads are visible as peaks, indicating Bach2 binding within the Bcl-6 promoter. (B) Bach2 binding sites (arrows) in the Bcl-6 promotor were predicted using rVISTA (57). (C) Visualization of ChIP sequencing data (https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE39756) of Batf KO, Irf4 KO (black), or WT (gray) CD4 T cells cultured in the presence of IL-21 and precipitated with an anti-Batf or anti–Irf-4 Ab. (D) Smarta T cells were stimulated under Tfh cell–promoting conditions and Downloaded from retrovirally transduced with an Irf-4/FLAG or WT Irf expression vector in combination with a Bach2 or empty control vector. Double-transduced cells (eGFP+mAmetrine+) were sorted. ChIP was performed with an anti-Flag Ab, followed by RT-PCR using a primer pair spanning the binding site determined in (A)and(C). Binding was deter- http://www.jimmunol.org/ mined by calculating the percent of input relative to the nonflagged control. (E) EL4 cells were transfected with a luciferaseexpressionplasmiddrivenbytheBcl-6promoter together with a Bach2 or empty expression vector in com- bination with or without Batf/Irf-4 expression vectors. Cells were stimulated with PMA and ionomycin for 5 h before analysis of luciferase activity (relative to Renilla) normalized to the empty vector control. Pooled data from two inde- pendent experiments. ****p , 0.0001. by guest on September 30, 2021

of Bach2 and functionally depend on these low levels to maintain binding to its promotor (42–44). In contrast, only STAT5 and their phenotype. This is in stark contrast to GC B cells, which share Foxo1 were demonstrated to suppress Bcl-6 by direct binding to Bcl-6 with Tfh cells as lineage-defining transcription factor regulatory elements (45, 46). With Bach2, we have identified a and require high Bach2 levels to maintain their phenotype by novel potent negative regulator of Bcl-6 that is critical for Tfh cell preventing upregulation of the antagonizing transcription factor maintenance. Blimp-1 (32). A KO of Bach2 does not only result in autoinflammatory How can the different function of Bach2 for the transcriptional symptoms in mice, but genetic polymorphisms within the Bach2 network in Tfh versus GC B cells be explained? Our data show that, locus were associated with several autoimmune and allergic dis- contrary to the regulation in GC B cells, Bach2 in Tfh cells does not eases in humans (47–54). Moreover, a recent study demonstrated regulate Bcl-6 indirectly via repression of Blimp-1. Instead, Bach2 that haploinsufficiency of Bach2 resulted in a multiorgan auto- is downregulated to prevent direct inhibition of Bcl-6 by binding to immune disease (55). Because autoimmune diseases are generally its promotor region. The mechanism responsible for the suppres- associated with increased frequencies of Tfh cells (3), it is sive effect of Bach2 appears to be the displacement of an activating tempting to speculate that reduced Bach2 levels might be related Batf/Irf4 complex at the same promoter element. This model also to this disease phenotype. provides an explanation why Bach2 is able to exert different Our investigation demonstrates that overexpression of Bach2 as functions in GC B cells compared with Tfh cells. In B cells, Irf4 a single factor is sufficient to completely reverse the phenotype of induces Bcl-6 expression not only in a complex with Batf, but is established Tfh cells, resulting in the breakdown of the GC re- also able to form activating complexes with PU.1, a factor that is action. This provides a mechanism for how a physiological Tfh cell not abundant in T cells (41). The PU.1/Irf4 complexes bind to response may be limited on a transcriptional level. Further in- distinct motifs in the promotor that are not susceptible to com- vestigations will be required to determine the external and internal petitive displacement by Bach2. Consequently, B cells are in contrast cues directing upregulation of Bach2 in a physiological immune to Tfh cells, able to coexpress Bach2 and Bcl-6. reaction. In the context of dysregulated Tfh cell responses, the Considering that Bcl-6 is the lineage-defining and functionally underlying cause of most Ab-driven autoimmune diseases, this is central transcription factor of Tfh cells, surprisingly little is known an interesting pathway to be targeted in therapeutic strategies. about its direct regulation. Although several transcription factors Affected patients require therapeutic strategies not only to prevent have been identified to promote early Tfh cell generation, only the de novo generation of Tfh cells, but rather to eliminate already Tcf-1 and Bob1 have been shown to directly regulate Bcl-6 by existing autoreactive Tfh cells. 10 Bach2 IN T FOLLICULAR HELPER CELL REGULATION

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