Bach2 Negatively Regulates T Follicular Helper Differentiation and Is Critical for CD4 + T Cell Memory

This information is current as Jianlin Geng, Hairong Wei, Bi Shi, Yin-Hu Wang, Braxton of September 27, 2021. D. Greer, Melanie Pittman, Emily Smith, Paul G. Thomas, Olaf Kutsch and Hui Hu J Immunol published online 10 April 2019 http://www.jimmunol.org/content/early/2019/04/10/jimmun

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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 10, 2019, doi:10.4049/jimmunol.1801626 The Journal of Immunology

Bach2 Negatively Regulates T Follicular Helper Cell Differentiation and Is Critical for CD4+ T Cell Memory

Jianlin Geng,* Hairong Wei,* Bi Shi,* Yin-Hu Wang,* Braxton D. Greer,† Melanie Pittman,* Emily Smith,* Paul G. Thomas,‡ Olaf Kutsch,† and Hui Hu*

T follicular helper (Tfh) cells are essential for germinal center B cell responses. The molecular mechanism underlying the initial Tfh cell differentiation, however, is still incompletely understood. In this study, we show that in vivo, despite enhanced non–Tfh cell effector functions, the deletion of transcription factor Bach2 results in preferential Tfh cell differentiation. Mechanistically, the deletion of Bach2 leads to the induction of CXCR5 expression even before the upregulation of Ascl2. Subsequently, we have identified a novel regulatory element in the murine CXCR5 locus that negatively regulates CXCR5 promoter activities in a Bach2- dependent manner. Bach2 deficiency eventually results in a collapsed CD4+ T cell response with severely impaired CD4+ T cell

memory, including Tfh cell memory. Our results demonstrate that Bach2 critically regulates Tfh cell differentiation and CD4+ Downloaded from T cell memory. The Journal of Immunology, 2019, 202: 000–000.

follicular helper (Tfh) cells are a unique CD4+ T cell In lymphocytes, the transcription factor Bach2 was initially subset that plays an essential role in the formation of discovered as a key player in Ab class switching (9). Subse- T germinal centers (GCs) and the generation of high-affinity quently, in T cells, Bach2 has been found to play a critical role in

Abs (1, 2). The molecular mechanism underlying the Tfh cell maintaining regulatory T cell function and homeostasis (10, 11); http://www.jimmunol.org/ differentiation, in particular, the initial stage involving the regu- Bach2 suppresses CD4+ T effector functions by constraining Th1, lation of CXCR5 expression, is still not well understood. Th2, and Th17 cell differentiation (10, 12). In addition, studies Induction of CXCR5 expression is a hallmark of Tfh cell dif- have also shown that Bach2 is required to suppress effector ferentiation (3, 4). For activated conventional CD4+ T cells to memory–related to maintain the naive T cell state (13). move out of the T cell zone and migrate into the B cell follicle, Without Bach2, CD8+ T cell memory is severely impaired (14). In studies have shown that the induction of CXCR5, as well as the Tfh cell differentiation, Bcl6 has been demonstrated to play a downregulation of CCR7, is critical for this process (5). Although central role (15–17), and Blimp-1 has been shown to antagonize cytokine IL-21 and costimulatory molecule ICOS have been the function of Bcl6 in Tfh cell development (15). Interestingly, shown to be important for CXCR5 induction (6, 7), the molecular Blimp1 has been shown to be a direct target of Bach2, and Bach2 by guest on September 27, 2021 mechanism underlying the regulation of CXCR5 expression is not negatively regulates Blimp1 expression (13, 18, 19). The function clear. An early report on transcription factor Ascl2 has shown that, of Bach2 in Tfh cell differentiation (20, 21), however, has not been remarkably, Ascl2 alone is sufficient to induce CXCR5 expres- studied. sion, and the study suggests that the Ascl2 induction initiates the In this study, we report that, surprisingly, Bach2 is a negative Tfh cell programming (8). regulator of Tfh cell differentiation. Despite the increased effector functions and Blimp1 expression, the deletion of Bach2 in CD4+ T cells leads to preferential Tfh cell differentiation. We find that Bach2 negatively regulates CXCR5 expression, and the enhanced *Department of Microbiology, School of Medicine, University of Alabama at Birmingham, CXCR5 upregulation in the absence of Bach2 occurs before the Birmingham, AL 35294; †Department of Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; and ‡Department of Immunology, induction of Ascl2. Bach2 deficiency eventually results in a col- St. Jude Children’s Research Hospital, Memphis, TN 38105 lapsed CD4+ T cell response, and Bach2 is important for CD4+ ORCIDs: 0000-0001-5642-9330 (J.G.); 0000-0002-3637-8852 (Y.-H.W.); 0000- T cell memory, including Tfh cell memory. 0002-7780-909X (B.D.G.); 0000-0002-9787-397X (E.S.); 0000-0001-7955- 0256 (P.G.T.); 0000-0002-1853-8023 (O.K.). Materials and Methods Received for publication December 19, 2018. Accepted for publication March 18, Mice 2019. This work was supported by National Institutes of Health Grants AI095439, AI103162, All mice were maintained in specific pathogen-free barrier facilities and AI130232 (to H.H.) and AI116188, AI122842, and AI133679 (to O.K.), and Uni- and were used in accordance with protocols approved by the Institu- versity of Alabama at Birmingham Center for AIDS Research Grant P30AI027767-26. tional Animal Care and Use Committee of the University of Alabama at + YFP The RNA sequences presented in this article have been submitted to the Birmingham. ACTB:FLPe B6J, Cre-ERT2 , Rosa , OT-II, and B1-8i Expression Omnibus under accession number GSE123350. transgenic mice were from The Jackson Laboratory. Cd4-Cre transgenic mice and Ly5.1+ (CD45.1) C57BL/6 congenic mice were from Taconic Address correspondence and reprint requests to Dr. Hui Hu, University of Alabama at Biosciences. Bach2tm1a(EUCOMM)WTsi (Bach2f/f) mice were recovered by Birmingham, Bevill Biomedical Research Building, Room 859, 845 19th Street f/f South, Birmingham, AL 35205. E-mail address: [email protected] The Jackson Laboratory from cryopreserved sperms. Bach2 mice were bred with ACTB:FLPe B6J to delete the neocassette, then were bred with The online version of this article contains supplemental material. Cre-ERT2+, RosaYFP, OT-II, and Cd4-Cre mice to generate various control Abbreviations used in this article: Bach2f/f, Bach2tm1a(EUCOMM)WTsi; GC, germinal and experimental mice. center; MIT, MSCV-IRES-Thy1.1; NP-OVA, 4-hydroxy-3-nitrophenyl acetyl–OVA; PR8, A/Puerto Rico/8/34; PR8-OVA, A/Puerto Rico/8/34-OVA323–339; RV, retrovirus; Flow cytometry, cell sorting, and intracellular staining Tfh, T follicular helper; WT, wild-type; YFP, yellow fluorescent . These procedures were carried out as described previously (22, 23). The Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 sorted population were .98% pure. Abs were as follows: Alexa Fluor

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801626 2 Bach2 IN Tfh CELL DIFFERENTIATION AND CD4+ T CELL MEMORY

647–anti-mouse Bcl6 (K112-91), Alexa Fluor 647–hamster anti-mouse pseudo-count of 1 for each gene. The Venn diagram was built by gener- CD95 (Fas) (Jo2) from BD Biosciences; Brilliant Violet 421–anti-mouse ating a list of genes that differed by at least 1.5-fold and had an adjusted CD185 (CXCR5) (L138D7), PE-Cy7–anti-mouse PD-1 (29F.1A12), p value ,0.01, which was compared with a list of 145 Tfh cell–associated PE-Cy7–anti-mouse CD38 (90), APC–Cy7–anti-mouse CD4 (GK1.5), genes (22). Pathway analysis was performed using the Gene Set Enrich- Alexa Fluor 700–anti-mouse CD45.2 (104), Brilliant Violet 510–anti- ment Analysis program available from the Broad Institute. mouse/ CD45R/B220 (RA3-6B2), PerCP-cy5.5–anti-mouse/rat CD90.1 (Thy1.1) (OX-7), APC–anti-human CD271 (NGFR) (ME20.4) Accession codes from BioLegend. For multicolor flow cytometry analysis, cells were first RNA sequencing data has been deposited to Omnibus gated on size and singularity, followed by excluding dead cells using under the accession number GSE123350 (https://www.ncbi.nlm.nih.gov/ LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Invitrogen). Donor OT-II geo/query/acc.cgi?acc=GSE123350). cells were gated on the congenic marker CD45.2 and/or yellow fluorescent protein (YFP), and donor B1-8i B cells were gated on the congenic marker Histology CD45.2 for further analysis. Flow cytometry results were analyzed using FlowJo software (Tree Star). These procedures were carried out as described previously (22, 23). The following Abs were used for staining: purified rat anti-mouse CD35 T cell stimulation and retroviral transduction (8c12; BD Biosciences), biotin–anti-CD45.2 (104; BD Biosciences), T cell stimulation and retrovirus (RV) transduction were done as described Alexa Fluor 555–conjugated goat polyclonal anti-rat (Invitrogen), previously (22, 23). The open reading frames of Bach2 were subcloned Alexa Fluor 488–streptavidin (Invitrogen), and Alexa Fluor 647– conjugated rat Ab to mouse IgD (11-26C.2a; BioLegend). Mounted into the retroviral vector MSCV-IRES-Thy1.1 (MIT). The plasmid MigR1- 3 3 CXCR5 was a gift from Dr. H. Qi. The plasmid MIT-Bach2-DZIP was a sections were imaged with 10 and 20 objective on a Nikon Eclipse gift from Dr. R. Roychoudhuri. The self-inactivating retroviral CXCR5 Ti-S microscope. promoter reporter was a gift from Dr. D. Yu. The CXCR5 59 regulatory

Statistics Downloaded from element site 1 (chr9:44370828-44371436) and site 2 (chr9:44345810- 44346556) were subcloned into the CXCR5 promoter reporter vector. RVs Unpaired two-tailed Student t test, one-way ANOVA, and two-way containing sequences encoding Bach2, CXCR5, and CXCR5 reporter were ANOVA were used for calculation of p values. Statistical analysis was produced in Plat-E cells (a gift from Dr. M. Pipkin) by cotransfection with performed with GraphPad Prism 6. retroviral vectors and helper plasmids. Adoptive transfer Results Bach2 negatively regulates Tfh cell differentiation Six- to ten-week-old mice were treated with tamoxifen (Sigma-Aldrich), as http://www.jimmunol.org/ described previously (22, 23). YFP+ or YFP2 CD44loVa2hiCD4+ naive To study how Bach2 may regulate Tfh cell differentiation, we first OT-II T cells were sorted with a BD FACSAria III sorter (BD Biosciences). examined the Bach2 expression in CD4+ T cells after activation 6 6 A total of 0.5 3 10 naive T cells alone or together with 10 3 10 total in vivo in the OT-II/NP-OVA model system (22, 23). We found B cells (∼5% Igl+) from B1-8i mice were transferred into age- and sex-matched SMARTA recipient mice through tail vein (i.v.) injection, that, compared with naive OT-II T cells, the activated non–Tfh followed by immunization by i.p. injection of 100 mg 4-hydroxy-3- OT-II T cells had reduced Bach2 mRNA expression, with nitrophenyl acetyl–OVA (NP-OVA; Biosearch Technologies) emulsified PD-1+CXCR5+ OT-II Tfh cells having the lowest levels (Fig. 1A). in alum adjuvant (Thermo Fisher Scientific). For mixed transfer experi- To verify such results, we employed a PR8 influenza virus in- 3 6 ments, 0.5 10 naive OT-II Bach2–wild-type (WT) T cells mixed with fection model in C57BL/6 mice. We found that on day 14 after 0.5 3 106 naive YFP+Bach2–deficient OT-II (OT-II Bach2-cKO) T cells by guest on September 27, 2021 were cotransferred into the SMARTA recipient mice through i.v. injection, PR8 intranasal infection, the Bach2 mRNA levels were also + + followed by immunization by i.p. injection of 100 mg NP-OVA in alum. the lowest in PD-1 CXCR5 Tfh cells (Fig. 1B), suggesting For the transfer of activated T cells, 1 3 106 T cells were cotransferred that the Bach2 expression needs to be suppressed for Tfh cell 6 + with 10 3 10 total B cells (∼5% Igl ) from B1-8i mice into age- and differentiation. sex-matched SMARTA recipient mice, followed by immunization with 100 mg NP-OVA in alum. For the rechallenge experiments, the recipient We obtained from the European Conditional Mouse Mutagenesis mice were first immunized with 100 mg NP-OVA in alum after cell Program the cryopreserved sperms of the mouse line with Bach2 transfer; 30 d later, mice were rechallenged by i.p. injection of 100 mg allele conditionally targeted and generated the Bach2f/f mice ac- NP-OVA in PBS. cordingly (Supplemental Fig. 1A–C). Studies have shown that Infection with influenza virus Bach2 is expressed at high levels in single-positive thymocytes (13). To circumvent potential thymocyte development issue, we Mice were immobilized with isoflurane and were infected intranasally generated OT-IITgBach2f/fCre-ERT2+RosaYFP mice, in which the with mouse-adapted influenza virus strain A/Puerto Rico/8/34-OVA323–339 (PR8-OVA; H1N1) at a dose of 200 VFU or mouse-adapted influenza virus Cre recombinase expressed from the gene encoding a tamoxifen- strain A/Puerto Rico/8/34 (PR8) at a dose of 15000 VFU. sensitive estrogen receptor variant (Cre-ERT2) will induce con- ditional deletion of both the Bach2 alleles and the stop codon Real-time RT-PCR in front of the gene encoding YFP inserted in the Rosa locus Total RNA purification and some PCR primers were as described previously (22, (22, 23); the Bach2-deleted OT-II cells will express YFP from the 23). Expression of mRNA was normalized to Rpl32 expression and presented as + ubiquitously expressed Rosa26 locus. relative to WT naive OT-II or CD4 T cells. The new primers were as fol- low + lows: for Bach2 (forward, 59-ACTGGTTGGACAGACGAAAG-39,andreverse, We sorted CD44 YFP Bach2-deficient naive OT-II (OT-II Bach2- 59-AGTAACAGCTTGGCAGTGTAG-39); for Ascl2 (forward, 59-CGCTGC- cKO) or WT naive OT-II (OT-II Bach2-WT) T cells from tamoxifen- CCAGACTCATGCCC-39,andreverse,59-GCTTTACGCGGTTGCGCTCG- treated OT-IITgBach2f/fCre-ERT2+RosaYFP mice or from control 39); and for CXCR5 (forward, 59-GACCTTCAACCGTGCCTTTCTC-39,and OT-IITgBach2f/fRosaYFP (or OT-IITgBach2+/+Cre-ERT2+RosaYFP)mice, 9 9 reverse, 5 -GAACTTGCCCTCAGTCTGTAATCC-3 ). respectively. Sorted cells were then cotransferred with B1-8i B cells RNA sequencing analysis into lymphocytic choriomeningitis virus epitope gp66-77–specific (SMARTA) TCR transgenic recipient mice, followed by immunization Total RNA was isolated by using miRNeasy Mini Kit (QIAGEN). Illumina library preparation and sequencing were performed at La Jolla Institute with NP-OVA in alum. SMARTA mice were used as recipients to sequencing center. Sequencing adapters were trimmed from reads using reduce the competition between transferred donor OT-II T cells and Trim Galore! (Babraham Bioinformatics). Trimmed reads were aligned to host T cells in response to NP-OVA challenge (22, 23, 26). B1-8i BCR mouse mm10 using STAR (24), and reads mapping to individual genes is derived from the 4-hydroxy-3-nitrophenyl acetyl–binding Ab B1-8i, were counted using HTSeq-Count. Raw read counts were normalized, and differential expression was analyzed using DESeq2 (25). The scatterplot and the B1-8i B cells will receive help exclusively from the donor comparing expression between WT and Bach2-deficient T cells was con- OT-II T cells after NP-OVA immunization in the SMARTA recipient structed by plotting the log2 of the mean of normalized counts plus a mice (23, 27). The Journal of Immunology 3 Downloaded from

FIGURE 1. Bach2 negatively regulates Tfh cell differentiation. (A) Real-time PCR analysis of Bach2 mRNA in WT naive OT-II T cells, day 7 splenic donor PD-12CXCR52 non–Tfh OT-II, and PD-1+CXCR5+ Tfh OT-II T cells from the Ly5.1+SMARTA recipient mice given transfer of naive OT-II T cells, followed by immunization with NP-OVA in alum. (B) Real-time PCR analysis of Bach2 mRNA in naive CD4+ T cells, day 14 CD44+PD-12CXCR52 non–

Tfh cells, and CD44+PD-1+CXCR5+ Tfh cells from the mediastina lymph nodes of Ly5.1+ mice infected with influenza virus PR8. (C–F) Naive OT-II http://www.jimmunol.org/ Bach2-WT or OT-II Bach2-cKO T cells were cotransferred with B1-8i B cells into the Ly5.1+SMARTA recipient mice, followed by immunization with NP-OVA in alum. The number of total splenic donor OT-II T cells was analyzed on days 3, 5, and 7 (C). The Tfh cell differentiation of donor OT-II T cells and the number of total splenic donor Tfh cells (D and E), and the GC B cell differentiation of B1-8i cells and the number of total splenic GC B1-8i cells (F) were analyzed 5 d after immunization. Bars in (C) represent average 6 SD (day 3, n = 4; days 5 and 7, n = 6). Bars in (D)–(F) represent average 6 SD (n = 6). (G) Naive OT-II Bach2-WT and OT-II Bach2-cKO T cells were cotransferred into the Ly5.1+SMARTA mice, followed by immunization with NP-OVA in alum. The Tfh cell differentiation of donor T cells was analyzed 4 d later. Bars in (G) represent average 6 SD (n = 3). Data in (A)–(G) are representative (or pooled results) of two independent experiments. * p , 0.05, ** p , 0.01, *** p , 0.001.

Whereas OT-II Bach2-cKO T cells and OT-II Bach2-WT T cells approach as well. The retroviral overexpression of Bach2 in WT by guest on September 27, 2021 expanded with similar kinetics for 5 d after NP-OVA challenge, the OT-II T cells led to increased total donor T cell recovery, but almost response of OT-II Bach2-cKO T cells seemed to collapse by day 7 completely abolished the Tfh cell differentiation and the subse- (Fig. 1C). On day 5, when the total donor OT-II T cell numbers quent GC B cell responses after Ag challenge in the OT-II/NP-OVA were at the same levels, Bach2-deficient OT-II T cells contained model (Fig. 2A, 2B, Supplemental Fig. 2C). In a PR8-OVA in- significantly higher frequency and number of PD1+CXCR5+ Tfh fluenza virus infection model in which the PR8 viruses express cells than those of the OT-II Bach2-WT cells (Fig. 1D). Consis- OVA (28), we also found that the retroviral overexpression of tently, Bach2-deficient OT-II T cells also contained higher fre- Bach2 drastically suppressed an otherwise very strong Tfh cell quency of Bcl-6+CXCR5+ Tfh cells (Fig. 1E), suggesting that response of OT-II T cells to influenza virus infection (Fig. 2C). Bach2 is a negative regulator of Tfh cell differentiation. In For transcriptional repression, Bach2 is dependent upon a DNA- agreement with the enhanced Tfh cell responses for the first 5 d, binding basic leucine zipper region (14). We found that the over- we found that both the frequency and number of GC (CD38loFAS+) expression of Bach2-DZIP, which lacks the DNA-binding basic Bi-8i B cells in the recipient mice that received OT-II Bach2-cKO leucine zipper region, failed to suppress the Tfh cell differenti- T cells were also higher than those in mice that received OT-II ation (Fig. 2D), suggesting that the DNA binding of Bach2 is Bach2-WT T cells (Fig. 1F), suggesting that Bach2-deficient Tfh important for its regulation of Tfh cell development, although it cells do function like Tfh cells. Published studies have shown that is not clear how Bach2-DZIP may seem to induce enhanced Tfh Bach2 deletion causes more T cell death (14). By day 7, although cell differentiation. Taken together, our results demonstrate that the Tfh cell frequency of donor OT-II Bach2-cKO T cells appeared Bach2 functions as a critical negative transcriptional regulator of to be at the same level of OT-II Bach2-WT T cells (Supplemental Tfh cell differentiation. Fig. 2A), which is likely due to the cell death of Bach2-deficient OT-II T cells (Fig. 1C), the Tfh cell number of OT-II Bach2-cKO Bach2 deletion leads to CXCR5 upregulation before the T cells was much lower than that of OT-II Bach2-WT T cells induction of Ascl2 (Supplemental Fig. 2A). And correspondingly, the GC B cell To understand the molecular mechanism underlying Bach2-mediated percentage and number were also lower (Supplemental Fig. 2B). suppression of Tfh cell differentiation, we performed the transcriptome The experiments of mixed cotransfer of OT-II Bach2-cKO profiling by RNA sequencing in naive and day 3 OT-II Bach2-cKO and T cells and OT-II Bach2-WT T cells in the same recipient mice OT-II Bach2-WT T cells after NP-OVA challenge. We found 590 showed that Bach2-deficient T cells still contained higher fre- genes differentially expressed between day 3 Bach2-deficient and WT quency of Tfh cells (Fig. 1G), demonstrating that Bach2-mediated donor OT-II T cells (Supplemental Fig. 3A, Supplemental Table I). regulation of Tfh cell differentiation is cell intrinsic. When compared with the 145 Tfh cell–associated genes previously To further verify the role of Bach2 in Tfh cell differentiation, in described (22), 33 of the 590 differentially expressed genes overlapped addition to the loss-of-function approach, we used the gain-of-function (Supplemental Fig. 3A). The signaling pathway analysis showed that 4 Bach2 IN Tfh CELL DIFFERENTIATION AND CD4+ T CELL MEMORY Downloaded from FIGURE 2. Bach2 overexpression suppresses Tfh cell differentiation and Bach2 ZIP domain is important for the inhibition of Tfh cell differentiation. (A–C) WT naive OT-II T cells were activated in vitro and infected with control RV (RV-Thy1.1) or RV-expressing Bach2 (RV-Bach2). (A and B) Retrovirally infected OT-II T cells were sorted and cotransferred with B1-8i B cells into the Ly5.1+SMARTA recipient mice, followed by immunization with NP-OVA in alum 1 d later. The Tfh cell differentiation and the total number of splenic donor OT-II T cells (A), and the GC B cell differentiation and the total number of splenic GC B1-8i cells (B), were analyzed 7 d after immunization. (C) Retrovirally infected OT-II T cells were sorted and transferred into the Ly5.1+SMARTA recipient mice, followed by infection with influenza virus PR8-OVA 1 d later. The Tfh cell differentiation of donor OT-II T cells in D

the mediastinal lymph nodes was analyzed 7 d postinfection. ( ) WT naive OT-II T cells were activated in vitro and infected with control RV (RV-Thy1.1) http://www.jimmunol.org/ and RV-Bach2 or Bach2 (DZIP) (RV-Bach2 [DZIP]). Retrovirally infected OT-II T cells were sorted and transferred into the Ly5.1+ SMARTA recipient mice, followed by immunization with NP-OVA in alum 1 d later. PD-1+CXCR5+ Tfh and Bcl6+CXCR5+ Tfh cell percentage of donor OT-II T cells was analyzed 7 d later. Data in (A)–(D) are representative (or pooled results) of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. the overall T cell activation, metabolism, and oxidative responses were their functions in regulating the CXCR5 promoter activity enhanced in the absence of Bach2 (Supplemental Fig. 3B). (Fig. 3E, right panel). The CXCR5 reporter vector uses en- Studies have shown that Bach2 suppresses the effector functions hanced GFP for infection indication, which is difficult to be of CD4+ T cells (10, 12). We found that in the OT-II/NP-OVA separated from the YFP marker of OT-II Bach2-cKO T cells in model, the mRNA levels of Tbx21, Ifng, and Gzmb were increased staining. Thus, in the reporter assay experiments, we used the by guest on September 27, 2021 in the Bach2-deficient OT-II T cells (Fig. 3A, Supplemental Fig. naive CD4+ T cells from the WT and Bach2f/fCd4-Cre mice. 3D), suggesting that without Bach2, the CD4+ T cell effector We found that in vitro, although there was no CXCR5 induction function is enhanced. Interestingly, on day 3, an early time point in the activated WT or Bach2-deficient CD4+ T cells (data not of the OT-II T cell response to NP-OVA challenge, we found that shown), a significant proportion of the activated CD4+ T cells the mRNA levels of CXCR5 already started to be higher in OT-II were positive for the CXCR5 promoter reporter activities, and Bach2-cKO T cells than in control WT OT-II T cells (Fig. 3B). A no difference was observed between the WT and Bach2-deficient higher proportion of activated Bach2-deficient OT-II T cells were groups (Fig. 3F). The site 2 element, 11 kb upstream of the found in the B cell follicles by day 4 (Fig. 3C). By gating on transcription start site of murine CXCR5, reduced the CXCR5 PD-1+CXCR5+ OT-II Tfh cells on day 5, the cell surface ex- promoter reporter activities, but in a Bach2-independent manner. pression levels of CXCR5 of OT-II Bach2-cKO Tfh cells were The site 1 element, 36 kb upstream of murine CXCR5 transcrip- higher than those of OT-II Bach2-WT Tfh cells (Fig. 3D). These tion start site, also helped suppress the promoter reporter activities results suggest that despite the enhanced effector functions, the (Fig. 3F). In the absence of Bach2, however, the site 1 element deletion of Bach2 preferentially leads the activated CD4+ T cells lost its suppression function (Fig. 3F), suggesting that Bach2 toward Tfh cell differentiation, and Bach2 may directly regulate likely suppresses CXCR5 upregulation via its control of the site CXCR5. 1 element. Ascl2 has been reported to initiate the Tfh cell differentiation and directly regulate CXCR5 expression (8). Surprisingly, we found CXCR5 overexpression does not rescue Bach2-mediated that at the early time point of the response, the increased CXCR5 suppression of Tfh cell differentiation expression in both WT and Bach2-deficient activated CD4+ T cells To examine whether Bach2 may regulate Tfh cell differentiation by seemed to occur in the absence of Ascl2 expression (Fig. 3B, controlling genes other than CXCR5, we retrovirally overexpressed Supplemental Fig. 3C). Studies of Bach2 global binding in the both CXCR5 and Bach2 in WT OT-II T cells (Fig. 4A). murine genome have been carried out (10). We performed Bach2 overexpression alone in OT-II T cells almost completely bioinformatics analysis of this published dataset (GSE45975) and suppressed the generation of PD-1+CXCR5+ Tfh cells (Fig. 2A, found that Bach2 binds to the promoter and two potential regu- 2C). In the OT-II T cells overexpressing both CXCR5 and Bach2, latory regions upstream of the murine CXCR5 locus (Fig. 3E, left the CXCR5 levels were restored to the levels of the OT-II T cells panel), in which the site 2 element has been reported to have a overexpressing CXCR5 alone (Fig. 4A). Thus, we wanted to de- Blimp1-mediated suppression function on the CXCR5 promoter termine whether the retroviral CXCR5 expression is sufficient to (29). After obtaining the murine CXCR5 promoter reporter con- rescue Bach2-mediated suppression of Tfh cell differentiation. struct (29), we cloned the site 1 and site 2 elements containing the We found that the PD-1+CXCR5+ and Bcl6+CXCR5+ Tfh cell Bach2 binding sites and performed the reporter assays to examine percentages of the OT-II T cells overexpressing both CXCR5 and The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. Bach2 deletion leads to enhanced CXCR5 expression before the induction of Ascl2. Naive OT-II Bach2-WT or OT-II Bach2-cKO T cells were transferred into the Ly5.1+SMARTA mice, followed by immunization with NP-OVA in alum. (A) Transcriptome analysis was performed in day 3 donor OT-II T cells. Scatterplot of the average signal of day 3 donor OT-II Bach2-cKO versus OT-II Bach2-WT T cells is shown. Expression of Th1 cell–associated genes including Tbx21, Ifng, Prdm1,andGzmb and Tfh cell–associated genes including Ascl2, CXCR5,andBcl6 is listed. The dash lines indicate gene expression change by 1.5-fold. Data shown in (A) are normalized from two replicates. (B) Real-time PCR analysis of Ascl2 and CXCR5 mRNA in naive, day 3 splenic donor OT-II T cells, and day 7 donor WT OT-II non–Tfh cells and Tfh cells. (C) Confocal microscopy of B cell follicles (IgD+ B cells and CD35+ follicular dendritic cells) and localization of donor (CD45.2+) OT-II T cells in the recipient mice 4 d after im- munization. Scale bar, 100 mm. (D) Histogram of CXCR5 cell surface expression on day 5 donor OT-II Bach2-WT Tfh and OT-II Bach2-cKO Tfh cells. by guest on September 27, 2021 CXCR5 expression on endogenous T cells was used as control. (E) Bach2 binding to the promoter and two potential regulatory regions upstream of the murine CXCR5 locus by bioinformatics analysis of published dataset (GSE45975) (left panel). Diagrams of retroviral Thy1.1 reporter vectors containing CXCR5 promoter (CXCR5P), CXCR5p plus 59 regulatory site 1 region (CXCR51+P), and CXCR5P plus 59 regulatory site 2 region (CXCR52+P) (right panel). (F) Naive Bach2-WT or Bach2-cKO (from Bach2f/fCd4-Cre mice) T cells were activated in vitro and infected with RV expressing

CXCR5P, CXCR51+P, or CXCR52+P, and the reporter activities were analyzed on day 4 by flow cytometry. Data in (B)–(F) are representative (or pooled results) of at least two independent experiments. ***p , 0.001.

Bach2 were still significantly lower than those of the OT-II T cells Bach2 is important for Tfh cell memory overexpressing CXCR5 only (Fig. 4B), suggesting that CXCR5 Studies have shown that Bach2 is important for CD8+ T cell overexpression is not sufficient to rescue Bach2-mediated sup- memory (29). In our study, we found that the number of Bach2- pression of Tfh cell differentiation. Interestingly, whereas the deficient OT-II T cells decreased dramatically on day 7 (Fig. 1C), Tfh cell percentage of the OT-II T cells overexpressing both suggesting that Bach2 may regulate CD4+ T cell memory for- CXCR5 and Bach2 was significantly higher than that of the control RV-Thy1.1 OT-II T cells, there was almost no GC B cell mation as well, including Tfh cell memory. To test this idea, we formation (Fig. 4B), which was also reflected by the almost transferred OT-II Bach2-WT and OT-II Bach2-cKO T cells into exclusive T cell zone localization of activated OT-II T cells the SMARTA recipient mice, followed by NP-OVA immunization overexpressing CXCR5 and Bach2 (Fig. 4C). These results in alum; 30 d after the first Ag challenge, we rechallenged the suggest that Bach2 likely regulates Tfh cell differentiation by recipient mice and examined the CD4+ T cell memory responses controlling genes other than CXCR5 as well, and potentially, cell and Tfh cell differentiation. migration is involved. We found that after the Ag rechallenge, the OT-II Bach2-cKO Studies have shown that Blimp1 is a direct target of Bach2, and T cells still had higher percentages of PD1+CXCR5+ and Bach2 negatively regulates Blimp1 expression (13, 18, 19). We Bcl6+CXCR5+ Tfh cells than did the OT-II Bach2-WT T cells found that in day 3 Bach2-deficient OT-II T cells, the mRNA (Fig. 5A). However, the numbers of total donor OT-II Bach2-cKO levels of Blimp1 were increased (Fig. 4D), and at the same time, Tcells,PD1+CXCR5+ Tfh cells, and PD12CXCR52 non–Tfh cells, the mRNA levels of Bcl6 were also increased (Fig. 4D). The si- were all significantly lower than those of the OT-II Bach2-WT multaneous increase of both Blimp1 and Bcl6 in the absence T cells (Fig. 5B). To confirm the role of Bach2 in CD4+ Tcell of Bach2 became more obvious in the day 4 Bach2-deficient memory, we also used the overexpression approach and found that OT-II T cells (Fig. 4D), suggesting that the Tfh cell differentia- the retroviral overexpression of Bach2 helped generate more robust tion of Bach2-deficient CD4+ T cells may be resistant to Blimp1- OT-II T cell memory responses with continued suppressed Tfh cell mediated inhibition. differentiation (Fig. 5C, 5D). Taken together, these results suggest 6 Bach2 IN Tfh CELL DIFFERENTIATION AND CD4+ T CELL MEMORY Downloaded from http://www.jimmunol.org/

FIGURE 4. CXCR5 overexpression does not rescue Bach2-mediated suppression of Tfh cell differentiation. (A) WT naive OT-II T cells were activated in vitro and infected with control RV (RV-Thy1.1), or RV-expressing CXCR5 (RV-CXCR5) or CXCR5 plus Bach2 (RV-CXCR5+RV-Bach2). The CXCR5 expression was analyzed on day 4 by flow cytometry. (B and C) WT naive OT-II T cells were activated and infected with different RVs. Subsequently, retrovirally infected T cells were sorted and cotransferred with B1-8i B cells into the Ly5.1+SMARTA recipient mice, followed by immunization with NP-OVA in alum 1 d later. On day 7, Tfh cell differentiation of donor OT-II T cells and the GC B cell differentiation of donor B1-8i B cells were analyzed by guest on September 27, 2021 by flow cytometry (B), and the localization of donor OT-II T cells in the spleens of the recipient mice was examined by fluorescence confocal microscopy (C). Scale bar, 50 mm. (D) Naive OT-II Bach2-WT or OT-II Bach2-cKO T cells were transferred into the Ly5.1+SMARTA mice, followed by immunization with NP-OVA in alum. Bcl6 and Prdm1 mRNAs in day 3 and day 4 splenic donor OT-II T cells were examined by real-time PCR analysis. Data in (A)–(D) are representative (or pooled results) of at least two independent experiments. **p , 0.01, ***p , 0.001. that whereas Bach2 still functions as a negative regulator of Tfh cell enhanced basal activity, which is suppressed by a Bach2-mediated differentiation in memory CD4+ T cell responses, the deletion of regulation via the site 1 negative regulatory element. The down- Bach2 is overall detrimental to CD4+ T cell memory, including Tfh regulation of Bach2 seems to be necessary, yet not sufficient, for cell memory. the CXCR5 expression. The mechanisms underlying the Bach2 downregulation and the induction of CXCR5 expression before Discussion Ascl2 expression in activated CD4+ T cells are still not clear. Previous studies have shown that Bach2 inhibits CD4+ T effector That CXCR5 overexpression alone is not sufficient to rescue functions of multiple lineages and is critical for regulatory T cell Bach2-mediated suppression of Tfh cell differentiation suggests that homeostasis (10). Surprisingly, in our study, we have found that there are other important genes regulated by Bach2 in Tfh cell despite the enhanced effector functions of Bach2-deficient con- development. Studies have shown that Bcl6 and Blimp1 antagonize ventional CD4+ T cells, the deletion of Bach2 leads the conven- each other in regulating Tfh cell differentiation, and Blimp1 nega- tional CD4+ T cells to a preferential Tfh cell differentiation. tively regulates Tfh cell differentiation (10). It is very intriguing that Bach2-deficient Tfh cells have increased CXCR5 expression in day 3 and day 4 Bach2-deficient CD4+ T cells after Ag challenge, levels, and the induction of CXCR5 expression occurs before the both Bcl6 and Blimp1 expression levels are increased, indicating Ascl2 expression. that the Tfh cell differentiation of Bach2-deficient CD4+ Tcells The regulation of CXCR5 expression after naive CD4+ T cell may somehow be resistant to Blimp1-mediated suppression. activation is complicated. Although we have identified a regula- Whether it is Bcl6 expression that is resistant to Blimp1-mediated tory element 36 kb upstream of the murine CXCR5 locus that suppression or Blimp1 may suppress Tfh cell differentiation via suppresses the CXCR5 promoter activity in a Bach2-dependent additional mechanisms (which presumably are abolished in the manner in the reporter assays in vitro, neither WT, nor Bach2- Bach2-deficient situation) is not clear. Further investigation is deficient, OT-II T cells upregulated CXCR5 expression in vitro warranted for this interesting phenomenon. despite the reporter activities, suggesting that there are other In our study, we found that the impact of Bach2 deletion on mechanisms involved in regulating the CXCR5 expression in vivo. CD4+ T cell memory is quite dominant, likely due to a cell sur- At the same time, the results also suggest that during the activation vival mechanism that is particularly manifested at the stages after of naive CD4+ T cells, the CXCR5 promoter may have acquired the peak of the primary response. Our loss-of-function and The Journal of Immunology 7 Downloaded from

FIGURE 5. Bach2 is important for CD4+ T cell memory. (A and B) Naive OT-II Bach2-WT or OT-II Bach2-cKO T cells were transferred into the Ly5.1+SMARTA mice, followed by immunization with NP-OVA in alum. Thirty days later, the recipient mice were rechallenged by immunization with NP-OVA in PBS. On day 5 after rechallenge, the Tfh cell differentiation of donor OT-II T cells was analyzed by flow cytometry (A), and the numbers of total splenic donor OT-II T cells, OT-II Tfh cells, and non–Tfh cells were calculated (B). (C and D) WT naive OT-II T cells were activated in vitro and http://www.jimmunol.org/ infected with control RV (RV-Thy1.1) or RV-expressing Bach2 (RV-Bach2). Retrovirally infected OT-II T cells were sorted and transferred into the Ly5.1+SMARTA recipient mice, followed by NP-OVA immunization and rechallenge as in (A). On day 5 after rechallenge, the Tfh cell differentiation of donor OT-II T cells was analyzed by flow cytometry (C), and the numbers of total splenic donor OT-II T cells, OT-II Tfh cells, and non–Tfh cells were calculated (D). Data in (A)–(D) are representative (or pooled results) of at least two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. gain-of-function experiments show that Bach2 greatly regulates T follicular helper cells is mediated by interleukin-21 but independent of + T helper 1, 2, or 17 cell lineages. Immunity 29: 138–149. the magnitude of the CD4 T cell memory responses, including 7. Choi, Y. S., R. Kageyama, D. Eto, T. C. Escobar, R. J. Johnston, L. Monticelli, both non-Tfh and Tfh cell responses. Taken together, our study C. Lao, and S. Crotty. 2011. 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Nature 429: 566–571. moter reporter plasmid, Dr. Rahul Roychoudhuri (Babraham Institute) 10. Roychoudhuri, R., K. Hirahara, K. Mousavi, D. Clever, C. A. Klebanoff, for the MIT-Bach2-DZIP plasmid, Dr. Matthew Pipkin (Scripps Research) M. Bonelli, G. Sciume`, H. Zare, G. Vahedi, B. Dema, et al. 2013. BACH2 re- for the Plat-E cells, and Drs. Frances Lund and Troy Randall (University presses effector programs to stabilize T(reg)-mediated immune homeostasis. Nature 498: 506–510. of Alabama at Birmingham) for the PR8 and PR8-OVA influenza viruses. 11. Kim, E. H., D. J. Gasper, S. H. Lee, E. H. Plisch, J. Svaren, and M. Suresh. 2014. We thank Ryan J. McMonigle, Andrew R Schroeder, and Zhaoqi Yan Bach2 regulates homeostasis of Foxp3+ regulatory T cells and protects against for scientific discussion. fatal lung disease in mice. J. Immunol. 192: 985–995. 12. Kuwahara, M., W. Ise, M. Ochi, J. Suzuki, K. Kometani, S. Maruyama, M. Izumoto, A. Matsumoto, N. Takemori, A. Takemori, et al. 2016. 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Table S1. Differentially expressed genes between D3 Bach2-deficient and Wild-type donor OT-II Cells

Up-regulated in OT-II Bach2-cKO Down-regulated in OT-II Bach2-cKO 1500009L16Rik 1600014C10Rik 2700094K13Rik 2410089E03Rik 2810417H13Rik A430078G23Rik 6430562O15Rik A430088P11Rik A430005L14Rik A630023P12Rik Acot7 Abca1 Adam8 Abcc10 Ahcy Abcc4 Ak6 Abcg1 Akap6 Abcg3 Alad Acvrl1 Alcam Adamts6 Alyref Adcy7 Angptl2 Adgre5 Anxa1 Aff3 Anxa2 Akap12 Apitd1 Akap8l Asb2 Als2cl Asf1b Ar Atad2 Arhgap31 Atf6 Arhgap9 Atl2 Arhgef18 Atp1a3 Arrb1 Atp5j2 Asb13 Atp5o Atg9a Atpif1 Atp10d Aurka Atp8a1 Birc5 BC021614 Brca1 Bcl2 Brip1 Btbd11 Bspry Btg1 Bub1 Btg2 Bub1b Bzrap1 C1d Cacna2d4 C330027C09Rik Capn3 Cacna1i Card6 Cacybp Ccdc125 Calm2 Ccr7 Casc5 Cd96 Casp1 Chd2 Casp3 Chd3 Ccna2 Chd6 Ccnb1 Chn2 Ccne2 Chst15 Ccr2 Col11a2 Ccr5 Cpm Cct6a Cyp2s1 Cdc25c Cyth3 Cdc45 D430042O09Rik Cdc6 Dapl1 Cdca3 Dennd1c Cdca7 Dlg4 Cdk1 Dlgap4 Cdk6 Dnah8 Cdkn1a Dopey2 Cdkn2c Dzip1 Cdkn3 Evl Cebpa Faah Cenph Fam101b Cenpk Fam102a Cenpl Fam160b1 Cenpn Fam168a Cenpw Fam169b Cep55 Fam189b Cep83 Fam214a Chaf1b Fbrs Chek1 Fbxl20 Cisd1 Fbxo32 Cish Fcrl1 Ckap2 Filip1l Cks1b Fn1 Clspn Fos Cmc2 Fyco1 Cops3 Gbp8 Coro2a Gigyf1 Coro2b Gm15800 Cox17 Gramd1a Cox7a2 Grina Creld2 Hectd2 Crip1 Helz2 Cse1l Hsdl1 Csrp2 Hspa1a Cxcr4 Hspa1b Cxcr5 Igfbp4 Cxcr6 Igflr1 D030056L22Rik Ikbke Dbi Il6ra Dctpp1 Il6st Dek Il9r Dhfr Inadl Dlgap5 Itga4 Dmwd Itga9 Dna2 Itgb3 Dpy30 Itpr2 Dsn1 Izumo1r Dtl Jun Dtymk Kbtbd11 Dut Kdm5b Egln3 Kmt2a Ehd4 Kmt2c Eif1ax Kmt2d Eif2s1 Letmd1 Eif2s2 Lpin1 Eif4ebp1 Lrig1 Eny2 Macf1 Ercc6l Maml2 Errfi1 Mdn1 Esco2 Mfhas1 Esr1 Ms4a4c Exosc8 Mtss1 F2r Mxd4 F2rl2 N4bp2l1 Fabp5 Nckap5l Fam111a Ndor1 Fam129b Ndrg3 Fam83g Neurl3 Fancd2 Nlrc3 Fanci Nod1 Far1 Nrip1 Fbxo5 Nsg2 Fen1 Nxpe3 Fignl1 Oas2 Fkbp4 Orai2 Ftsj1 Parp3 Fut8 Parp8 Gbp3 Pbxip1 Gcnt1 Pdcd4 Ggt1 Pde1b Gins1 Pdzk1ip1 Gins2 Pear1 Glipr1 Pglyrp2 Glrx Phf1 Gm14005 Phf21a Gmnn Pik3ip1 Gna15 Pim2 Gpr183 Pkd1 Gsr Plcb2 Gtf2h5 Plcg1 Gyk Polm Gzmb Ppp1r15a H2-Q2 Prickle3 Hat1 Prr33 Haus5 Ranbp10 Havcr2 Rapgef4 Hells Rasgrp4 Hmgb2 Rassf2 Hmgn1 Rcan3 Hmmr Rere Hmox1 Rgp1 Hsp90b1 Rgs3 Hyls1 Rin3 Ica1l Rnf123 Ifng Rnf167 Ifrd1 Satb1 Il10 Scml4 Il12rb1 Sema4f Il18rap Sesn3 Il21 Sidt1 Il2ra Slc12a7 Irf8 Slc28a2 Itgb1 Slc2a4rg-ps Kcnk5 Slfn5 Kif11 Slfn8 Kif15 Smad7 Kif18a Smarca2 Kif18b Sorl1 Kif20a Ssh1 Kif20b St8sia1 Kif2c St8sia6 Kpna2 Stoml1 Kpna3 Syne1 Lamc1 Synpo Lgals1 Syt11 Lgals3 Szt2 Lig1 Tcp11l2 Lmnb1 Tdrp Lmo4 Tecpr1 Lrr1 Tet1 Lrrc40 Tlr1 Lsm3 Tnfrsf25 Lsm8 Tom1l2 Ly6a Treml2 Mad2l1 Trib2 Mafg Trio Magoh Txnip Manf Ube2h Map2k3 Utrn Marcks Uvssa Marcksl1 Vipr1 Mastl Xdh Matn2 Ypel3 Mbnl3 Zc3h6 Mcm10 Zcchc11 Mcm2 Zer1 Mcm3 Zfhx2 Mcm5 Zfp287 Mcm6 Zfp395 Mcm7 Zfp579 Mcm8 Zfp831 Med30 Znf512b Melk Znfx1 Metap2 Znrf1 Metrn Zscan2 Mis18a Mis18bp1 Mrpl12 Mrpl18 Mrpl20 Mrpl35 Mrpl42 Mrpl46 Mrps14 Mrps18c Mrps22 Mtfr2 Mtx2 Myadm Mydgf Myo1b N4bp3 Nasp Ncapg Ncapg2 Ndc1 Ndc80 Ndrg1 Ndufa12 Ndufa4 Neb Nek2 Neto2 Nhp2 Nme1 Nop58 Npm1 Nqo1 Nsmce4a Nudcd1 Nudcd2 Nudt5 Nuf2 Nup107 Nup43 Nup62 Nusap1 Nxt1 Oat Orc1 Orc6 Osbpl3 Ostc Padi4 Paics Pbdc1 Pbk Pcna Pdcd1 Pdia6 Phf19 Phgdh Pim1 Pkm Plac8 Plek Plekhg3 Plk4 Plxdc1 Pmm1 Pno1 Pola1 Pold1 Pole2 Pole3 Pomp Pop4 Ppa1 Ppia Ppih Prc1 Prdm1 Prdx1 Prdx4 Prim1 Prim2 Prmt3 Prpf38a Psat1 Psma1 Psma5 Psmb7 Psmc3ip Psmc6 Ptgr1 Ptma Ptpn13 Pycrl Rad51 Rad51ap1 Rad54b Ranbp1 Rbbp8 Recql4 Rfc3 Rfc4 Rgs1 Rnase4 Rnaseh2b Rpa2 Rpa3 Rpp30 Rps27l Rrm1 Rrm2 Runx2 S100a4 S100a6 Samsn1 Sccpdh Sdf2l1 Sdhd Sema7a Serpinb9 Sgol1 Sgol2a Sh2d1a Shcbp1 Siva1 Ska1 Ska2 Slbp Slc25a10 Slc43a3 Slmo2 Smc2 Smco4 Smim3 Snrpd1 Snrpd2 Snx2 Spc24 Spc25 Spdl1 Sptssa Srgn Stoml2 Stra13 Syce2 Tbc1d31 Tbx21 Tceb1 Tcf19 Tcp1 Tex30 Tigit Timm17a Timm50 Tipin Tk1 Tmem258 Tmem97 Top2a Tpi1 Tpm4 Tpx2 Traip Trim37 Trip13 Ttc39c Ttk Tuba1b Tuba1c Tubb2b Txn1 Txnl1 Tyms Ube2e3 Ube2t Uqcr10 Usp1 Wls Xpo1 Xrcc6 Ybx3 Ywhaq Zdhhc2 Zfp367 Zwint