Bach2 Negatively Regulates T Follicular Helper Cell Differentiation and Is Critical for CD4 + T Cell Memory
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Bach2 Negatively Regulates T Follicular Helper Cell 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 ol.1801626 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/04/10/jimmunol.180162 Material 6.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 genes 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 Gene 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 protein. 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/human 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 Gene Expression 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).