Cyclooxygenase-1 Regulates the Development of Follicular Th Cells

Cyclooxygenase-1 Regulates the Development of Follicular Th Cells via Prostaglandin E 2 Ting Liu, Qiong Yang, Ying-Jiao Cao, Wei-Ming Yuan, Ai-Hua Lei, Pan Zhou, Wei Zhou, Yong-Dong Liu, This information is current as Mao-Hua Shi, Quan Yang, Jin-Yi Tang, Hai-Kun Wang, Hui of October 2, 2021. Zhang, Ying Yu and Jie Zhou J Immunol published online 26 June 2019 http://www.jimmunol.org/content/early/2019/06/25/jimmun ol.1801674 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2019/06/25/jimmunol.180167 Material 4.DCSupplemental http://www.jimmunol.org/

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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 June 26, 2019, doi:10.4049/jimmunol.1801674 The Journal of Immunology

Cyclooxygenase-1 Regulates the Development of Follicular Th Cells via Prostaglandin E2

Ting Liu,*,†,1 Qiong Yang,‡,1 Ying-Jiao Cao,*,1 Wei-Ming Yuan,x,1 Ai-Hua Lei,‡ Pan Zhou,‡ Wei Zhou,x Yong-Dong Liu,{ Mao-Hua Shi,‖ Quan Yang,# Jin-Yi Tang,** Hai-Kun Wang,** Hui Zhang,‡ Ying Yu,†† and Jie Zhou*,†

Cyclooxygenase (COX)-1, one of the critical enzymes required for the conversion of arachidonic acid to PGs, has been demonstrated to play an important role not only in the cardiovascular system but also in the immune system. COX-1 has been found to regulate early B cell differentiation, germinal center formation, and Ab production of B cells. However, the underlying mechanisms of COX-1–mediated B cell activation remains not fully understood. In this study, we reported that COX-1 is a potential regulator 2/2 for the development of follicular Th (TFH) cells. COX-1–deﬁcient (COX-1 ) mice displayed a signiﬁcant reduction of TFH cells

upon inﬂuenza infection or immunization with keyhole limpet hemocyanin, which led to a severe impairment of germinal center Downloaded from

responses. We further demonstrated that COX-1–derived PGE2, via binding with its receptors EP2/EP4, represents the underlying mechanism. The administration of EP2/EP4 agonists or PGE2 almost completely rescued the defective TFH cell generation in 2/2 COX-1 mice. Taken together, our observations indicate that COX-1 plays an important role in the development of TFH cells. The Journal of Immunology, 2019, 203: 000–000. + ollicular Th (TFH) cells, a subset of CD4 T cells spe- the master regulator of TFH cells (6, 7). The process of TFH cell cialized for the provision of help to B cells, play important differentiation starts from the priming of naive CD4+ T cells with http://www.jimmunol.org/ F roles in the formation of germinal center (GC) and long- dendritic cells (DC) (8). If the chemokine receptor CXCR5 is term humoral immunity (1–3). The differentiation of TFH cells expressed after DC priming, early TFH cells will migrate to the is a multistage process and involves multiple signals (4, 5). border of the T–B zone and undergo further TFH cell differentia- Transcription factor B cell lymphoma 6 (Bcl-6) was thought to be tion (8). The molecules of IL-6, ICOS-ICOSL, IL-2, and TCR signaling orchestrate the early phase of TFH cell differentiation during DC priming via the regulation of CXCR5 and Bcl-6 genes *Joint Program in Immunology, Department of Internal Medicine, Affiliated Guangz- hou Women and Children’s Medical Center, Zhongshan School of Medicine, Sun (9–11). After the migration to the T–B border of the lymph node † Yat-sen University, Guangzhou 510623, China; Key Laboratory of Immune Micro- and spleen, TFH cells interact with Ag-experienced B cells via by guest on October 2, 2021 environment and Disease (Ministry of Education), Department of Immunology, binding between ICOS and ICOS ligand, which further drives the School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, ‡ China; Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen localization of TFH cells in GC and their differentiation into University, Guangzhou 510080, China; xDepartment of Neonatology, Guangzhou GC-TFH cells (12, 13). GC-TFH cells provide help and enhance Women and Children’s Medical Centre, Guangzhou 510623, China; {Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou B cell affinity maturation in GC (10, 14, 15). GC-TFH cells de- ‖ # 510080, China; First People’s Hospital of Foshan, Foshan 528000, China; Key velop into memory TFH cells after exiting GC (14, 16, 17). Al- Laboratory of Immunology, Sino-French Hoffmann Institute, School of Basic Med- though the critical roles of T cells in the clearance of infections ical Sciences, Guangzhou Medical University, Guangzhou 511436, China; FH **Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, and vaccination responses have been well documented, the de- †† China; and Department of Pharmacology, School of Basic Medical Sciences, tailed mechanisms underlying TFH cell differentiation remain to Tianjin Medical University, Tianjin 300070, China be fully understood. 1 T.L., Qiong Yang, Y.-J.C., and W.-M.Y. contributed equally to this work. Cyclooxygenases (COXs) are enzymes that catabolize arachi- ORCIDs: 0000-0001-9234-0050 (W.-M.Y.); 0000-0002-2548-7252 (Quan Yang); donic acid to produce prostanoids, including PGs and thrombox- 0000-0003-3593-4324 (J.-Y.T.); 0000-0002-4714-4672 (H.-K.W.); 0000-0003-2427- 0411 (Y.Y.); 0000-0001-5964-1599 (J.Z.). anes (18). PGs act through G protein–coupled receptors to elicit a diversity of intracellular signaling pathways, which displays Received for publication December 27, 2018. Accepted for publication June 7, 2019. distinct functions in multiple cell types (19). Two COX isoforms, This work was supported by the following grants to J.Z.: the High-level Talent Start- up Funding of Tianjin Medical University, the National Natural Science Foundation COX-1 and COX-2, have been identified. It is generally thought of China (91542112; 81571520, 81771665, and 81742002); the National Natural that COX-1 is constitutively expressed in most cell types, whereas Science Foundation of Guangdong (2017B030311014), and the Science and Tech- COX-2 is rapidly inducible in response to inflammatory stimuli nology Program of Guangzhou (201605122045238). This work was also supported by the National Natural Science Foundation of China (31600717) and the Natural (20). COX-1 and COX-2 are therefore regulated by distinct Science Foundation of Guangdong (42030118) to Qiong Yang. mechanisms and play distinct roles, despite that they both catalyze Address correspondence and reprint requests to Prof. Jie Zhou, Department of the same enzymatic reaction (21). Compared with the extensive Immunology, School of Basic Medical Sciences, Tianjin Medical University, studies about COX-2, the function of COX-1 is less understood. 22 Qixiangtai Road, Tianjin 300070, China. E-mail address: [email protected] Interestingly, accumulating evidence have indicated the potential The online version of this article contains supplemental material. roles of COX-1 in the immune system (22–25), especially in the Abbreviations used in this article: Bcl-6, B cell lymphoma 6; BM, bone marrow; COX, cyclooxygenase; CT, cycle threshold; Cxcr5, C-X-C motif chemokine receptor development and biological function of B cells. COX-1 has been 5; DC, dendritic cell; dLN, draining lymph node; GC, germinal center; KLH, keyhole identified as an important regulator of early B cell development limpet hemocyanin; mLN, mediastinal lymph node; MS, mass spectrometry; TFH, via thromboxane A2-mediated JAK/STAT5 signaling (23). In ad- follicular Th; Treg, regulatory T; WT, wild-type. dition, COX-1 has been demonstrated to orchestrate GC formation Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 and Ab class switch via the regulation of IL-17 (26). And PGE2

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801674 2 COX-1 REGULATES TFH CELL DEVELOPMENT was reported to drive IL-21–dependent B cell death (27). Consid- previous report (28). On day 7 after immunization, mice were sacrificed, and T and GC B cells were analyzed by flow cytometry. ering the importance of TFH cells in the facilitation of B cell acti- FH vation, we investigate the potential roles of COX-1 in TFH cells. Mixed BM chimeric In this study, it was found that COX-1 was preferentially expressed Six- to ten-weeks-old congenic, sex-matched C57B/L6-Ly5.1/Ly5.2 mice in mouse TFH cells. Deficiency of COX-1 dramatically impaired the (CD45.1.2+) were treated daily for 7 d with 0.5 mg/ml neomycin. BM cells development of T cells in both influenza infection and keyhole 2/2 FH from COX-1 mice (CD45.2+) and congenic wild-type (WT) mice limpet hemocyanin (KLH) immunization models. Mechanistic (CD45.1+) were mixed at ratio of 1:1 (5 3 106/mouse) before i.v. trans- + studies showed that PGE2 mediated the effect of COX-1 on TFH ferred into lethally irradiated WT recipients (CD45.1.2 ). The lethal irra- cells via EP2/EP4 receptors. These observations therefore provide diation was 9 Gy (in 2 bursts: 4.5 Gy/dose administered 3 h apart). new insights about the important roles of COX-1 in humoral im- Peripheral blood was collected for reconstitution evaluation at 4 wk posttransplantation. Recipients were infected with H1N1 influenza virus munity and protection against infections. 6 wk posttransplantation. Mice were sacrificed 7 wk postinfection; the frequencies of TFH and GC B cells in spleen and lung mLNs were analyzed by flow cytometry. CD4+ T cells from spleen were used as internal controls Materials and Methods in mixed BM chimeras. Mice Adoptive transfer All animals were on the C57BL/6 background and used in accordance with protocols approved by the institutional Animal Care and Use Committee of Naive CD4+ T cells (CD4+CD62LhiCD44lo) from the spleen of COX-12/2 2/2 Sun Yat-sen University and Tianjin Medical University. COX-1 mice mice and WT littermates were purified by FACSAria III Cell Sorter with C57BL/6 background were generously provided by Dr. Y. Yu at (BD Biosciences). Cells (2.5 3 106) were then transferred into Tcra2/2 Tianjin Medical University. C57B/L6-Ly5.1 mice were kindly provided by mice by injection via tail vein. Two weeks later, mice were intranasally Dr. H. Wang from Institut Pasteur of Shanghai, Chinese Academy of infected with H1N1 virus after the formation of GC. Mice were sacrificed, Downloaded from 2/2 tm1Mom Sciences. Tcra mice (002116; B6.129S2-Tcra /J) were obtained and the frequencies of TFH and GC B cells in the spleens and mLNs were from The Jackson Laboratory. All animals were housed in a specific analyzed by flow cytometry at day 7 postinfection. pathogen-free barrier facility, according to the guidelines from Sun Yat-sen University and Tianjin Medical University, and sex- and age-matched ELISA littermates were used as controls. Influenza-specific ELISA was performed by precoating 96-well plates with heat-inactivated virus. Serum samples were diluted in a 3-fold serial di-

Isolation of cells from tissues http://www.jimmunol.org/ lution, and the bound Ab was detected using HRP-conjugated anti-mouse Bone marrow (BM) cells were obtained by flushing tibias and femurs with IgG (Invitrogen). Data were presented as an OD value at 450 nm. one syringe containing RPMI 1640 media. Then, RBCs were lysed with ammonium-chloride-potassium buffer. Single cells were obtained from Measurement of PGs by HPLC/mass spectrometry spleen, draining lymph nodes (dLN), and mediastinal lymph nodes (mLN) 3 6 by mechanical disruption on 70-mmcellstrainers. 1.5 10 TFH cells from the spleen of influenza-infected mice were purified by FACSAria III Cell Sorter (BD Biosciences). Cells were then Flow cytometric analysis and sorting resuspended with 500 ml ice-cold PBS, and the amounts of PGs profiles were measured as previously reported (29). The samples were spiked Cells were resuspended in PBS and were stained with fluorescence- immediately with 5 ng PGD2-d4, PGE2-d4, TxB2-d4, 6-keto PGF1a-d4, conjugated Abs. All flow cytometry data were analyzed with FlowJo and PGF2a-d4, followed by 40 ml 1 M citric acid and 5 ml 10% butylated V10.0.7 after being collected with a BD LSRFortessa flow cytometer (BD hydroxytoluene administration. All the chemicals were obtained from by guest on October 2, 2021 Biosciences). The fraction of labeled cells was analyzed with a minimum Cayman Chemical. Purification by solid phase extraction with Strata event of 100,000. Before incubating with fluorescence-labeled mAbs at 4˚C X C18 cartridges (Phenomenex) was followed. The solid phase extraction for 30 min, cells were blocked with anti-FcR (anti-CD16/32; BD Phar- cartridge was conditioned with acetonitrile (1 ml) and equilibrated with mingen) for 20 min. For the staining of TFH cells, cells were washed with water (1 ml). Then, samples were applied to the cartridge, which was PBS and stained with anti-CXCR5 biotin (551960; BD Biosciences) for 50 washed with 1 ml of 5% acetonitrile in water and dried with vacuum for min at 4˚C. After washed with PBS, cells were stained with streptavidin– ∼15 min. The analyte and internal standards were eluted from the cartridge allophycocyanin, anti-CD4-FITC, and anti-PD-1–PE for 30 min at 4˚C. by 1 ml of 5% acetonitrile in ethyl acetate. The eluate was collected and GC B cells were determined by staining with anti–B220-PECy7, anti–IgD– then dried under a gentle stream of nitrogen. The resulting residue was allophycocyanin, anti–Fas-PE, and anti–GL-7–FITC. COX-1 polyclonal then reconstituted in 10% acetonitrile in water and filtered by centrifu- Ab (160109; Cayman Chemical) and allophycocyanin–goat anti-rabbit IgG gation (12,000 3 g, 1 min) using 0.2 mm Nylon MicroSpin Filters (Alltech (H+L) secondary Ab (A-21244; Invitrogen) were used for intracellular Associates) and then quantitated using HPLC/mass spectrometry (MS) staining of COX-1 inpermeabilized cells. For the flow cytometric sorting, analyses as described previously (30). a BD FACSAria III Cell Sorter (BD Biosciences) was used. The following Abs were used: anti–CD4-FITC (GK1.5; eBioscience), anti–CD44- In vitro differentiation of T cell subsets Percp-cy5.5 (IM7; eBioscience), anti–CD44-eFlour450 (IM7; eBioscience), Differentiation of distinct T cells subsets in vitro followed the previous anti–CD62L-PE-cy7 (MEL-14; eBioscience), anti–CD8-PE (53-6.7; eBio- + reports (31). Purified naive CD4 T cells were sorted from C57BL/6 mice science), anti–CD25-PE-cy7 (PC61.5; eBioscience), anti–GITR(CD357)-PE and then were stimulated with anti-CD3 (0.5 mg/ml; eBioscience) and anti- (DTA-1; eBioscience), anti–PD-1 (CD279)-PE (J43; eBioscience), anti– CD28 (1 mg/ml; eBioscience) on plates for 48 h in DMEM medium in the PD-1–Pacific Blue (J43; eBioscience), anti–B220-PE-cy7 (RA-3-6B2; presence of recombinant human IL-2 (100 U/ml) and cytokine mixture as BioLegend), anti–IgD–allophycocyanin (11-26c; eBioscience), streptavi- follows: for T cell–like: 20 ng/ml IL-6, IL-21 (R&D Systems), 10 mg/ml din–allophycocyanin (eBioscience), anti–GL-7-FITC (GL-7; eBioscience), FH anti–IFN-g (Bio X Cell), and 10 mg/ml anti–IL-4 (R&D Systems); for anti–CD95-PE (15A7; eBioscience), anti–CD45.1–Pacific Blue (A20; T 1: anti–IL-4 10 mg/ml and IL-12 10 ng/ml; for T 2: 10 mg/ml anti–IL- BioLegend), and anti–CD45.2-Percp-cy5.5 (104; eBioscience). H H 12 (C17.8; eBioscience),10 ng/ml IL-4 (R&D Systems), and 10 mg/ml Infection with influenza virus anti–IFN-g; for TH17: 20 ng/ml IL-6 (R&D Systems), 1 ng/ml TGF-b (R&D Systems),10 mg/ml anti–IL-4, and 10 mg/ml anti–IFN-g; and for Mice were anesthetized with isoflurane and infected intranasally with a 0.5 regulatory T (Treg) cell: 5 mg/ml anti–IL-4, 10 mg/ml anti–IFN-g, 5 ng/ml lethal dose to 50% of animals tested of influenza virus H1N1 (50 ml per TGF-b (R&D Systems), and 200 U/ml IL-2. After a 5-d culture, cells were mouse), according to the previous report (17). The weight loss was harvested for expression analysis of signature genes by quantitative recorded daily. On day 7 after H1N1 infection, TFH and GC B cells from RT-PCR (qRT-PCR) to validate the success of culture conditions. spleen and lung mLN were analyzed by flow cytometry. Lung tissue was immersed in 4% paraformaldehyde for H&E staining. PGs treatment 2/2 KLH immunization COX-1 mice (6–8-wk-old) and WT mice (6–8-wk-old) were randomly divided into three groups, were i.p. injected once daily with vehicle (PBS), Mice were injected with KLH protein (0.5 mg/ml; Calbiochem) emulsified 0.3 mg/kg PGD2, 0.3 mg/kg PGE2 after H1N1 virus infection (50 PFU), in CFA (0.5 mg/ml; Calbiochem) s.c. (100 ml per mouse), according to the and PBS was used as vehicle control. The dosages used were according to The Journal of Immunology 3 the previous studies (32, 33). Seven days later, mice were sacrificed, and different groups was analyzed with the Student t test: ns, no significance; flow cytometry was performed. p . 0.05. The p values , 0.05 were considered statistically significant: *p , 0.05, **p , 0.01, ***p , 0.001, and ****p , 0.0001. PG agonists treatment Age- and sex-matched COX-12/2 mice (6–8-wk-old) and WT (6–8-wk- Results old) were used. Mice were i.p. injected once daily with the indicated ag- COX-1 is preferentially expressed in mouse TFH cells onists for PGs after H1N1 virus infection (50 PFU). The dosages used were according to the previous studies (33–39), including butaprost (EP2 ago- To explore the roles of COX-1 in the regulation of TFH cells, nist, 1.0 mg/kg), L-902,688 (EP4 agonist, 1.0 mg/kg), U46619 (TP agonist, C57BL/6 mice were infected with H1N1 influenza virus, 10 mg/kg), DK-PGD2 (DP2 agonist, 30 mg/kg), and sulprostone (EP1/3 and mRNA expression of COX-1 was measured in different agonist, 1.0 mg/kg). The same volume (200 ml) of PBS was used as a T cell subsets from spleen on day 7 postinfection by qRT-PCR, control. Seven days later, mice were sacrificed, and flow cytometry was + + hi lo performed. All agonists were bought from Cayman Chemical. including naive CD4 T cells (CD4 CD62L CD44 ), acti- + + lo hi vated CD4 Tcells(CD4CD62L CD44 ), TFH cells Real-time RT-PCR (CD4+CD62LloCD44hiCXCR5+PD-1+), CD8+ T cells (CD8+), + + + + Total RNA was isolated from sorted or fractionated cells using TRIzol Treg cells (CD4 GTIR CD25 ), as well as total B cells (B220 ). reagent (Invitrogen), and cDNAwas synthesized with High-Capacity cDNA Fig. 1A shows that whereas expression of COX-1 mRNA in Reverse Transcription Kit (Takara Bio). Real-time PCR was performed on + TFH cells was significantly greater than seen in naive CD4 T cells a Bio-Rad CFX96 using Power SYBR Green PCR Mix (Takara Bio), + according to the manufacturer’s instructions. For the RT-PCR, duplicates and activated CD4 T cells, COX-1 mRNA expression in were performed and analyzed using the DDCt method (40). Cycle threshold other lymphocyte subsets did not differ significantly from naive (CT) values for analyzed genes were normalized to CT values of the CD4+ T cells and activated CD4+ T cells (Fig. 1A). Further- housekeeping gene b-actin. Gene expression was normalized against b-actin Downloaded from more, TFH cells expressed a much higher level of COX-1 compared internal control (CtTarget 2 Ctb-actin = DCt), and the data represent + lo hi 2 2 2 2 with activated non-T cells (CD4 CD62L CD44 CXCR5 PD-1 ) 2 (DCtexperimental DCtcontrol = DDCt). The relative fold changes were based on the FH corresponding controls. Primers used were listed in Supplemental Table I. (Fig. 1B). No clear differences in COX-1 expression between naive + CD4 T cells and activated non-TFH cells were found, indicating that Statistical analysis COX-1 may not uniformly affect the activation of CD4+ T cells Data analysis was performed with GraphPad Prism 8.0 software. Results (Fig.1B).Asexpected,TFH cells highly expressed their signature were expressed as mean 6 SEM, and the comparison between two genes, including Bcl-6, C-X-C motif chemokine receptor 5 (Cxcr5), http://www.jimmunol.org/

FIGURE 1. COX-1 is preferentially expressed in by guest on October 2, 2021 mouse TFH cells. (A–C) C57BL/6 mice (n = 6) were infected with PR8H1N1 inﬂuenza virus for 7 d. (A) The indicated populations were sorted from spleen, and COX-1 expression was measured by qRT-PCR. The markers used for ﬂow cytometric sorting are as follows: naive CD4+ T cells (CD4+CD62LhiCD44lo), + + lo hi activated CD4 T cells (CD4 CD62L CD44 ), TFH cells (CD4+CD62LloCD44hiCXCR5+PD-1+), CD8+ T cells (CD8a+), Treg cells (CD4+GTIR+CD25+), and B cells (B220+). (B)mRNAexpressionof

COXs and TFH cell signature genes, TFH cells, + naive CD4 T cells, and activated non-TFH cells (CD4+CD62LloCD44hiCXCR52PD-12) were exam- ined by qRT-PCR. (C) Intracellular COX-1 protein was + evaluated in TFH cells, naive CD4 T cells, and activated non-TFH cells by ﬂow cytometry. (D) qRT-PCR analysis of Cox-1 expression in different CD4+ Tcell subsets generated under in vitro culture conditions described in Materials and Methods. In Fig. 1A and 1B, naive CD4+ T cells were considered to be 1, and in

Fig. 1D, TH0 cells were considered to be 1; the values in other samples were fold changes compared with them. In all plots, mean 6 SEM are shown. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ****p , 0.0001, using two-tailed Student t test. 4 COX-1 REGULATES TFH CELL DEVELOPMENT and Il-21 (IL-21), whereas they expressed a lower level of C-C motif and bronchiolar hyperplasia and metaplasia were aggravated in the chemokine receptor 7 (Fig. 1B) (10, 41, 42). Unlike for COX-1, the lung from COX-12/2 mice (Fig. 2B). Further flow cytometric expression of Cox-2 mRNA did not differ between TFH cells with analysis showed that both the frequencies and the absolute numbers + 2/2 active non-TFH cells or naive CD4 T cells (Fig. 1B). The prefer- of TFH cells were significantly decreased in COX-1 mice when ential expression of COX-1 in TFH cells was further confirmed by compared with WT controls in both lung mLNs and spleen flow cytometry (Fig. 1C). In addition, Cox-1 expression was also (Fig. 2C). No noticeable differences were found in total CD4+ + + significantly higher in TFH-like cells when compared with other T cells, naive CD4 T cells, activated CD4 T cells, or Treg cells 2/2 T cell subsets under in vitro culture conditions, including TH0, TH1, between COX-1 mice and WT controls (Supplemental Fig. 1B– TH2, TH17, and Treg cells (Fig. 1D). The highly expressed signa- D). In line with the reduced responses of TFH cells, both the fre- ture genes in distinct Th cells were confirmed by qRT-PCR quencies and absolute numbers of GC B cells in COX-12/2 mice (Supplemental Fig. 1A). were significantly lower than those of WT littermates (Fig. 2D). Consistent with the impaired GC reaction, the production of Mice with COX-1 deficiency differ from WT mice in their influenza-specific Ab was much lower in COX-12/2 mice (Fig. 2E). responses to H1N1 influenza virus infection These observations suggest that the deletion of COX-1 diminishes To explore the potential roles of COX-1 in TFH cells, COX-1 the responses of TFH cells in influenza infection model. deficiency mice (COX-12/2) and WT controls were intranasally infected with PR8 H1N1 influenza virus (A/Puerto Rico/8/34). COX-1 deficiency impairs the development of TFH cells during Results showed that COX-12/2 mice displayed a significantly KLH immunization greater loss of body weight when compared with WT littermate For further confirmation, COX-12/2 mice and WT littermates were controls during 7-d influenza infection (Fig. 2A). Histological s.c. immunized with KLH emulsified with CFA, as described pre- Downloaded from analysis revealed that the perivascular and interstitial inflammation viously (43, 44). Mice were sacrificed on day 7 after immunization. http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 2. Mice with COX-1 deficiency differ from WT mice in their responses to H1N1 influenza virus infection. (A–E) COX-12/2 mice and WT littermate controls were infected intranasally with H1N1 influenza virus (n = 6). (A) The body weight was monitored daily postinfection. (B) Representative H&E staining of lungs from COX-12/2 mice and WT controls on day 7 after influenza infection. (C and D) Flow cytometric analysis of TFH and GC B cells in lung mLN and spleen from influenza-infected WT and COX-12/2 mice. Both representative and statistical results were included. (E) The concentrations of influenza-specific IgG in sera were measured by ELISA. In all plots, mean 6 SEM from all mice are shown. Data are representative of three independent experiments. *p , 0.05, **p , 0.01 using two-tailed Student t test. The Journal of Immunology 5

Flow cytometric analysis showed that COX-12/2 mice exhibited peripheral blood was evaluated 4 wk after transplantation + comparable frequencies and absolute numbers of naive CD4 (Supplemental Fig. 2A).TFH cell development was assessed in T cells and activated CD4+ T cells with WT controls in both dLNs reconstituted recipients infected with H1N1 virus 6 wk after and spleen (Fig. 3A). In line with the observations from the in- transplantation (Fig. 4A). Results from the flow cytometry of the fluenza model, the frequencies and absolute numbers of TFH cells spleen and mLN cells on day 8 postinfection are shown in Fig. 4B. 2/2 2/2 in dLNs and spleen were dramatically diminished in COX-1 The reconstitution of TFH cells from COX-1 donors was sig- mice compared with WT controls after immunization (Fig. 3B). nificantly lower than those from WT donors in both mLN and 2/2 + Moreover, TFH cells from spleen of COX-1 mice expressed spleen, whereas the total CD4 T cells were equally derived from 2/2 much lower levels of TFH cell signature genes, including Bcl-6, WT and COX-1 donors (Fig. 4B, Supplemental Fig. 2B) The Cxcr5, and Il-21 (Fig. 3C). The frequencies and absolute numbers reconstitution of GC B cells did not differ significantly between of GC B cells in COX-12/2 mice were consistently decreased WT and COX-12/2 donors (Supplemental Fig. 2C). These obser- when compared with WT littermates (Fig. 3D). These results vations indicated that the reduced frequencies of TFH cells from support the hypothesis that COX-1 plays an important role in the COX-12/2 BM may be caused by defective development of CD4+ development of TFH cells. T cell lineage into TFH cells upon COX-1 deficiency. To test the above possibility, naive CD4+ T cells from either Regulation of TFH cells by COX-1 is T cell intrinsic COX-12/2 or WT mice were adoptively transferred into Tcra2/2 Using mixed BM chimeras, we investigated whether differences in recipients, and H1N1 infection was performed after 2 wk recon- 2/2 + TFH cell development in COX-1 and WT mice reflected the stitution (Fig. 4C) (31, 47). Naive CD4 T cells displayed a function of radiation-sensitive cells from a BM lineage (Fig. 4A). comparable proliferation rate between WT and COX-12/2 mice as BM cells from COX-12/2 mice (CD45.2+) and their congenic WT measured by Ki-67 staining (Supplemental Fig. 2D). Flow cyto- Downloaded from + controls (CD45.1 ) were mixed at 1:1 ratio, before being trans- metric analysis showed that the frequencies of TFH cells in lung ferred into lethally irradiated WT recipients (CD45.1.2+) (45, 46). mLNs and spleen were much lower in recipients receiving No clear differences were found in BM reconstitution between COX-12/2 donor cells when compared with those receiving CD45.1+ WT and CD45.2+ COX-12/2 donor mice when the WT controls (Fig. 4D). The frequencies of GC B cells in lung http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 3. COX-1 deficiency impairs the development of TFH cells during KLH immunization. (A–D) WT and COX-12/2 mice were immunized with KLH emulsified in CFA (n = 6). (A) Flow cytometric analysis of naive CD4+ T cells and activated CD4+ T cells from dLNs and spleen at day 7 postimmunization. (B)

Flow cytometric analysis of TFH cells from dLN and spleen. Both representative and statistical results were shown. (C) mRNA expression of TFH cell marker genes 2/2 in TFH cells from WT and COX-1 mice spleen was measured by qRT-PCR, including Bcl-6, Cxcr5, and Il-21. Expression of individual genes in WT was con- 2/2 sidered to be 1; their levels in COX-1 TFH cells were fold changes over WT controls. (D) Flow cytometric analysis of GC B cells from dLN and spleen. Both representative and statistical results were shown. In all plots, mean 6 SEM are shown. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, using two-tailed Student t test. activated CD4+ T cells, CD4+CD62LloCD44hi; GC B, B220+IgDloGL-7+Fas+; naive CD4+ T cells, CD4+CD62LhiCD44lo; ns, no + + + signiﬁcance; TFH cells, CD4 CXCR5 PD-1 . 6 COX-1 REGULATES TFH CELL DEVELOPMENT

FIGURE 4. Regulation of TFH cells by COX-1 is T cell intrinsic. (A) The experimental schematics of mixed BM chimera were indicated. BM cells from COX-12/2 mice (CD45.2+) and their congenic WT controls (CD45.1+) were mixed at a 1:1 ratio before transferred into lethally irradiated WT recipients (CD45.1.2+). The chimeric mice were infected with influenza after 6 wk reconstitution (n = 6). (B) Flow + cytometric analysis of donor-derived CD4 T cells in Downloaded from spleen (upper) and TFH cells in mLN and spleen (middle and lower) from influenza-infected chimeric mice. Both representative and statistical results were shown. CD4+ T cells from spleen were used as an internal control. (C) The experimental schematics of naive CD4+ T cells (CD4+CD62hiCD44lo) transfer + were indicated. Naive CD4 T cells from WT or http://www.jimmunol.org/ COX-12/2 donor mice were transferred into Tcra2/2 recipient mice, followed by H1N1 infection for 7 d after 2 wk (n = 6). (D) Flow cytometric analysis of the proportions of donor-derived TFH cells in lung mLN and spleen. In all plots, mean 6 SEM are shown. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, using two-tailed Student t test. ns, no significance. by guest on October 2, 2021

mLNs and spleen were consistently lower in recipients re- spleen (Fig. 5B, 5C). Consequently, the GC reaction was sub- 2/2 2/2 ceiving COX-1 donor cells (Supplemental Fig. 2E). Alto- stantially enhanced by PGE2 administration in either COX-1 gether, these results indicated that the expression of COX-1 in mice or WT controls (Fig. 5D, 5E). These results indicated that + CD4 T cells facilitates the development of TFH cells. COX-1–derived metabolite PGE2 mediates the effect of COX-1 on TFH cells. PGE2-EP2/4 mediates the effect of COX-1 on TFH cells To explore which receptor(s) mediates the effect of COX-1/PGE2 To investigate the potential mechanism underlying COX-1–mediated on TFH cell development, mRNA expression of receptors for TFH cell development, TFH cells were sorted from the spleen of COX-1–derived prostanoids was evaluated. Results showed that influenza-infected WT and COX-12/2 mice, and the amounts of several receptors, including EP1, EP2, EP4, and TP, were highly COX-1 metabolites were measured by HPLC/MS. Results showed expressed in TFH cells (Fig. 6A). These were similarly expressed that COX-1–deficient TFH cells produced lower levels of PGD2 in COX-1–sufficient and COX-1–deficient TFH cells as indicated 2/2 and PGE2 when compared with COX-1–sufficient TFH cells by qRT-PCR (Fig. 6B). Subsequently, COX-1 mice and WT 2/2 (Fig. 5A). Subsequently, COX-1 mice and their control litter- controls were administrated with agonists for PGE2 receptors mates were injected i.p. with PGD2 or PGE2 during infection with during H1N1 infection, including butaprost (EP2 agonist) and H1N1 influenza virus. Flow cytometry analysis was performed 7 d L-902,688 (EP4 agonist). Flow cytometry was performed 7 d postinfection. Results showed that the defective generation of TFH postinfection. Results showed that butaprost and L-902,688 ef- 2/2 cells from COX-1 mice could be significantly rescued by the fectively enhanced the frequencies of TFH cells in mLN and spleen 2/2 administration of PGE2, but not PGD2, in both lung mLN and in both COX-1 mice and WT controls (Fig. 6C, 6D). The Journal of Immunology 7

FIGURE 5. Administration of PGE2 rescues the 2/2 impaired TFH cells in COX-1 mice. (A) The concentrations of prostanoids in TFH cells from inﬂuenza- infected WT and COX-12/2 mice spleen were measured by HPLC/MS (n =6).(B–E) COX-12/2 mice and their

WT littermates were i.p. administered with PGD2 or Downloaded from PGE2 during H1N1 influenza infection; PBS was used as vehicle control (n = 6). (B and C) The proportions of TFH cells (CD4+CXCR5+PD-1+)inCD4+ T cells in lung mLN (B) and spleen (C)fromPGD2-orPGE2-treated WT and COX-12/2 mice were analyzed by flow cytometry. (D and E)FlowcytometricanalysisofGC B cells (B220+IgDloGL-7+Fas+) in lung mLN and spleen 2/2 http://www.jimmunol.org/ from PGD2-orPGE2-treated WT and COX-1 mice. In all plots, mean 6 SEM are shown. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, using two-tailed Student t test. ns, no significance. by guest on October 2, 2021

The increased TFH cells by butaprost and L-902,688 adminis- cultured murine B cells resulted in decreased eicosanoid production tration consequently enhanced the GC response (Fig. 6E, 6F). andalteredAbproduction(26).Mice lacking COX-1, but not COX-2, However, other agonists for PG receptors displayed minor ef- displayed a defect in Ig class-switching and a lack of Borrelia-specific fects, including sulprostone (EP1/3 agonist), U46619 (TP ago- IgG production, which could be fully recovered by IL-17 adminis- nist), and DK-PGD2 (DP2 agonist). (Supplemental Fig. 3A–D). tration (26). This study indicates that IL-17 mediates the effect of These observations indicated that EP2 or EP4 mediates the effect COX-1 in GC formation and Abs production. Interestingly, it was + of COX-1/PGE2 on TFH cells. reported that IL-17 is an extrinsic stop signal that acts on IL-17RA TFH cells to enable its interaction with B cells in the GC light zone Discussion (50, 51). Blocking IL-17 signaling disrupts the interaction between Prostanoids and their rate-limiting enzymes, COX-1 and COX-2, CD4+ T cells and B cells, which caused impaired GC formation (52). have pleotropic effects on the immune system (48, 49). The de- These reports raise the possibility that defective IL-17 production from tailed mechanisms, however, remain incompletely understood. In COX-1–deficient B cells may contribute to the impaired responses of this study, we demonstrated an important role of COX-1 in the TFH cells observed in this study, which deserves further investigation. development of TFH cells, in which PGE2 and its receptors EP2/EP4 Compared with other T cell subsets and B cells, TFH cells may participate. expressed a much higher level of COX-1, which supports their The contribution of COXs in adaptive immunity has been reported. physiological importance in TFH cells. The deficiency of COX-1 Murine B cells constitutively expressed COX-1 and upregulated ex- significantly impaired the development of TFH cells during influ- pression of both COX-1 and COX-2 after stimulation with Borrelia enza infection or KLH immunization. The responses of GC were burgdorferi or anti-CD40 (26). Inhibition of COX-1 and/or COX-2 in subsequently diminished. Adoptive transfer of naive T cells into 8 COX-1 REGULATES TFH CELL DEVELOPMENT

FIGURE 6. EP2 and EP4 mediate the effect of

COX-1/PGE2 on TFH cells. (A)mRNAexpressionofre- ceptors for prostanoids in TFH cells from inﬂuenza- infected mice spleen was measured by qRT-PCR. Results are representative of three independent experiments. (B) mRNA expression of receptors for prostanoids in TFH cells from inﬂuenza-infected WT and COX-12/2 mice spleen was determined by qRT-PCR (n =3).(C–F) 2/2

COX-1 mice and WT littermates were i.p. adminis- Downloaded from tered with butaprost (EP2 agonist) and L-902,688 (EP4 agonist) during inﬂuenza infection; PBS was used as vehicle control (n =6).(C and D) The frequencies of TFH cells (CD4+CXCR5+PD-1+)inlungmLN(C) and spleen 2/2 (D)fromPGD2-orPGE2-treated WT and COX-1 mice were analyzed by ﬂow cytometry. Both representative and statistical results were shown. (E and F) Flow cytometry http://www.jimmunol.org/ analysis of GC B cells (B220+IgDloGL-7+Fas+)inlung mLN (E)andspleen(F)fromPGD2-orPGE2-treated WT and COX-12/2 mice. Both representative and statistical results were shown. In all plots, mean 6 SEM are shown. Data are representative of three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, using two-tailed Student t test. by guest on October 2, 2021

recipient mice showed that T cells with COX-1 deficiency dis- application of the COX-1 inhibitor may have side effects on hu- played a compromised capability in their differentiation into TFH moral immunity and the development of related disorders. cells, which indicates that the effect of COX-1 on TFH cells is intrinsic to T cells. Administration of PGE2 or EP2/EP4 agonist Acknowledgments significantly rescued the defective TFH cells responses in COX-1– We thank Dmitry I. Gabrilovich for helpful advice and comments during deficient mice. However, we need to point out that these observa- preparation of this manuscript. tions do not exclude the possibility that prostanoids (or other cytokines) from activated B cells might drive the upregulation of Disclosures COX-1 and its downstream prostanoids in TFH cells. It was reported that COX-1 mRNA was upregulated by arachidonic acid and The authors have no financial conflicts of interest. prostanoids in keratinocytes (53). It is therefore possible that the proximity of TFH cells to activated B cells might drive the COX-1 References elevation in TFH cells. Use of the conditional knockout of COX-1 in 1. Schaerli, P., K. Willimann, A. B. Lang, M. Lipp, P. Loetscher, and B. Moser. CD4+ T cells would help to distinguish the roles of COX-1 in 2000. CXC chemokine receptor 5 expression defines follicular homing T cells T cells and B cells. with B cell helper function. J. Exp. Med. 192: 1553–1562. 2. Breitfeld, D., L. Ohl, E. Kremmer, J. Ellwart, F. Sallusto, M. Lipp, and In summary, this study has demonstrated that COX-1 represents a R. Fo¨rster. 2000. Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production. J. Exp. Med. novel regulator of TFH cells, which provides important evidence 192: 1545–1552. about the potential roles of COX-1 in the protection against virus 3. Crotty, S. 2011. Follicular helper CD4 T cells (TFH). Annu. Rev. Immunol. 29: infection, vaccination response, and autoimmune diseases. Clinical 621–663. The Journal of Immunology 9

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Cyclooxygenase-2-dependent prostacyclin formation and nucleotide-linked prostaglandin receptors. J. Lipid Res. 41: 873–881. Supplemental Figure 1. Documentation of in vitro T cell subset differentiation and evidence that in vivo COX-1 has a minor role in other CD4+ T cells. (A) RT-PCR for subset-defining gene expression following in vitro subset differentiation of T cells. (B-D) COX-1-/- mice and their littermate WT controls were infected intranasally with influenza virus A/PR8 (n=6). Flow cytometry analysis of total CD4+ T cells (CD4+) (B) Naïve CD4+ T cells (CD4+CD62LhiCD44lo) and activated CD4+ T cells (CD4+CD62LloCD44hi) (C) Treg cells (CD4+Foxp3+) (D) In all plots, mean ± SEM are shown. ns: no significance, using two tailed Student’s test. Data are representative of three independent experiments. Supplemental Figure 2.The reconstitution efficiency of mixed bone marrow chimera and GC B cell profiles in BMT experiment and naïve CD4+ T cell adoptive transfer experiment. (A) Peripheral blood was collected and labelled with CD45.1 and CD45.2 at 4 weeks after mixed bone marrow transplantation to analysis the reconstitution of chimeric mice. (B) Flow cytometric analysis of donor-derived CD4+ T cells in mLN from influenza-infected chimeric mice. (C) Flow cytometric analysis of donors-derived GC B (B220+GL-7+Fas+) cells in mLN and spleen from mixed bone marrow chimeric Tcrα-/- mice. (D) The proliferation of naïve CD4+ T cells in spleen of WT and COX-1-/- mice under steady state condition was measured by Ki-67 staining. (E) Flow cytometric analysis of GC B (B220+GL-7+Fas+) cells in mLN and spleen in naïve CD4+ T cell transfer model. In all plots, mean ± SEM are shown. ns: no significance, *P < 0.05, using two tailed Student’s test. Data are representative of three independent experiments.

Supplemental Figure 3. EP1/3, TP and DP2 agonists had minor role in TFH cell development. (A-D) COX-1-/- mice and their WT littermates were administrated with Sulprostone (EP1/3 agonist), U46619 (TP agonist) or DK-PGD2 (DP2 agonist) during influenza virus infection, and PBS was used as vehicle control. TFH cells (CD4+CXCR5+PD-1+) (A, B) and GC B (B220+IgDloGL-7+Fas+) cells (C, D) in lung mLN and spleen were evaluated by flow cytometry. In all plots, mean ± SEM are shown. ns: no significance, using two tailed Student’s test. Data are representative of three independent experiments. Table S1. Sequences of primers used this study Gene Forward primer Reverse primer β-actin 5'-CGGATGTCAACGTCACACTT-3' 5'-GGCCAGGTCATCACTATTGG-3' Bcl-6 5'-CCTGTGAAATCTGTGGCACTCG-3' 5'-CGCAGTTGGCTTTTGTGACG-3'

Cxcr5 5’-ATGAACTACCCACTAACCCTGG -3’ 5’-TGTAGGGGAATCTCCGTGCT-3’

Ccr7 5’-TGTACGAGTCGGTGTGCTTC-3’ 3’-GGTAGGTATCCGTCATGGTCTTG-5’

Il-21 5’-GGCTGCCTTACTCCTGCTG-3’ 5’-TCATCTTGCCAGGTGAGACTG-3’

Cox-1 5'-ACAGTATCACCTGCGGCTCT-3' 5'-GGAAGCAACCCAAACACCT-3'

Cox-2 5'-TCTTTGCCCAGCACTTCAC-3' 5'-ACACCTCTCCACCAATGACC-3'

DP1 5'-GCTTTCTGTGCGCTCCCCTTTG-3' 5'-CATCCGGAATACTGAAGTCCTG-3'

DP2 5'-CATGTGCTACTACAACTTGC-3' 5'-GCAGACTGAAGATGTGGTAGG-3'

EP1 5'-CGCAGGGTTCACGCACACGA-3' 5'-CACTGTGCCGGGAACTACGC-3' EP2 5'-AGGACTTCGATGGCAGAGAGAC-3' 5'-CAGCCCCTTACACTTCTCCAATG-3'

EP3 5'-AGGGAAATGATGGCACGA-3' 5'-GCTGTCCGTCTGTTGGTC-3'

EP4 5'-TGGCTGAGGTTGGAGGTA-3' 5'-GAAGTAGGCGTGGTTGAT-3'

FP22 5'-TATCAACGGAGGCATAGC-3' 5'-CGACTGGCAAGTTTATACAGG-3'

TP 5'-CTCGGGACACAGCGCGGTGAC-3' 5'-GATATAGACCCAGGGGTCCAAG-3'

IP 5'-GGCACGAGAGGATGAAGTTT-3' 5'-GTCAGAGGCACAGCAGTCAATGG-3'