Published June 2, 2014, doi:10.4049/jimmunol.1300429 The Journal of Immunology

Lysophosphatidic Acid Receptor 5 Inhibits B Cell Antigen Receptor Signaling and Antibody Response

Jiancheng Hu,*,1,2 Shannon K. Oda,*,2,3 Kristin Shotts,* Erin E. Donovan,* Pamela Strauch,* Lindsey M. Pujanauski,*,4 Francisco Victorino,* Amin Al-Shami,† Yuko Fujiwara,‡ Gabor Tigyi,‡ Tamas Oravecz,† Roberta Pelanda,* and Raul M. Torres*

Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, traf- ficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature

murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Ga12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5- triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity. The Journal of Immunology, 2014, 193: 000–000.

ignals transmitted by the B cell AgR are not only required bound by IgG signals simultaneously via the BCR and FcgRIIB; for the Ab response but also for the development and signals transmitted by FcgRIIB dampen BCR signaling, thus at- S survival of B lymphocytes (1, 2). BCR signaling by mature tenuating the Ab response (3, 6). B cells can be either positively or negatively regulated by addi- In addition to these established BCR coreceptors, lymphocytes tional surface coreceptors depending on the developmental stage express G –coupled receptors (GPCRs) for chemokines of the B cell, the nature of Ag, and the microenvironment where and lysophospholipids, and both of these ligands were reported to the Ag is encountered (3, 4). For example, BCR signaling is more modify lymphocyte AgR signaling (7, 8). GPCRs signal primarily effective when complement-decorated Ag simultaneously engages via associated abg heterotrimeric G , and a pertussis toxin– the BCR and CD21/CD19 coreceptor complex compared with insensitive Ga subunit has long been known to regulate BCR sig- BCR signaling alone (5). In contrast, a B cell encountering Ag naling (9, 10). However, neither the identity of this Ga protein nor

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. the mechanism(s) by which any of these GPCRs regulate AgR *Integrated Department of Immunology, University of Colorado Denver and National signaling in B lymphocytes has been established. Jewish Health, Denver, CO 80206; †Lexicon Pharmaceuticals, Inc., The Woodlands, Sphingosine-1-phosphate (S1P) and lysophosphatidic acid TX 77381; and ‡Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163 (LPA) are biologically active serum lysophospholipids that can signal extracellularly by engaging cognate GPCRs expressed by 1Current address: Department of Pathology and Immunology, Washington University, St Louis, MO. diverse cell types (11). Notably, S1P has emerged as an important 2J.H. and S.K.O. contributed equally to this work. chemoattractant that guides leukocytes during development, ho- 3Current address: Division of Basic Sciences, Fred Hutchinson Cancer Research meostasis, and inflammation (12–16). LPA is another major lyso- Center, Seattle, WA. phospholipid that can signal via six established LPA GPCRs,

http://classic.jimmunol.org 4 Current address: Laboratory of Infectious Diseases, National Institute of Allergy and LPA1–6, each capable of associating with members from distinct Infectious Diseases, National Institutes of Health, Bethesda, MD. Ga families (17, 18). These LPA GPCRs are encoded by two Received for publication February 12, 2013. Accepted for publication May 5, 2014. clusters that include LPA1-2-3 in the endothelial differentiation This work was supported by the National Institutes of Health (Grants AI052157 to gene family, whereas LPA4-5-6 are closely related to the purinergic R.M.T., AI052310 to R.P., and AI08405 to G.T.), the Cancer League of Colorado (to GPCR subfamily (18). R.M.T.), Cancer Research Institute Special Emphasis Program in Tumor Immunology awards (to J.H., S.K.O., and K.S.), and National Institute for Allergy and Infectious The plasma and tissue concentrations of LPA are in the hun-

Downloaded from Diseases Training Grant T32-AI07405 (to E.E.D. and L.M.P.). dred nanomolar range, whereas, in serum, inflammatory exudates Address correspondence and reprint requests to Dr. Raul M. Torres, University of or tumor cell effusates can reach as high as 10 mM (11, 17, 19, 20). Colorado Denver and National Jewish Health, 1400 Jackson Street, Denver, CO The biological function of LPA has received the most attention 80206. E-mail address: [email protected] with respect to cancer; it was shown to promote cell migration, The online version of this article contains supplemental material. proliferation, and survival of a number of diverse cancer cell types Abbreviations used in this article: A20-shG12/13, A20 cell line constitutively dox- (18, 21, 22). Moreover, LPA levels were found to be significantly ycyclineinducible expressing Ga12 and Ga13 shRNA; A20/shGaq, A20 cell line constitutively expressing Gaq sh RNA; ER, endoplasmic reticulum; ES, embryonic elevated with infection (23), inflammation (24), and particular stem; faf, fatty acid free; FO, follicular; GPCR, G protein–coupled receptor; IP3, cancers (19, 22). These data led to the notion that LPA contributes inositol 1,4,5-triphosphate; IP3R, inositol 1,4,5-triphosphate receptor; LPA, lysophos- phatidic acid; MZ, marginal zone; NP, 4-hydroxy-3-nitrophenylacetic acid; OTP, to the promotion and metastasis of cancer (22) and have focused octadecenyl thiophosphate; PLCg2, phospholipase C-g2; shRNA, short hairpin attention on modulating LPA in vivo as a possible therapeutic 2+ RNA; SOC, store-operated Ca channel; S1P, sphingosine-1-phosphatase; TI-2, T approach (18). independent, type 2. In this study, we show that the LPA5 GPCR expressed by mature Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 B lymphocytes negatively regulates BCR signaling by inhibiting

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300429 2LPA5 NEGATIVELY CONTROLS BCR SIGNALING AND Ab RESPONSE

calcium release from intracellular stores via an LPA5–Ga12/13– MA) for the indicated times and quenched in ice water. Cells were lysed in Arhgef1 pathway. We further demonstrate that LPA also dimin- RIPA buffer and used to prepare whole-cell lysates, as described previously 3 ishes the activation and Ab response of Ag-specific B cells upon (29). Briefly, 30 ml whole-cell lysates was mixed with 6 ml2 SDS loading buffer and run on SDS-PAGE after boiling at 90–95˚C for 5 min. After engaging cognate Ag. Cumulatively, these data show that LPA SDS-PAGE, proteins were transferred to nitrocellulose membranes (Bio-Rad, directly regulates B lymphocyte activation and function via the Hercules, CA) and blocked with 5% nonfat milk–TBS solution at room LPA5 GPCR that serves as a negative coreceptor for the BCR. temperature for 30 min. Membranes were incubated with primary Ab in 5% nonfat milk–TBS solution for 1 h at room temperature or overnight at 4˚C, washed three times with Tween 20 and Tris-buffered saline, incubated with Materials and Methods HRP-labeled secondary Ab in 5% nonfat milk–TBS solution for 45 min, and Mice washed four times with Tween 20 and Tris-buffered saline. Target proteins C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME), C57BL/6-IghB1-8/B1-8 were detected by standard chemiluminescence and quantified by densitometric mice (25) (gift of Dr. Klaus Rajewsky, Harvard University, Boston, MA), analysis using ImageJ software (National Institutes of Health, Bethesda, MD). Arhgef12/2 mice (26), Lpar22/2 mice (27) (gift of Dr. Jerold Chun, Scripps Research Institute, La Jolla, CA), B6.C20 mice (C57BL/6 mice Intracellular calcium measurements congenic for Igha; gift of Dr. Leonore Herzenberg, Stanford University, 3 7 2/2 Approximately 0.5–1 10 cells/ml were resuspended in IMDM (Invi- Stanford,CA),andLpar5 mice were bred and maintained within the trogen, Grand Island, NY) with 2.5% fatty acid–free (faf) BSA plus 5 mM Biological Resource Center at National Jewish Health and used in accor- Indo-1-AM (Molecular Probes, Grand Island, NY) and incubated at 37˚C dance with the regulations of the Institutional Animal Care and Use 2/2 for 30 min. Subsequently, cells were washed twice with IMDM with faf Committee. Lpar5 mice were backcrossed to C57BL/6 mice for at least 2.5% BSA and resuspended at 2 3 106 cells/ml in IMDM with faf 2.5% three or four generations before these analyses. BSA. Cell aliquots were stored at room temperature for analysis. The Indo- Generation of Lpar52/2 mice 1-AM 400/475 fluorescence ratio was acquired as a function of time, using an LSR (BD Biosciences, Sparks, MD) or MoFlo (Beckman Coulter, Brea, The Lpar5-targeting vector was derived using the L KOS system (28). CA) cytometer. After a baseline reading of 30 s, A20 or splenic B cells The L KOS phage library was screened by PCR using Lpar5-specific were stimulated with anti-IgG F(ab9)2 (2 mg/ml) or anti-IgM F(ab9)2 primers: forward 59-GTTCTGCCTGGGCGTGTG-39 and reverse 59- (20 mg/ml), respectively, with or without 1-20 mM LPA (16:0, 1-palmitoyl- GCCAGCAGGAGGCGCAC-39. Five pKOS genomic clones, (pKOS-28, 2-hydroxy-sn-glycero-3-phosphate; Avanti Polar Lipids; critical micelle pKOS-58, pKOS-68, pKOS-81, and pKOS-92) were isolated and confirmed concentration = 0.54 mM), and readings were continued for an additional by sequence and restriction analysis. Gene-specific arms (59-CTACGAT- 5–6 min. We note that the maximum concentration of LPA used in GCCTCAGACTAATTTCTCTTCCCACCTGGACAT-39 and 59-CTGCA- experiments, 20 mM, is .25-fold below the critical micelle concentration. CACTAGAGCTGGAGTTGTTTCAAAGTCCAAGTAC-39) were appended In some experiments 18:0, 18:1, and 20:4 species of LPA, phosphatidic by PCR to a yeast-selection cassette containing the URA3 marker. The acid 16:0 (1,2-dihexadecanoyl-sn-glycero-3-phosphate), or phosphatidyl- yeast selection cassette and pKOS-58 were cotransformed into yeast, glycerol 16:0 (1,2-dipalmitoyl-sn-glycero-3-phospho-(19-rac-glycerol)) were and clones that had undergone homologous recombination to replace a used (Avanti Polar Lipids, Alabaster, AL). To distinguish endoplasmic 1200-bp region containing the single coding exon with the yeast selection reticulum (ER) calcium release from extracellular calcium influx, 4 mM cassette were isolated. The yeast cassette was subsequently replaced with a EGTA was added to the cell solution before stimulation, and 8 mM Ca2+ LacZ/Neo selection cassette to complete the Lpar5-targeting vector. The was added 210 s after stimulation. When used to discharge intracellular NotI linearized targeting vector was electroporated into 129/SvEvBrd (Lex-1) calcium stores, 1 mM thapsigargin (Alomone Labs, Jerusalem, Israel) was embryonic stem (ES) cells. G418/FIAU-resistant ES cell clones were added to cells to empty ER calcium stores. isolated, and correctly targeted clones were identified and confirmed by In some experiments, A20 cells were loaded with caged-IP3 (cat. no. cag- Southern blot analysis. Five targeted ES cell clones were identified and iso-2-145; SiChem, Bremen, Germany) and Indo-1-AM and resuspended 2+ by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. microinjected into C57BL/6 (albino) blastocysts to generate chimeric in IMDM supplemented with 2.5% faf BSA and 4 mMEGTA.Ca animals that were bred to C57BL/6 (albino) females; the resulting hetero- concentration was recorded for 45 s to establish basal Ca2+ levels, after zygous offspring were interbred to produce homozygous Lpar5-deficient which samples were exposed to UV light (330 6 25 nm) for 1 s to release +/2 mice. Mating of Lpar5 mice generated pups of the three possible gen- IP3, and cells were monitored for an additional 3 min. otypes with ratios that fit well with normal Mendelian frequencies. Lpar52/2 mice exhibited no substantial difference in growth rate and size. LPA receptor expression Abs and flow cytometry Lpar mRNA expression was determined in mature follicular (FO) B cells identified by surface phenotype as CD23+CD21intermediate and purified Rabbit anti-mouse IgG F(ab9) (Zymed Laboratories, San Francisco, CA) 2 using a MoFlo (Beckman Coulter) cell sorter. RNA was isolated using and goat anti-mouse IgM F(ab9) (SouthernBiotech, Birmingham, AL) 2 TRIzol reagent (Invitrogen Life Technologies), and DNA was removed were used to signal via the BCR on A20 B lymphoma cells and primary http://classic.jimmunol.org using a DNA-free kit (Ambion, Grand Island, NY). cDNA was prepared B cells, respectively. Abs used for Western blot analyses were anti–b- from equivalent amounts of RNA using a SuperScript III First-Strand (AC-40; Sigma, St. Louis, MO); anti-Syk, anti-Btk, anti–phospholipase C-g2 Synthesis System for RT-PCR (Invitrogen Life Technologies). Quantita- (PLCg2), anti–phospho-Syk (mouse Tyr519/520 or Tyr323), anti–phospho-Btk tive PCR amplification was performed using Platinum SYBR Green qPCR (Ser180; 3D3), and anti–phospho-PLCg2(Tyr759) (Cell Signaling Technol- SuperMix-UDG (Invitrogen Life Technologies) and detected on an MJ ogy, Beverly, MA); and rabbit anti-Ga12 (S-20), anti-Ga13 (A-20), anti- Research DNA Engine Opticon 2 Real-Time PCR machine. Primers for the Gaq (E-17), HRP-labeled goat anti-mouse IgG, and donkey anti-rabbit IgG LPA receptors were the following: LPA : forward, 59-CTGTGGTCATT- (Santa Cruz Biotechnology, Santa Cruz, CA). 1 GTGCTTGGTG-39,LPA1: reverse, 59-CATTAGGGTTCTCGTTGCGC-

Downloaded from The following fluorescently labeled mAbs were used for flow cyto- 39;LPA: forward, 59-GGCTGCACTGGGTCTGGG-39,LPA: reverse, 59- metric analysis: B220 (RA3-6B2; BD Pharmingen, San Jose, CA and 2 2 GCTGACGTGCTCCGCCAT-39;andLPA:forward,59-GCGCACAGGA- eBioscience, San Diego, CA), CD69 (H1.2F3; eBioscience), CD86 (GL1; 3 ATGGGAGAG-39,LPA: reverse, 59-GAGCTGGAGGATGTTGGGAG-39. eBioscience), Igl (JC5-1; SouthernBiotech), CD23 (B3B4; BD Pharmingen), 3 Primers from LPA and LPA were purchased from Super Array Bio- and CD21 (7G6; BD Pharmingen). 4-Hydroxy-3-nitrophenylacetic acid 4 5 sciences (Frederick, MD). The cycle threshold (Ct) was determined for (NP)-specific B cells were gated on 7-aminoactinomycin D–negative viable each experiment based on background from control samples (no cDNA). cells and identified by flow cytometry after staining with Alexa Fluor 647– Expression of Lpar mRNA of interest was normalized to HPRT mRNA and coupled NP-CGG (Biosearch Technologies, Petaluma, CA). Flow cyto- 2 calculated using the equation 2 DCt 3 100. metric analyses were performed on either a FACSCalibur or a BD LSR II (both from BD Bioscience, Sparks, MD) and analyzed with FlowJo v8.8.6 software (TreeStar, Ashland, OR). Cell culture and transfection Western blot analysis A20 B lymphoma cells (American Type Culture Collection; TIB-208), HEK-293FT cells (Invitrogen), and Phoenix retroviral packaging cells Splenic B cells were isolated by negative enrichment using anti-CD43 were maintained in DMEM (Mediatech, Herndon, VA) supplemented with magnetic beads (Miltenyi Biotec, San Diego, CA), resuspended in FBS- 10% FCS, 1000 U/ml penicillin, 100 mg/ml streptomycin, and GlutaMAX. free DMEM, and incubated for 15 min at 37˚C. Cells were stimulated HEK-293FT cells were transfected with retroviral short hairpin RNA with 20 mg/ml anti-IgM F(ab9)2 in the presence of 20 mMLPAor,insome (shRNA) vectors and packaging vectors using PolyFect transfection reagent experiments, with 1 mM Syk inhibitor, BAY61-3606 (EMD Millipore, Billerica, (QIAGEN). Approximately 12 h later, fresh medium was added, and retroviral- The Journal of Immunology 3

containing supernatant was collected 24 h later (36 h after transfection) and at day 0 before immunization; NP-specific (allotype-specific) IgM ELISAs used to transduce A20 cells. and ELISPOTs were performed 7 d later, as described previously (32). To establish stable A20 cell lines constitutively expressing either Gaq shRNA (A20/shGaq) or doxycycline-inducible Ga12 and Ga13 shRNA Statistical and data analyses (A20-shG12/13), cells were infected using HEK-293FT supernatants previ- ously transfected with the appropriate lentiviral vectors. Fluorescent-positive Data were graphed and analyzed using Prism v5.0a software (GraphPad, cells were sorted by flow cytometry on day 5 postinfection and cultured in La Jolla, CA). Statistical significance was assessed with a Student t test standard medium. After another 5 d, cells were sorted again and cultured with unequal variance, and the appropriateness of one-tailed or two-tailed + a + significance was determined on an individual-experiment basis. The until cells were 100% GFP (A20/shG q) or Venus (A20-shG12/13) posi- , tive. These stable cell lines were used for further experiments. To inhibit the p values 0.05 were considered significant. expression of Ga12 and Ga13, these corresponding stable cell lines were cultured in medium containing 1 mg/ml doxycycline (Sigma) for 5 d. Plas- mids (and source) used in these experiments were pLX_mU6-Gq-I-UGIH Results (ATCC; 10629198) for Gaq knockdown and pSLIK-Venus-TmiR-G12-G13 LPA inhibits AgR-induced calcium mobilization (30) (gift of Drs. Iain Fraser and Mel Simon, California Institute of Tech- nology, Pasadena, CA) for simultaneous doxycycline-induced knockdown of The S1P and LPA lysophospholipids were shown to modulate B Ga12 and Ga13. The control shRNA vector pLX-mU6-I-UGIH for pLX- and T cell AgR proliferation and cytokine secretion (7, 33–35). mU6-Gq-I-UGIH was constructed by deleting the Gaq targeting sequence However, the mechanism(s) by which these lysophospholipids by site-directed mutagenesis (Stratagene, Santa Clara, CA). regulate AgR signaling is not established, and we questioned To generate LPA receptor shRNA vectors, the following shRNA sequences were cloned into the pMSCV-LTRmiR30-PIG retroviral vector whether LPA could influence early BCR-signaling events. LPA obtained from Open Biosystems (Huntsville, AL). Lpar5 sense oligo: 59- alone, at concentrations as high as 20 mM, did not mobilize in- TGCTGTTGACAGTGAGCGAGCGAGATACACATCGTTTGCATGTAGT- tracellular Ca2+ in either the A20 B lymphoma cell line or murine GAAGCCACAGATGTACATGCAAACGATGTGTATCTCGCGTGCCTAC- splenic B lymphocytes (Fig. 1A), similar to a previous report (35). TGCCTCGGA-39 or luciferase control oligo: 59-TGCTGTTGACAGTGAGC- However, the ability of the BCR to signal an increase in intra- GAGCTTACGCTGAGTACTTCGAGTAGTGAAGCCACAGATGTACTCG- 2+ AAGTACTCAGCGTAAGCGTGCCTACTGCCTCGGA-39. These vectors cellular Ca was inhibited when B cells were simultaneously contain an internal ribosomal entry site with GFP as a reporter and a puro- treated with LPA in a dose-dependent manner including at path- mycin resistance gene. For virus production, Phoenix cells were transfected ophysiological concentrations (Fig. 1A). Multiple molecular spe- with 10 mg retroviral constructs and 2 mg pCL-ECO retroviral packaging cies of LPA exist in biological fluids (19), and we found that 16:0, construct/plate using Lipofectamine (Invitrogen). Medium was replaced after 24 h, and supernatant was collected 48 and 72 h after transfection. 18:0, 18:1, and 20:4 LPA all similarly inhibited BCR-mediated 2+ Immature B cells expressing LPA5 or control shRNA were produced by increases in intracellular Ca (E.E. Donovan and R.M. Torres, retroviral transduction of 3-83Igi, H-2d bone marrow cells (31) using spin unpublished observations). LPA 16:0 was used for all subsequent infection. 3-83Igi, H-2d bone marrow cells were isolated (day 0) and cul- experiments; we note that the critical micelle concentration for tured in complete IMDM supplemented with 50–100 U/ml IL-7 for 2 d, 6 16:0 LPA is 0.54 mM and considerably higher than 20 mM, the as described (13). On days 2 and 3, 8 3 10 cells/well, in a 24-well plate, were retrovirally transduced with 1 ml Phoenix cell supernatants, 0.5 ml highest LPA concentration used in our studies. LPA inhibition complete IMDM, 3.2 mg/ml Polybrene, and IL-7 during centrifugation at of anti-IgM–stimulated intracellular calcium mobilization by sple- 2300 rpm at ∼22˚C for 120 min. Two hours after spin infection, medium nic B cells was also specific, inasmuch as phosphatidic acid or was replaced with complete IMDM supplemented with IL-7. Cells were phosphatidylglycerol was not able to alter BCR calcium signaling cultured with IMDM supplemented with IL-7 on days 4 and 5. On day 5, by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. before cells were used in calcium assays, they were washed of IL-7 and (Supplemental Fig. 1A). In the presence of 20 mM LPA, peak incubated for 2 h in calcium medium for serum starvation. intracellular calcium levels of wild-type splenic B cells were routinely and significantly (n =5,p , 0.001) reduced to 75% of In vitro B cell activation the levels observed in the absence of LPA. Furthermore, if B cells Single-cell suspensions were prepared from the spleens of 8-wk-old C57BL/ were pretreated with LPA for .30 min in advance, this inhi- 6-IghB1-8/B1-8 mice after RBC lysis, and B cells were purified using negative bition was lost (Supplemental Fig. 1B), presumably as a result of selection with anti-CD43 magnetic MicroBeads and an autoMACS (Miltenyi GPCR desensitization. Thus, LPA negatively and specifically regu- Biotec). B cell purity, as determined by flow cytometric analysis, was always 2+ 6 lates B cell AgR Ca signaling, presumably by engaging its cog- .95%. B cells were resuspended at 2 3 10 cells/ml and stimulated with nate GPCR(s) expressed on the B cell surface. 10 ng/ml NP28-Ficoll (Biosearch Technologies) in the presence of 10 ng/ml 2+ http://classic.jimmunol.org rBAFF (R&D Systems, Minneapolis, MN), to promote B cell survival, and The increase in intracellular Ca levels after BCR stimulation with or without 20 mMLPAor50mM sterile-filtered octadecenyl thio- ultimately results from the activation of PLCg2 (36, 37). PLCg2 phosphate (OTP). After 24 h, cells were collected, stained, and analyzed for is activated upon tyrosine phosphorylation and hydrolyzes the expression of Igl,CD86,andCD69byflowcytometry. phosphatidylinositol-4,5-bisphosphate to produce inositol 1,4,5-tri- Mixed bone marrow chimeras phosphate (IP3), which subsequently engages IP3 receptors (IP3Rs). 2+ IP3Rs are ligand-gated Ca -release channels expressed on the Mixed bone marrow adoptive transfers were performed as described (32). 2+ Downloaded from Briefly, bone marrow cells were isolated from 10–12-wk-old Lpar52/2 (Ighb), surface of the ER that mediate the release of intracellular Ca +/+ b a Lpar5 littermates (Igh ) and B6.C20 (Igh ) donor mice, mixed at a ratio of stores on binding IP3 (reviewed in Refs. 36–38). In B cells, Syk, either 1:1 or 1:4 (Ighb:Igha), and a total of 1 3 106 cells was injected i.v. into Lyn, and Btk tyrosine kinase activities are required for the activation 6–8-wk-old C57BL/6 recipients that had been lethally irradiated (2 3 500 of PLCg2 (36). To assess whether LPA regulates BCR-mediated rad). At 5–6 wk postreconstitution, PBLs from chimeric mice were ana- Ca2+ mobilization by altering the activation of PLCg2 or by acti- lyzed by flow cytometric analysis to ensure engraftment of both IgMb-and IgMa-expressing B cells and to measure reconstitution frequencies in the vating tyrosine kinases, splenic B cells were isolated and stimulated experimental chimeras compared with the control chimeras. Recipient mice with anti-IgM F(ab9)2 in the absence or presence of LPA. Fig. 1B 2 2 reconstituted with IgMb-expressing Lpar5 / and Lpar5+/+ B cells at fre- and Supplemental Fig. 2 show that tyrosine phosphorylation of Syk, quencies between 25 and 35% (1:1 transferred ratio) or 5 and 25% (1:4 Btk, and PLCg2 was similar in the absence or presence of 20 mM transferred ratio) were immunized at 6–7 wk postreconstitution; serum NP– specific Ab responses were measured by ELISA 7 d later, and the total number LPA, suggesting that Syk, Btk, or PLCg2 activation is not influ- and frequency of splenic IgMb-andIgMa-expressing B cells were enumerated. enced by LPA signaling. These data further suggest that LPA in- hibition of BCR Ca2+ signaling is downstream of PLCg2 activation. Immunizations, ELISA, and ELISPOT IP3 concentration is tightly regulated by inositol lipid kinases Mice were immunized i.p. as indicated with 5 mg NP hapten conjugated to and phosphatases (39), and decreased IP3 production can lead to 2+ NP28-Ficoll (Biosearch Technologies, Petaluma, CA). Serum was obtained impaired intracellular Ca mobilization. To address whether LPA 4LPA5 NEGATIVELY CONTROLS BCR SIGNALING AND Ab RESPONSE

2+ FIGURE 1. LPA inhibits BCR-mediated intracellular Ca mobilization. (A) A20 B cells or primary splenic B cells were stimulated (arrow) with F(ab9)2 anti-IgG (2 mg/ml) or anti-IgM (20 mg/ml), respectively, in the absence or presence of increasing concentrations of LPA; intracellular Ca2+ levels were measured by flow cytometry over time. LPA (20 mM) in the absence of BCR signaling is shown as a thin dotted line. Data are representative of four independent experiments. (B) LPA does not influence anti-Ig–induced phosphorylation of Syk, Btk, or PLCg2. Primary splenic B cells were stimulated with

F(ab9)2 anti-IgM (20 mg/ml) in the presence or absence of 20 mM LPA for the indicated times. After stimulation, total and phosphorylated Syk, Btk, and PLCg2 were measured in whole-cell lysates. Numbers below Western blots indicate fold induction over normalized time 0 protein levels. Data are rep- 2+ resentative of three experiments. (C) LPA inhibits IP3-induced intracellular Ca release. A20 cells were loaded with caged-IP3; in the presence of 4 mM EGTA, Ca2+ concentration was recorded for 45 s to establish basal Ca2+ levels. Samples were exposed to UV light (330 6 25 nm) for 1 s to release caged

IP3 and monitored for an additional 3 min (solid line). Dashed line represents samples that were treated with 5 mM LPA prior to UV exposure. Data are representative of two experiments with similar results.

2+ inhibition of Ca mobilization resulted from altered IP3 produc- was inhibited by LPA correlated with the amount of subsequent 2+ tion, we exploited a membrane-permeable ester of IP3 to introduce extracellular Ca influx. 2+ by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. exogenous photoactivatable IP3 into the cytosol after UV expo- Ca concentration within the ER is monitored by STIM1; upon sure. A20 B cells were loaded with the membrane-permeable es- depletion of ER Ca2+ stores, STIM1 activates the plasma mem- 2+ 2+ ter of IP3, and the release of Ca from intracellular stores was brane SOCs, facilitating entry of extracellular Ca (38, 40, 41). 2+ measured after liberating IP3 in the absence of BCR stimulation. Although both Ca store release and extracellular influx appeared As expected, introduction of exogenous IP3 into A20 cells resulted attenuated by LPA, we asked whether the reduced extracellular in a rapid and transient increase in intracellular Ca2+ (Fig. 1C). Ca2+ entry might be an indirect consequence of reduced store However, in the presence of 5 mM LPA, this Ca2+ increase was release. ER Ca2+ stores can be artificially depleted with thapsi- reduced considerably, providing strong evidence that LPA inhi- gargin, a pharmacologic inhibitor of the ER resident Ca2+ ATPase 2+ bition of intracellular Ca mobilization is independent of IP3 (42) that functions to refill ER stores by transporting cytosolic 2+ http://classic.jimmunol.org production. These data indicate that LPA regulates BCR signaling Ca back into the ER. To assess whether LPA can directly in- 2+ downstream of PLCg2 activation and IP3 generation. hibit SOC activity, intracellular Ca concentration was measured in A20 cells treated with thapsigargin in the absence or presence of LPA inhibits calcium release from intracellular stores 1, 5, or 20 mM LPA. Thapsigargin-treated A20 cells slowly dis- Elevated intracellular Ca2+ levels mediated by BCR signaling are charge Ca2+ from intracellular stores, resulting in elevated cyto- a consequence of both the release of Ca2+ from intracellular stores solic Ca2+ and subsequent SOC activation and extracellular Ca2+ 2+

Downloaded from and the entry of extracellular Ca following activation of store- entry (Fig. 2B). However, under these same conditions, LPA was operated Ca2+ channels (SOCs) in the plasma membrane. We next unable to inhibit extracellular Ca2+ influx at any concentration. evaluated whether LPA regulated either or both of these Ca2+- These data suggest that LPA does not directly inhibit SOCs fol- mobilization events. lowing BCR signaling. To measure Ca2+ release from intracellular stores, A20 cells Thus far, our data indicate that LPA inhibits BCR Ca2+ signaling 2+ were stimulated with anti-IgG F(ab9)2 in the presence of EGTA by reducing the total amount of Ca stores release and the length 2+ 2+ to prevent extracellular Ca influx. Once Ca concentrations of time in which it is released (Fig. 2A). Upon BCR-mediated IP3 returned to baseline levels after BCR stimulation, extracellular production, ER Ca2+ concentration is determined by the opposing 2+ 2+ 2+ Ca was restored, and plasma membrane SOC activity was activities of the IP3RreleasingCa into the cytosol and the Ca measured as Ca2+ entry. These initial experiments revealed that ATPase transporting cytosolic Ca2+ back into the ER. Thus, we rea- both the release of Ca2+ from intracellular stores and extracellular soned that the inhibition of Ca2+ store release by LPA could manifest 2+ 2+ 2+ Ca entry were inhibited by LPA in a dose-dependent manner either by inhibiting IP3R-induced Ca release or by enhancing Ca (Fig. 2A). Specifically, increasing LPA concentration progres- ATPase activity. To discriminate between these possibilities, we sively diminished both the magnitude and duration of intracellular tested whether LPA inhibition of BCR Ca2+ signaling required Ca2+ Ca2+ release. Furthermore, the degree to which Ca2+ store release ATPase activity. Thus, A20 cells were treated with thapsigargin in The Journal of Immunology 5

FIGURE 2. LPA inhibits intracellular Ca2+ stores release. (A) A20 cells were loaded with Indo-1-AM and resuspended in 4 mM EGTA, and intracellular 2+ Ca concentration was measured. At the time point indicated by the first arrow, cells were stimulated with 2 mg/ml F(ab9)2 anti-IgG alone (black line) or with the indicated increasing concentrations of LPA. Once Ca2+ concentration returned to baseline levels, extracellular Ca2+ was restored (second arrow), and Ca2+ entry was measured for approximately three additional minutes. (B) Indo-1–loaded A20 cells were resuspended in EGTA and treated with thapsigargin (1 mM) alone (black line) or with 1 mM (blue line), 5 mM (red line), or 20 mM (green line) LPA (first arrow). Once intracellular Ca2+ levels returned to baseline, extracellular Ca2+ was again restored (second arrow), and SOC activity was measured as Ca2+ entry. (C) Indo-1–loaded A20 cells were by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. resuspended in EGTA and treated (first arrow) with thapsigargin alone (red line). As intracellular Ca2+ stores were discharging, some cells were additionally treated (second arrow) with anti-Ig alone (black line) or together with 5 mM LPA (green line). Once intracellular Ca2+ levels returned to baseline, ex- tracellular Ca2+ was restored (third arrow).

2+ the presence of EGTA to inhibit the Ca ATPase and initiate ER of BCR signaling. To determine whether LPA5 participated in this stores depletion. During thapsigargin treatment, A20 cells were regulation, we initially used RNA interference to inhibit LPA5 ex- stimulated via the BCR in the presence or absence of LPA. These pression. As shown, stable expression of an LPA5 shRNA in the A20 results showed that, in the absence of LPA, thapsigargin-mediated B cell line specifically reduced LPA5 expression by 67% relative to 2+ http://classic.jimmunol.org release of Ca from intracellular stores was increased further by a control shRNA and parental cells (Fig. 3B). Furthermore, BCR BCR stimulation, presumably as a consequence of the additional Ca2+ signaling was no longer attenuated by LPA in either A20 2+ ER Ca release mediated by the IP3Rs engaging BCR-generated cells or primary B cells expressing shRNAs for LPA5 (Fig. 3C). IP3 (Fig. 2C). However, in the presence of LPA, this additional in- These findings show that LPA5 expressed by mature B cells is crease in cytosolic Ca2+ was inhibited. These findings demonstrate responsible for repressing BCR signaling. that LPA can still reduce BCR Ca2+ signaling when the Ca2+ ATPase 2+ AGa12/13-associated LPA receptor inhibits BCR calcium

Downloaded from is inhibited, excluding a direct effect of LPA on the Ca ATPase. Considered together, the results from the above experiments signaling 2+ provide compelling evidence that LPA regulates BCR Ca sig- The LPA5 receptor signals by associating with either Gaqor 2+ naling by inhibition of IP3R-induced Ca store release. Ga12/13 heterotrimeric subunits (17, 18, 43). To investigate which of these Ga families was re- LPA signals via LPA5 to inhibit BCR-mediated calcium sponsible for LPA5 inhibition of BCR signaling, we again used signaling RNA interference. We initially stably expressed A20/shGaq that Of the five initially established LPA receptors (17, 18), mature FO led to an ∼98% reduction in Gaq expression (Fig. 4A). Despite B lymphocytes and A20 B cells predominantly express LPA2 and this significant reduction in Gaq expression, 5 mM LPA still 2+ LPA5, as determined by quantitative PCR (Fig. 3A, 3B). To deter- suppressed BCR-mediated ER Ca release in A20/shGaq cells to mine which of these LPA receptors regulates BCR signaling, we first a level comparable to the control shRNA cell line or parental A20 examined LPA inhibition of BCR signaling in B cells from LPA2- cells (Fig. 4B). These data indicate that LPA5 does not associate knockout mice (27) and found that LPA maintained the ability to with Gaq to regulate B cell AgR Ca2+ signaling. inhibit BCR Ca2+ signaling in Lpar22/2 B cells (Supplemental Fig. The Ga12 family is composed of Ga12 and Ga13 (44), and 3). This indicates that LPA2 does not contribute to LPA regulation the participation of these Ga subunits in LPA regulation of BCR 6LPA5 NEGATIVELY CONTROLS BCR SIGNALING AND Ab RESPONSE

FIGURE 3. The LPA5 receptor inhibits BCR signaling. (A) FO B cells were evaluated for the expression of LPA1–5, as measured by quantitative PCR. Dashed line represents background limit of detection. (B)LPA5 and LPA2 expression was measured by quantitative PCR in parental A20 cells (white bars) 2+ or cells that were transduced with a retrovirally encoded control shRNA (gray bars) or LPA5 shRNA (black bars). (C) BCR-mediated Ca mobilization was measured in A20 cells (upper panel) and in primary B cells (lower panel) that had been transduced with retroviruses encoding either a control (black lines)

or LPA5 (gray lines) shRNA and in the absence (solid lines) or presence (dashed lines) of 20 mM LPA. Arrows indicate time of anti-Ig 6 LPA treatment.

signaling initially was evaluated individually. However, constitutive To examine the contribution of Arhgef1 in the signaling pathway repression of Ga12 or Ga13 expression in A20 cells could not be from LPA receptor to regulation of intracellular stores Ca2+ con- maintained for more than two passages (J. Hu and R.M. Torres, centration, we evaluated B lymphocytes from Arhgef1-deficient unpublished observations). Thus, we exploited an inducible knock- mice (26). Fig. 4E shows that LPA is unable to inhibit BCR Ca2+ down platform to conditionally repress Ga12 and Ga13 simulta- signaling in Arhgef1-deficient primary splenic B cells. This dem- neously upon treatment with doxycycline (30). Doxycycline-regu- onstrates that Arhgef1 is also required for LPA inhibition of BCR lated expression of microRNA-like shRNAs that target Ga12 and signaling. Therefore, we conclude that LPA5 inhibits BCR signaling Ga13 were introduced into A20 cells with a single lentiviral infec- through a Ga12/13-associated heterotrimeric G protein and Arhgef1. tion, and transduced cells (A20-shG12/13) were monitored by the expression of the Venus fluorescent protein. Fig. 4C shows that, upon Ag-mediated B cell activation and Ab response are inhibited by LPA by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. doxycycline treatment, Ga12 and Ga13 expression was reduced by 92 and 88%, respectively, compared with untreated cells. To assess the We next asked whether LPA inhibition of BCR-induced Ca2+ sig- contribution of Ga12 and Ga13 to LPA5 receptor inhibition of BCR naling had more distal biological consequences on B cell activation signaling, a mix of Venus+ transduced cells and Venus2 parental A20 and Ab response after Ag-specific BCR signaling. To investigate cells were treated or not with 1 mg/ml doxycycline for an additional this we used IghB1-8/B1-8 mice, whose B cells express the same VDJ 5 d. Subsequently, parental A20 (Venus2) and A20-shG12/13 (Venus+) (B1-8) rearrangement inserted by gene targeting where rearrange- cells were simultaneously evaluated for Ca2+ stores release following ments naturally occur at the Igh loci (25, 32). Thus, all IghB1-8/B1-8 BCR stimulation (Fig. 4D). In the absence of doxycycline, both pa- B cells express a B1-8 IgH that, when paired with an Igl Lchain, rental A20 and A20-shG12/13 cells released Ca2+ from intracellular generates specificity for NP hapten (46) (Fig. 5A). Stimulation of B1-8/B1-8 http://classic.jimmunol.org stores to the same level when stimulated through the BCR with anti- splenic Igh B cells in vitro with 10 ng/ml of NP-Ficoll IgG; this level was similarly inhibited by LPA, indicating that lenti- resulted in increased intracellular Ca2+ levels in IghB1-8/B1-8 Igl+ viral transduction and/or expression of Venus did not alter BCR or B cells that was again blunted in the presence of 20 mMLPA 2+ LPA5 signaling. In contrast, similar BCR stimulation of doxycycline- (Fig. 5B). Thus, LPA inhibits BCR-mediated intracellular Ca treated cells abolished the ability of LPA to inhibit BCR Ca2+ sig- mobilization after Ag-specific stimulation. naling in A20-shG12/13 cells but not parental A20 cells (Fig. 4D, Ag-specific B cell activation was tested by evaluating the expres- B1-8/B1-8

Downloaded from right panels). Considered together, these data provide compelling sion of the CD69 and CD86 activation Ags on splenic Igh support that LPA5 associates with Ga12/13 heterotrimeric proteins to B cells treated with 10 ng/ml NP-Ficoll in the presence or absence negatively regulate BCR-mediated Ca2+ intracellular stores release. of LPA or a synthetic metabolically stable LPA analog, OTP. OTP is a potent agonist of LPA5 (47), with a 10-fold selectivity over Arhgef1 is required for LPA inhibition of BCR calcium LPA (48), and it also efficiently inhibits BCR-mediated intra- signaling cellular Ca2+ mobilization (S.K. Oda and R.M. Torres, unpublished Ga12/13-associated GPCRs stimulate a family of signaling mol- observations). As expected, 24 h after NP-Ficoll stimulation of ecules referred to as regulator of G protein signaling–containing splenic B cells in vitro, the expression of CD69 was increased on RhoGEFs, whose family members are Arhgef1 (also known as Ag-specific Igl+ IghB1-8/B1-8 cells but not on Ag nonspecific murine Lsc or human p115RhoGEF), Arhgef12 (LARG), and Igl2 B cells (Fig. 5C). Furthermore, when B cells were stimu- Arhgef11 (PDZ-RhoGEF) (45). In addition to functioning as lated with specific Ag, the expression of both CD69 and CD86 RhoA exchange factors, these family members activation Ags was inhibited in the presence of either LPA or harbor regulator of G-protein signaling domains that function as OTP (Fig. 5D). GTPase-activating proteins for GTP-bound Ga12/13 subunits, Thus far, these data demonstrate that LPA signaling via LPA5 effectively terminating signaling by the these Ga subunits. inhibits both BCR-induced intracellular calcium mobilization and The Journal of Immunology 7

FIGURE 4. LPA5 signals via a Ga12/13–Arhgef1–signaling pathway to repress BCR calcium signaling. (A) Western blot analysis of Gaq from stable A20 transfectants harboring a control vector (A20shCTL) or expressing a Gaq shRNA (A20shGaq). b-actin is shown as loading control. (B) Intracellular Ca2+ stores release after anti-Ig stimulation (arrow) in parental A20 cells (black solid line) or A20 shRNA transfectants (solid gray line) alone or in the presence of 5 mM LPA (dashed lines). Ca2+ mobilization for A20shCTL (left panel) and 20shGaq(right panel). Data are representative of three inde- pendent experiments. (C) Western blot analysis of Ga12 and Ga13 in A20-shG12/13 cells before (–) and after (+) doxycycline (DOX) treatment. b-actin is shown as loading control. (D) Intracellular Ca2+ stores release was measured in parental A20 cells (dashed lines) and A20-shG12/13 cells (solid lines) in the by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. presence of EGTA after anti-Ig stimulation alone (top panels) or with 5 mMLPA(middle panels)or20mMLPA(bottom panels). Intracellular Ca2+ stores release also was measured in the absence (left panels) or presence (right panels) of DOX. Data are representative of similar results from three independent experiments. (E) BCR-mediated intracellular Ca2+ mobilization was measured in splenic B cells from C57BL/6 mice (upper panel) and Arhgef12/2 mice (lower panel) in the absence (solid lines) or presence (dashed lines) of 20 mMLPA.

expression of activation Ags. To test whether LPA5 acts normally influence the magnitude of this response. Thus, to more directly in vivo to inhibit the Ab response, we obtained LPA5-deficient evaluate the role of LPA5 expressed by B cells in the Ab response, mice and initially evaluated the FO and marginal zone (MZ) we immunized mice that had been lethally irradiated and recon- 2/2 2/2 http://classic.jimmunol.org B cell populations in the spleen. Lpar5 MZ and FO B cells are stituted with a mixture of bone marrow cells from Lpar5 and present at normal frequencies (Fig. 6A) and appear to localize wild-type animals with NP-Ficoll. Specifically, bone marrow from normally within the spleen, as evidenced by the IgMhighIgD2 MZ Lpar52/2 (IgMb) and B6.C20 (IgMa) mice, or as control Lpar5+/+ B cells in the MZ surrounding the IgM+IgD+ FO B cells in the (IgMb) and B6.C20 (IgMa) mice, was used to reconstitute lethally follicles (Fig. 6B). Both FO and MZ B cells are inhibited in in- irradiated wild-type hosts, and the Ab response from each B cell tracellular Ca2+ mobilization by LPA after BCR stimulation population was measured using an ELISA that discriminates be-

Downloaded from (Fig. 6C); however, in the absence of LPA5, LPA is unable to tween IgM allotypes. Approximately 6 wk after adoptive transfer, suppress BCR-induced intracellular Ca2+ response (Fig. 6D). reconstituted mice were immunized with 5 mg NP-Ficoll, and These data indicate that FO and MZ B cell populations develop NP-specific IgMb Ab was measured in the serum of individual normally in the absence of LPA5 and confirm that LPA5 is the LPA recipient mice 7 d later. To ensure that the Ab response reflected receptor responsible for negatively regulating BCR signaling. a difference in B cell function and not a difference in the number We next measured the Ab response by Lpar52/2, Lpar5+/+ of reconstituted B cells, we normalized the Ab response to the num- littermate controls and wild-type mice to NP-Ficoll, a T- ber of reconstituted mutant and wild-type B cells (Supplemental independent type 2 (TI-2) Ag that elicits Ag-specific IgM essen- Fig. 4). Importantly, in these adoptive mixed bone marrow chime- tially exclusively from MZ B cells (32, 49–52). Four days after ras splenic lymphoid architecture develops normally, and IgMb+ immunization, NP-specific Ab was measured in serum by ELISA Lpar52/2 B cells localize to both the B cell follicle and MZ similar and revealed that Lpar52/2 mice produce twice the level of NP- to wild-type B cells. The results from these experiments revealed specific IgM compared with littermate control and wild-type that NP-specific IgMb produced by Lpar52/2 Bcellswassignifi- C57BL/6 mice (Fig. 6E). Although Ag-specific IgM production cantly increased relative to that produced by wild-type B cells and in in response to NP-Ficoll is independent of T cells, the presence of the presence of wild-type hematopoietic cells and host environment (noncognate) T cells, and possibly other hematopoietic cells, can (Fig. 6F). Furthermore, this elevated Ab response also was reflected 8LPA5 NEGATIVELY CONTROLS BCR SIGNALING AND Ab RESPONSE

FIGURE 5. LPA inhibits Ag-specific B cell activation. (A)Igl+ and Igl2 B220+ IghB1-8/B1-8 B cells labeled with FITC-NP hapten. (B)Igl+ NP-specific B cells were treated with 10 ng/ml of NP-Ficoll, and intracellular Ca2+ was measured over time in the presence (dashed line) or absence (solid line) of 20 mMLPA.(C) Primary splenic IghB1-8/B1-8 B cells were isolated and stimulated with 10 ng/ml NP-Ficoll for 24 h. CD69 expression was measured by flow cytometry on Ag-specific Igl+ and Ag-nonspecific Igl2 B cells. (D) Expression of CD69 (left panel) and CD86 (right panel) on viable Ag-specific Igl+ IghB1-8/B1-8 B cells 24 h after stimulation with NP-Ficoll alone or with 20 mM LPA or 50 mM OTP. Data in each panel are representative of at least four independent experiments.

2+ by the increased number of NP-specific Ab-secreting cells recovered inhibit BCR calcium signaling by impairing IP3R-mediated Ca in the spleen of immunized recipients 7 d after immunization (Fig. release from intracellular stores. This conclusion is supported by 6G). From these data, we conclude that, in response to endogenous experiments evaluating signaling events receptor-proximal and LPA concentrations, LPA5 suppresses the primary B cell Ab re- distal to IP3R activity in the presence of LPA. These results show sponse to a TI-2 model Ag. BCR-induced tyrosine phosphorylation of several membrane- proximal signaling intermediates to, and including, PLCg2is Discussion comparable in the presence or absence of LPA, indicating that

by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. Extracellular bioactive lysophospholipids signal through GPCRs LPA interferes with BCR signaling downstream of PLCg2. Con- expressed by diverse cell types to regulate a number of cell ac- sistent with this, we find that release is inhibited in the presence of tivities (11). In this article, we show that LPA signals via the LPA5 LPA intracellular stores, whereas SOC activity appears normal. receptor to inhibit Ag-specific BCR signaling, subsequent B cell The inhibition of Ca2+ stores release by LPA could, in principle, activation, and in vivo Ab responses. These findings extend the be accomplished by either suppressing IP3R activity or by en- influence of lysophospholipids on immune cell function to include hancing the calcium ATPase transport of calcium back into the regulating the activation and function of B lymphocytes. Our data ER. However, when the ATPase was inhibited by thapsagargin and further provide initial evidence that a GPCR can serve as a BCR ER calcium was being discharged, BCR signaling could further coreceptor that negatively regulates AgR signaling by B cells. increase calcium stores release, and LPA maintained the ability to http://classic.jimmunol.org Both LPA and S1P were shown to modulate AgR signaling by inhibit this increase. This suggests that LPA does not act by en- T lymphocytes (7, 33, 34), although the precise lysophospholipid hancing ATPase activity. Further evidence that LPA suppresses receptors or T cell–signaling pathways mediating this regulation IP3R activity was provided by the introduction of exogenous IP3 were not determined. Recently, we demonstrated that LPA sig- into B cells that led to a transient increase in cytosolic Ca2+ that naling via the LPA5 receptor expressed by tumor-specific CD8 could be inhibited in the presence of LPA (Fig. 1C). Considered T cells suppresses tumor immunity (53). Our findings in this study together, these findings provide strong evidence that LPA5 sig-

Downloaded from show that physiological and pathophysiological concentrations of naling impairs IP3R-induced calcium release. However, these LPA act on the LPA5 receptor to suppress BCR-mediated calcium results do not exclude that LPA also may inhibit IP3 production, release from intracellular stores. As a signaling second messenger further inhibiting intracellular stores release. Precisely how IP3R in lymphocytes, calcium is required for a number of cellular ac- activity is inhibited by an LPA5–Ga12/13–Arhgef1–signaling axis tivities that include the regulation of the NF-kBandNF-AT remains to be defined, although we note that Gbg dimers liberated transcription factors (54, 55), as well as lymphocyte differentia- from Gaibg heterotrimers can associate and gate IP3R (57), pro- tion and effector function (37, 56). We show that LPA inhibition viding precedence for regulation of IP3R activity by GPCR sig- manifests early by preventing complete intracellular calcium naling. mobilization; it later suppresses Ag-specific induction of CD69 Immunization of LPA5-deficient and -sufficient mice revealed and CD86 expression and ultimately limits the early primary Ab that LPA5 is able to repress the early primary Ab response to a response. TI-2 model Ag and presumably in response to physiological levels In B cells, the signaling pathway from the BCR leading to el- of LPA. However, LPA5 also is expressed by other hematopoietic evated intracellular calcium has been relatively well studied (36, and nonhematopoietic cell types, including T cells (43, 47), which 37). Our RNA interference experiments initially identified LPA5 are able to influence T-independent Ab responses (58). Thus, we and Ga12/13 as the LPA receptor and associated Ga subunits that further restricted our in vivo evaluation of LPA5 function to MZ The Journal of Immunology 9

FIGURE 6. LPA5 suppresses the TI-2 Ab response to NP-Ficoll. (A) Percentage of transitional T1, FO, and MZ B cell populations in the spleen of Lpar5+/+ control littermate (white bars) and Lpar52/2 (black bars) mice. (B) Representative immunofluorescence histology of Lpar5+/+ (left panel) and Lpar52/2 (right panel) spleen using Abs against IgM (red), IgD (blue), and CD3 (green). Scale bar, 50 mm. (C) Wild-type splenic B cells were isolated and 2+ treated with F(ab9)2 anti-IgM (10 mg/ml) in the presence (dotted line) or absence (solid line) of 20 mM LPA, and intracellular Ca levels were measured over time in cells gated based on CD21 and CD1d expression to identify FO (upper panel) and MZ (lower panel) B cells. (D) B220+ B cells from Lpar52/2 +/+ (lower panel)orLpar5 (upper panel) littermates were stimulated with 10 mg/ml F(ab9)2 anti-IgM without (solid line) or with (dotted line) 20 mMLPA, and intracellular Ca2+ levels were measured over time. (E) Lpar52/2, wild-type littermate, or C57BL/6 mice were immunized with 5 mg NP-Ficoll, and NP- specific IgM levels were determined by ELISA. Data are shown as day-4 NP-specific IgM over day-0 preimmune NP-specific IgM levels for 6–10 mice immunized in three experiments. Day-0 preimmune NP-specific IgM concentrations were 11.9 6 8.6 mg/ml for controls and 8.0 6 4.7 mg/ml for mutants. *p , 0.02, one-tailed Student t test with unequal variance. (F) NP-specific IgMb concentration was measured in the serum of mixed bone marrow chimeras 7 d after immunization with 5 mg/ml NP-Ficoll and normalized to the number of reconstituted IgMb+ Lpar52/2 (experimental chimeras) or Lpar5+/+ (littermate control chimeras) B cells. Data are combined from two independent experiments. *p , 0.05, Student t test with unequal variance. (G) NP- by guest on October 1, 2021. Copyright 2014 Pageant Media Ltd. specific IgMb ASCs enumerated in the spleen of adoptive mixed bone marrow chimeras 7 d after immunization from two independent experiments. Reconstitution frequencies and numbers of splenic IgMb+ B cells and day-0 and day-7 NP-specific IgMb concentrations are shown for two independent mixed bone marrow chimeras in Supplemental Fig. 4. *p , 0.05, Student t test with unequal variance. Symbols represent individual chimeric mice, and horizontal bars indicate mean. Data are representative of three independent experiments.

B cells with mixed bone marrow chimeras and found that Lpar52/2 on cells in the immediate microenvironment (61). Thus, local B cells mounted a heightened Ag-specific primary Ab response increased LPA production may have immunosuppressive activities compared with Lpar5+/+ B cells and in competition with wild- that are not necessarily reflected by systemic concentrations. http://classic.jimmunol.org type B cells and in the presence of a wild-type immune system. Furthermore, the elevated systemic LPA levels found in certain Because MZ B cells dominate this TI-2 Ab response (32, 49–52) pathological settings would almost certainly promote increased 2/2 and we show that Lpar5 MZ B cells develop at normal fre- LPA5 signaling by Ag-specific B cells and further impair Ab quencies and localize appropriately, these data provide compelling responses. Clearly, a better understanding is needed of how LPA evidence that LPA5 signaling by B cells normally inhibits the production is regulated in homeostatic and inflammatory settings. primary Ab response to TI-2 Ags. With regard to lysophospholipid regulation of lymphocytes, the

Downloaded from The physiological concentrations of LPA have been variably re- S1P lysophospholipid has received considerable attention for ported in the high nanomolar to low micromolar range (11, 17, 19, its role in regulating lymphocyte migration during development 20), and our in vivo findings suggest that B cell AgR signaling and and homeostatic trafficking (12–16). In contrast, relatively little is function are normally suppressed by these endogenous levels. In understood about how LPA, an additional major lysophospholipid, contrast, our in vitro experiments required LPA at 5–20 mM regulates lymphocyte function, although both T and B cells were concentrations to reveal a role for this lipid in regulating BCR reported to express LPA receptors (33, 62, 63). We show B cells signaling. However, we note that with regard to the concentration express LPA2 and LPA5 and this finding is consistent with the of LPA that promotes receptor signaling, it was proposed that previous reported expression of LPA2 on human B cells (7, 33–35), systemic LPA levels may be less relevant for LPA receptor sig- a finding obtained prior to the identification of LPA5. Our findings naling than are locally produced microenvironmental levels (59, demonstrate that the LPA5 GPCR negatively regulates BCR sig- 60). Specifically, based on the ability of autotaxin, the secreted naling and function and identify the release of calcium from in- that generates extracellular LPA from abundantly avail- tracellular stores as a likely point of inhibition in the BCR-signaling able lysophosphatidylcholine, to associate with integrins and the pathway. Although it is not yet clear why the LPA lysophospholipid recent autotaxin crystal structure (59, 60), it was suggested that functions to inhibit B cell activation and Ab response, a plausible newly produced LPA is immediately delivered to LPA receptors consequence of this suppression may be to prevent the activation of 10 LPA5 NEGATIVELY CONTROLS BCR SIGNALING AND Ab RESPONSE

weak-affinity autoreactive B cells that are known to be selected into 21. Liu, S., M. Umezu-Goto, M. Murph, Y. Lu, W. Liu, F. Zhang, S. Yu, L. C. Stephens, X. Cui, G. Murrow, et al. 2009. Expression of autotaxin and the mature peripheral B cell pool. Current experiments are ex- lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, ploring this possibility. and metastases. Cancer Cell 15: 539–550. Although attention on LPA has focused on its aberrant pro- 22. Mills, G. B., and W. H. Moolenaar. 2003. The emerging role of lysophosphatidic acid in cancer. Nat. Rev. Cancer 3: 582–591. duction by diverse cancer cell types and its ability to promote 23. Watanabe, N., H. Ikeda, K. Nakamura, R. Ohkawa, Y. Kume, J. Aoki, K. Hama, tumorigenesis (18, 21, 22), our data suggest that certain malig- S. Okudaira, M. Tanaka, T. Tomiya, et al. 2007. Both plasma lysophosphatidic nancies might exploit LPA production not only to promote tu- acid and serum autotaxin levels are increased in chronic hepatitis C. J. Clin. Gastroenterol. 41: 616–623. morigenesis but also as a mechanism to inhibit adaptive immune 24. Fourcade, O., M. F. Simon, C. Viode´, N. Rugani, F. Leballe, A. Ragab, responses (53). In the future, it will be important to consider that B. Fournie´, L. Sarda, and H. Chap. 1995. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed the pathological or therapeutic settings that alter LPA levels or from activated cells. Cell 80: 919–927. LPA receptor signaling also likely modulate adaptive humoral 25. Sonoda, E., Y. Pewzner-Jung, S. Schwers, S. Taki, S. Jung, D. Eilat, and immunity. K. Rajewsky. 1997. B cell development under the condition of allelic inclusion. Immunity 6: 225–233. 26. Rubtsov, A., P. Strauch, A. Digiacomo, J. Hu, R. Pelanda, and R. M. Torres. Acknowledgments 2005. Lsc regulates marginal-zone B cell migration and adhesion and is required for the IgM T-dependent antibody response. Immunity 23: 527–538. We thank Drs. Mel Simon and Ian Fraser for the kind gift of the pSLIK- 27. Contos, J. J., I. Ishii, N. Fukushima, M. A. Kingsbury, X. Ye, S. Kawamura, Venus-TmiR-G12-G13 expression vector, Dr. Jerold Chun for LPA2- J. H. Brown, and J. Chun. 2002. Characterization of lpa(2) (Edg4) and lpa(1)/lpa deficient mice, Dr. Dennis Voelker (National Jewish Health) for advice (2) (Edg2/Edg4) lysophosphatidic acid receptor knockout mice: signaling defi- and guidance with lipid handling, and Dr. Paul Waterman for technical help. cits without obvious phenotypic abnormality attributable to lpa(2). Mol. Cell. Biol. 22: 6921–6929. We also acknowledge Dr. John Cambier and the Torres and Pelanda labo- 28. Wattler, S., M. Kelly, and M. Nehls. 1999. 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