Loss of IP3 −Mediated Ca2+ Release in Mouse B Cells Results in Abnormal B Cell Development and Function

This information is current as Huayuan Tang, Hong Wang, Qingsong Lin, Feifei Fan, Fei of September 27, 2021. Zhang, Xiaohong Peng, Xi Fang, Jie Liu and Kunfu Ouyang J Immunol published online 14 June 2017 http://www.jimmunol.org/content/early/2017/06/13/jimmun ol.1700109 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2017/06/14/jimmunol.170010 Material 9.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

by guest on September 27, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 14, 2017, doi:10.4049/jimmunol.1700109 The Journal of Immunology

2+ Loss of IP3 Receptor–Mediated Ca Release in Mouse B Cells Results in Abnormal B Cell Development and Function

Huayuan Tang,* Hong Wang,* Qingsong Lin,* Feifei Fan,* Fei Zhang,* Xiaohong Peng,* Xi Fang,† Jie Liu,‡ and Kunfu Ouyang*

Intracellular calcium (Ca2+) mobilization after engagement of the BCR has been proposed to play an important role in B cell development and function. BCR activation causes an initial Ca2+ release from the endoplasmic reticulum that is mediated by 2+ 2+ inositol 1,4,5-trisphosphate receptor (IP3R) and then triggers store-operated Ca entry once endoplasmic reticulum Ca store is depleted. Store-operated Ca2+ entry has been shown to regulate B cell function but is dispensable for B cell development. By 2+ contrast, the function of IP3R-mediated Ca release in B cells remains to be determined. In this study, we generated a B cell– specific IP3R triple-knockout (IP3R-TKO) mouse model and revealed that loss of IP3Rs increased transitional B cell numbers and

reduced recirculating mature B cell numbers in bone marrow. In the peripheral tissues, the numbers of conventional B2 B cells Downloaded from

and B1 B cells were both significantly decreased in IP3R-TKO mice. Ablation of IP3Rs also dramatically reduced BCR-mediated B cell proliferation and survival. Furthermore, T cell–dependent and T cell–independent Ab responses were altered in IP3R-TKO mice. In addition, deletion of IP3Rs reduced IL-10–producing regulatory B cell numbers and led to defects in NFAT activation, which together resulted in decreased IL-10 secretion. Taken together, our study demonstrated for the first time, to our knowledge, 2+ that IP3R-mediated Ca release plays an essential role in regulating B cell development, proliferation, Ab production, and B cell regulatory function in vivo. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/

lymphopoiesis originates in the fetal liver and switches B cells give rise to two types of mature conventional B2 B cells: to bone marrow after birth. In the bone marrow, B cells follicular (FO) B cells and marginal zone (MZ) B cells (2). Naive B progress through a highly ordered series of develop- FO B cells, which constitute the majority of mature splenic B cells, mental stages: progenitor B cell, precursor B (pre-B) cell, and reside in the primary follicles of B cell zones and gain the ability to immature B cell stages (1). To prevent any further development of recirculate through second lymphoid tissues (3). With the assistance self-reactive cells, immature B cells undergo negative selection of T cells, naive FO B cells can be further activated and play a major with survivors developing into transitional B cells, the majority of role in T cell–dependent (TD) Ab response (3). Unlike FO B cells, by guest on September 27, 2021 which then migrate to the spleen, where they undergo further MZ B cells are noncirculating mature B cells located at the white maturation and differentiation (2). Within the spleen, transitional pule of the spleen and are responsible for T cell–independent (TI) Ab response (4). Besides conventional B2 B cells, there is another subclass of B cells, B1 B cells, which predominate in body cavities *Drug Discovery Center, Key Laboratory of Chemical Genomics, Peking University and provide “innate-like” protection against pathogens by producing Shenzhen Graduate School, Shenzhen, Guangdong 518055, China; †Department of Medicine, University of California, San Diego School of Medicine, La Jolla, CA natural Abs (5). In addition to these Ab-producing B cells, there is 92093; and ‡Department of Pathophysiology, School of Medicine, Shenzhen Univer- a special subset of B cells termed regulatory B cells, which nega- sity, Shenzhen 518060, China tively regulate autoimmunity by secreting anti-inflammatory cyto- ORCIDs: 0000-0002-5653-7654 (F.F.); 0000-0003-0292-375X (K.O.). kines such as IL-10 (6). Received for publication January 24, 2017. Accepted for publication May 17, 2017. Signals from BCRs are essential for B cell development, sur- This work was supported by the National Key Basic Research Program of China (Grant vival, and activation. One of the key signals in response to BCR 2013CB531200), the National Science Foundation of China (Grants 31370823 and 2+ 91439130), the Shenzhen Basic Research Foundation (Grants JCYJ20140509093817680 stimulation is intracellular calcium (Ca ) elevation, which leads to and JCYJ20160428154108239), and the Guangdong Province Basic Research Foun- activation of many transcription factors to control the expres- dation (Grant 2016A020216003). sion that mediates diverse genetic programs (7). In B lymphocytes, H.T., H.W., Q.L., F.F., F.Z., and X.P. performed the research; H.T., K.O., X.F., and BCR-mediated Ca2+ elevation occurs via two main mechanisms: J.L. designed the research; and H.T., K.O., X.F., and H.W. wrote the manuscript. Ca2+ release from endoplasmic reticulum (ER) stores and Ca2+ in- Address correspondence and reprint requests to Dr. Kunfu Ouyang, Drug Discovery Center, Key Laboratory of Chemical Genomics, Peking University Shenzhen Grad- flux from the extracellular space across the plasma membrane (8). uate School, 2199 Lishui Road, Xili University Town, Nanshan District, Shenzhen, Cross-linking of BCRs activates several tyrosine kinases Guangdong 518055, China. E-mail address: [email protected] and eventually results in activation of phospholipase C (PLC)-g2. The online version of this article contains supplemental material. Activated PLC-g2 hydrolyzes the membrane phospholipid, Abbreviations used in this article: B10, IL-10–producing B cell; Ca2+, intracellular phosphatidylinositol-4,5-bisphosphate, into two intracellular calcium; CGG, chicken g-globulin; CsA, cyclosporin A; DKO, double-knockout; ER, second messengers: inositol 1,4,5-trisphosphate (IP ) and diacyl- endoplasmic reticulum; FO, follicular; GC, germinal center; IP3, inositol 1,4,5- 3 trisphosphate; IP3R, IP3 receptor; IP3R-TKO, IP3R triple-knockout; MZ, marginal glycerol. IP3 binds to IP3 receptors (IP3Rs) in the ER membrane, zone; no., number; NP, 4-hydroxy-3-nitrophenyl; PI, propidium iodide; PLC, phos- leading to a transient release of Ca2+ from ER stores, which stands pholipase C; pre-B, precursor B; PSS, physiological saline solution; RT-PCR, real- 2+ time PCR; SOCE, store-operated Ca2+ entry; STIM1, stromal interaction molecule 1; for the first phase of BCR-induced Ca signal. Subsequently, the T1, transitional type 1; T2, transitional type 2; T3, transitional type 3; TD, T cell– decrease of ER luminal Ca2+ concentration is detected by stromal dependent; TG, thapsigargin; TI, T cell–independent. interaction molecule 1 (STIM1), which further triggers the opening 2+ 2+ Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 of Orai1 Ca channels, to induce a sustained Ca influx across the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700109 2IP3Rs REGULATE MOUSE B CELL DEVELOPMENT AND FUNCTION plasma membrane. These processes are called store-operated Ca2+ 80), Alexa Fluor 488–conjugated anti-CD1d (catalog no. 53-0011-80), entry (SOCE), which represents the second phase of BCR-induced PE-conjugated anti-IgM (catalog no. 12-5790), anti-CD21/CD35 (catalog no. Ca2+ signal (9). 12-0211-81), anti-Fas (catalog no. 12-0951-81), PE-cyanine5.5–conjugated 2+ anti-CD19 (catalog no. 35-0193-80), PE-cyanine7–conjugated anti-CD23 Evidence showing the importance of Ca signaling in B cells (catalog no. 25-0232-81), allophycocyanin-conjugated anti-B220 (catalog has mainly originated from studies of mutant mice in which SOCE no. 17-0452), allophycocyanin-eFluor 780–conjugated anti-CD21/CD35 was genetically abrogated. BCR-induced Ca2+ influx was almost (catalog no. 47-0211-80), and streptavidin PE (catalog no. 12-4317) were completely blocked in STIM1 and STIM2 double-knockout (DKO) purchased from eBioscience. Biotin-conjugated anti-CD5 (catalog no. 553019) and PE-conjugated anti-CD93 (catalog no. 558039) were purchased from B cells, whereas residual SOCE was present in STIM1- and Orai1- BD Biosciences. For immunoblotting, the primary Abs against JNK (catalog deficient B cells (10, 11). Accordingly, BCR-mediated proliferation no. 9258), ERK1/2 (catalog no. 9102), p-JNK (catalog no. 9251), and is most severely inhibited in STIM1/STIM2 DKO B cells and only p-ERK (catalog no. 9101), and NFAT1 (catalog no. 4389) were purchased partially decreased in B cells lacking either STIM1 or Orai1 (10, from Cell Signaling Technology. The Ab against b-actin was purchased 11). Furthermore, loss of both STIM1 and STIM2 in B cells dra- from Santa Cruz Biotechnology (catalog no. SC-47778). The Ab against IP R3 was purchased from BD Biosciences (catalog no. 610312). The Abs matically impairs IL-10 production and exacerbates the severity 3 against IP3R1 and IP3R2 were raised in our laboratory as previously de- of experimental autoimmune encephalomyelitis in vivo (11). scribed (22). For cell stimulation, anti-mouse IgM F(ab9)2 and anti-CD40 However, B cell populations in the bone marrow and secondary were purchased from Jackson Immunoresearch (catalog no. 115-006-075) lymphoid organs were shown to be normal in Orai12/2, Orai1R93W and BD Biosciences (catalog no. 550285), respectively. LPS (catalog no. f/f f/f L2630), cyclosporin A (CsA; catalog no. C3662), and FK506 (catalog no. knock-in, and Stim1 Stim2 Mb1-Cre mice (10–12). Human pa- F4679) were purchased from Sigma-Aldrich. tients with mutations in ORAI1 and STIM1 were also found to have normal numbers of B cells (13, 14). Collectively, these data suggest Sample preparation and flow cytometry Downloaded from that SOCE plays an important role in regulating B cell function but Single-cell suspensions were prepared from the bone marrow, spleen, in- is dispensable for B cell development. guinal lymph node, peritoneal cavity, and peripheral blood by passing through a cell strainer to remove clumps and debris. Cells were treated with Compared with the role of SOCE in B cells, the role of IP3Rs in B cell development and function is not well understood. In RBC lysis buffer (Beyotime) and washed with ice-cold PBS containing 0.1% BSA. Cells were then incubated with Abs for 30 min, washed twice, mammals, there are three different IP3R subtypes (IP3R1, IP3R2, and resuspended in an appropriate volume of PBS with 0.1% BSA. Data and IP3R3) encoded by three , Itpr1, Itpr2,andItpr3,respec- were acquired on FACSCalibur and FACSCanto II flow cytometers (BD http://www.jimmunol.org/ tively (15). Various studies utilizing IP3R knockout mice have Biosciences) and analyzed with FlowJo (Tree Star). demonstrated that IP3Rs regulate brain function (16), exocrine B cell isolation and calcium measurement secretion (17), perception (18), embryonic development (19, 20), vascular smooth muscle contractility (21), and T cell devel- Single-cell suspensions were obtained from spleens of control and IP3R- TKO mice. After RBCs were depleted by hypotonic lysis, splenic B cells opment (22). On the other hand, IP3R subtypes have also been + 2+ were purified by the negative selection of CD43 cells with anti-CD43 shown to play a redundant role in regulating BCR-induced Ca magnetic beads (Invitrogen). The purity of the B cells (B220+)was.95% mobilization and cell apoptosis in DT40 cells, a chicken pre-B cell determined by FACS analysis. 2+ line (23). However, the in vivo role of IP3Rs in B cell development For Ca imaging, purified splenic B cells were planted on glass cov- and physiological function remains unclear. In this study, we erslips coated with poly-D-lysine (Sigma), washed with regular physio- by guest on September 27, 2021 logical saline solution (PSS; in mM: NaCl 137, KCl 5.4, MgSO4 1.0, generated a B cell–specific IP3R triple-knockout (IP3R-TKO) glucose 10, CaCl 1.8, and HEPES 10 [pH 7.4]), and then incubated with 2+ 2 mouse model and found that loss of IP3R-mediated Ca release 5 mM Fluo4-AM (Invitrogen) for 30 min at 37˚C. Cells were imaged with a reduced conventional B2 and B1 B cell population. Deletion of all Zeiss LSM510 inverted confocal microscope. To separate BCR-induced Ca2+ release from SOCE, we perfused cells with a Ca2+-free PSS (zero IP3Rs also impaired BCR-mediated cell proliferation in vitro and [Ca2+] plus 1.0 mM EGTA) for a short time before the administration of altered TD and TI Ab responses in vivo. Furthermore, we found 2+ anti-mouse IgM F(ab9)2 (10 mg/ml). To measure IP3-induced Ca release, that loss of IP3Rs reduced regulatory IL-10–producing B cell (B10) we incubated splenic B cells with a membrane-permeant caged derivative numbers and caused defective activation of the calcineurin-NFAT of IP3, caged-IP3/PM (catalog no. cag-iso-2-145-100; SiChem), together signaling pathway, which together resulted in decreased BCR- with Fluo4-AM for 30 min at 37˚C. Photolysis of caged-IP3 in selected B cells was evoked by a 20-ms exposure of 405 nm UV laser. To measure mediated IL-10 production. In summary, we provide evidence for 2+ 2+ 2+ thapsigargin (TG)-induced Ca signal, we also perfused cells with a Ca -free the first time, to our knowledge, that IP3R-mediated Ca release PSS for a short time before the administration of 2 mM TG (Sigma). Image plays an essential role in regulating B cell development and function. processing and data analysis were performed as previously described (26). In vitro proliferation and survival Materials and Methods Mice Purified splenic B cells were incubated with 5 mM CFSE (Sigma) at a density of 1 3 107 cells/ml in PBS containing 0.1% BSA for 5 min at 37˚C.

The generation of floxed alleles of the genes encoding IP3R1, IP3R2, and The staining was quenched by the addition of 10 vol of ice-cold PBS con- IP3R3 has been described previously (22, 24). We crossed IP3R triple- taining 5% FBS. After three washes with PBS containing 0.1% BSA, CFSE- floxed (Itpr1f/fItpr2f/fItpr3f/f) mice with Cd19tm1(cre)Cgn/J (CD19-Cre) labeled B cells were resuspended in RPMI 1640 with 10% FBS and plated in mice (The Jackson Laboratory). CD19-Cre mice were maintained on the 96-well flat-bottom dishes at a density of 1 3 106 cells/ml. Thereafter, cells pure C57BL/6 background. IP3R triple-floxed mice were originally gen- were treated with vehicle (RPMI 1640 with 10% FBS), 10 mg/ml anti-IgM erated on a mixed genetic background of Black Swiss, 129, and C57BL/6, F(ab9)2,10mg/ml anti-CD40, 10 mg/ml LPS, or a combination of 10 mg/ml and had been backcrossed with C57BL/6 mice for more than seven gen- anti-IgM with 10 mg/ml anti-CD40 for 48 h, respectively. Cells were stained erations. CD19-Cre is constitutively expressed and has been frequently with anti-B220 to indicate B cells and propidium iodide (PI; eBioscience) used as a B cell–specific Cre (25). Itpr1f/fItpr2f/fItpr3f/f CD19-Cre+ mice for cell viability analysis. In some experiments, cells were stained with were used as B cell–specific IP R-TKO mice. Itpr1f/fItpr2f/fItpr3f/fCD19-Cre2 anti-B220, anti-CD93, anti-CD21, and anti-CD23 to separate different 3 2 mice were used as control mice. All mice used in this study were at the age of B cell subsets. The percentages of PI and CFSE-diluted B cell subsets were 8–10 wk. All mice were housed under specific pathogen-free conditions with assessed by FACSCalibur (BD Biosciences). a 12/12 h day/night cycle. All animal care and use procedures in this study were approved by the Institutional Animal Care and Use Committee. Immunoblot analysis Abs and reagents Purified splenic naive B cells or LPS-activated B cells (pretreated with 10 mg/ml LPS for 48 h) were stimulated with 10 mg/ml anti-IgM F(ab9)2 in For flow cytometry, FITC-conjugated anti-CD43 (catalog number [no.] 11- serum-free RPMI 1640 medium for indicated time periods and then lysed 0431-81), anti-CD93 (catalog no. 11-5892-81), anti-GL7 (catalog no. 53-5902- with lysis buffer (8 M urea, 2 M thiourea, 3% SDS, 75 mM DTT, 0.05 M The Journal of Immunology 3

Tris-HCl [pH 6.8], and 0.03% bromophenol blue). Cell lysates were sep- 1A). However, the expression level of each subtype varied between arated by standard SDS-PAGE and analyzed by Western blotting using the different stages. Interestingly, all three IP3R subtypes were highly Abs against IP3Rs, NFAT1, b-actin, ERK, p-ERK, JNK, and p-JNK. expressed at the transitional stage compared with mature B cell stage 2+ p-ERK analysis (Supplemental Fig. 1A), which suggests that IP3R-mediated Ca signaling could play a role in B cell development from the transi- Pervanadate treatment and flow cytometric analysis of p-ERK were per- formed as previously described (27). In brief, freshly isolated single-cell tional to mature stage. suspension from bone marrow of control and IP3R-TKO mice was placed To further explore the role of IP3Rs in B cell development and 2+ 2+ on ice for 1 h in dPBS with Ca and Mg containing 1% FBS. Cells were function, we generated a B cell–specific IP3R-TKO mouse model treated with 60 mM sodium pervanadate for 5 min at 37˚C, fixed in 4% using a CD19-Cre mouse line that has been shown to be consti- paraformaldehyde, and permeabilized in PBS containing 0.2% Triton-X 100, 5% BSA, and 2% horse serum. Cells were then washed with PBS and tutively expressed from the early progenitor B cell stage and stained with primary Abs against ERK1/2 (catalog no. 9102; Cell Sig- throughout all the later B cell developmental stages (25). Once naling Technology) and p-ERK1/2 (catalog no. 9101; Cell Signaling IP3R-TKO mice were generated, we first examined whether Technology) for 45 min at room temperature, respectively. Thereafter, cells CD19-Cre could efficiently delete IP3Rs in B cells. In purified were washed again and further stained with the Abs recognizing surface IP R-TKO splenic B cells, both mRNA and protein levels of all markers, as well as the secondary Ab FITC-conjugated anti-rabbit IgG 3 (catalog no. 406403; BioLegend). three IP3R subtypes were dramatically reduced compared with control B cells (Fig. 1A, 1B). In addition, we found that mRNA Quantitative real-time PCR analysis levels of all three IP3R subtypes were dramatically decreased in Total RNA was extracted using TRIzol reagent (Invitrogen) or RNeasy immature, transitional, and recirculating mature B cells purified

MiniKit (Qiagen), and cDNA was synthesized using TransScript One-Step Downloaded from from the bone marrow of IP3R-TKO mice compared with control gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech). mice (Supplemental Fig. 1B). We also found that deletion of IP Rs Quantitative real-time PCR (RT-PCR) was performed using TransStart Tip 3 Green qPCR SuperMix (TransGen Biotech) according to the manufac- was less efficient in pro/pre-B cells of IP3R-TKO mice (Supplemental turer’s instructions. The sequences for primers of Itpr1-3 and Gapdh were Fig. 1B), which is consistent with weaker expression of CD19-Cre used as previously described (21). The primer sequences for Il-10, Ccnd2, in pro/pre-B cells (28). Furthermore, we checked whether deletion Bip, GRP94, Calr, Baffr, Mef2c, Foxo1, Rag1, and Rag2 are presented as of IP Rs could efficiently abrogate IP -induced Ca2+ release. Splenic follows: Il-10 (forward: 59-TGCCTTCAGTCAAGTGAAGACT-39; reverse: 3 3 59-AAACTCATTCATGGCCTTGTA-39), Ccnd2 (forward: 59-CAGAGCTT- B cells were isolated and incubated with a membrane-permeant http://www.jimmunol.org/ CGATTTGCTCCT-39;reverse:59-ACACACTCACGTGTGATGCC-39), Bip caged derivative of IP3, and a UV laser was applied to selected (forward: 59-ACATGGACCTGTTCCGCTCTA-39;reverse:59-TGGCTCC- cells to release IP3 from the caged compound, which can thus trigger TTGCCATTGAAGA-39), GRP94 (forward: 59-GGGAGGTCACCTTCAA- the opening of IP3Rs. In control B cells, photolysis of caged-IP3 was GTCG-39;reverse:59-CTCGAGGTGCAGATGTGG G-39), Calr (forward: able to induce a rapid Ca2+ transient, which was totally absent in 59-AGCAGTTCTTGGACGGAGATG-39;reverse:59-TTCTCCAGGTCCC- CGTAAAAT-39), Baffr (forward: 59-CCTCCGCTCAAAGAAGATGCA-39; IP3R-TKO B cells (Supplemental Fig. 1C, 1D), further indicating reverse: 59-GTGGAGCCCAGTTCTGT-39), Mef2c (forward: 59-AGATCT- that all IP3R subtypes were successfully deleted in these IP3R-TKO GACATCCGGTGCAG-39;reverse:59-TCTTGTTCAGGTTACCAGGTG-39), B cells. We next investigated whether deletion of all three IP3Rs Foxo1 (forward: 59-TGCTGTGAAGGGACAGATTG-39;reverse:59-GAGT- could alter ER Ca2+ content and induce ER stress and the unfolded

GGATGGTGAAGAGCGT-39), Rag1 (forward: 59-CTGAAGCTCAGGGTA- by guest on September 27, 2021 protein response. We depleted ER Ca2+ store with TG, an ER Ca2+ GACGG-39;reverse:59-CA ACCAAGCTGCAGACATTC-39), Rag2 (forward: 2+ 59-CTTCCTGCTTGTGGATGTGA-39;reverse:59-AGTGACTCTTCCCCA- ATPase blocker, in the absence of extracellular Ca , and measured AGTGC-39). Relative transcript abundance was normalized to Gapdh. Each TG-induced Ca2+ signals in the cytosol. We found that deletion of all sample was run at least in duplicate. three IP3R subtypes did not significantly increase the amplitude 2+ ELISA of TG-induced Ca signals (Supplemental Fig. 1E, 1F), suggesting 2+ that the filling state of ER Ca stores between control and IP3R- To determine basal Ig titers, we prepared serum from unimmunized control TKO B cells was comparable. Consistently, we found that expres- and IP3R-TKO mice, and titers were measured using HRP-conjugated goat anti-mouse IgM, IgG1, IgG2b, IgG3, and IgA (Southern Biotech). sion of prototypical unfolded protein response target genes Bip, To determine TD responses, we immunized control and IP3R-TKO mice GRP94,andCalr was also not significantly changed in IP3R-TKO i.p. with 100 mg of 4-hydroxy-3-nitrophenyl (NP)-chicken g-globulin B cells compared with control B cells (Supplemental Fig. 1G). (CGG) (NP-CGG; Biosearch Technologies) in alum (Thermo Scientific). We further investigated the effects of IP3R deletion on BCR- To determine TI responses, we immunized mice i.p. with NP-LPS (TI-1 2+ Ag; Biosearch Technologies) or NP-Ficoll (TI-2 Ag; Biosearch Technol- induced Ca mobilization in B cells. To characterize the phase of 2+ ogies). Blood was collected at days 0, 7, 14, and 21 by orbital sinus blood Ca release and the following phase of SOCE separately, we first 2+ 2+ sampling. For detection of NP-specific Abs, ELISA plates were precoated applied anti-IgM in the absence of extracellular Ca (zero [Ca ]o) 2+ 2+ with 2 mg/ml NP29-BSA (Biosearch Technologies). Serum samples were to induce Ca release from the ER. When ER Ca store was de- serially diluted and relative anti-NP titers were measured using isotype- pleted, regular extracellular solution with 2 mM [Ca2+] was added specific HRP-conjugated secondary Abs (Southern Biotech). o For IL-10 production detection, LPS-activated splenic B cells were to induce SOCE. In control cells, application of anti-IgM was able 2+ stimulated with 10 mg/ml anti-IgM F(ab9)2 for 24 h. IL-10 in the cell to elicit a transient Ca elevation, and subsequent reintroduction culture medium was detected by ELISA according to the manufacturer’s of extracellular Ca2+ was able to induce a second phase of Ca2+ protocol (Invitrogen). elevation. However, both phases of Ca2+ increase were completely abolished in IP3R-TKO B cells (Fig. 1C, 1D), suggesting that IP3Rs Results are essential for BCR-induced Ca2+ release and subsequent activa- Deletion of all three IP3R subtypes abolished BCR-induced tion of SOCE. Ca2+ mobilization 2+ We first sorted B cells from control mouse bone marrow and ex- IP3R-mediated Ca release regulates B2 and B1 B cell development amined expression of all three IP3R subtypes in pro/pre-B cells + + 2 low + int 2+ (B220 CD19 IgM ), immature B cells (B220 CD19 IgM ), Next, we investigated the role of IP3R-mediated Ca signal in transitional B cells (B220intCD19+IgMhigh), and recirculating B cell development by characterizing distinct B cell subsets using mature B cells (B220highCD19+IgMint) using quantitative RT-PCR. FACS analysis. B cells were isolated from bone marrow and pe- + Consistent with expression in DT40 cells (23), all three IP3R subtypes ripheral secondary lymphoid tissues. First, total numbers of CD19 + were detected at all B cell developmental stages (Supplemental Fig. B220 B cells in bone marrow of IP3R-TKO mice were not significantly 4IP3Rs REGULATE MOUSE B CELL DEVELOPMENT AND FUNCTION Downloaded from

2+ FIGURE 1. B cell–specific deletion of IP3Rs abrogated BCR-mediated Ca mobilization. Splenic B cells were isolated from control and IP3R-TKO mice by negative selection of CD43+ cells with anti-CD43 magnetic beads. Quantitative RT-PCR (A) and Western blot analysis (B) were used to measure the RNA and protein levels of each IP3R subtype in control and IP3R-TKO B cells, respectively. n = 3–5 mice per group. Data represent mean 6 SEM. (C) 2+ Sequential confocal images of splenic B cells at the time point of t0, t1, t2, and t3 as indicated by the arrows in (D). Splenic B cells were loaded with Ca 2+ 2+ 2+ indicator Fluo4 and imaged using confocal microscopy. Intracellular Ca mobilization was first elicited by anti-IgM (10 mg/ml) in Ca -free (zero [Ca ]o) http://www.jimmunol.org/ 2+ 2+ buffer with subsequent reintroduction of regular buffer with normal extracellular Ca concentration (2 mM [Ca ]o). Scale bar, 100 mm. (D) Representative 2+ traces of averaged Ca signals in control (black) and IP3R-TKO (red) B cells. Data are representative of at least three independent experiments. Signif- icance was determined by the two-tailed, unpaired Student t test. ***p , 0.001 versus control B cells. changed compared with control mice (3.71 6 0.42 3 106 cells in to give rise to mature FO B cells and MZ B cells (2). Compared 6 control mice versus 3.45 6 0.33 3 10 cells in IP3R-TKO mice, with control mice, we found that both the number and the per- n = 11 mice for both groups). The numbers of pro/pre-B and im- centage of T2 B cells (25.6 6 1.1% in control mice versus 38.0 6 + + mature B cells were also not significantly changed in IP3R-TKO 1.6% in IP3R-TKO mice within B220 CD93 B cells gate, n =4 mice (Fig. 2A, 2B). By contrast, the number of transitional B cells mice for both groups) were significantly increased in the spleen of by guest on September 27, 2021 was significantly increased, whereas the number of recirculating IP3R-TKO mice (Fig. 2C, 2D). Consistent with what we found in mature B cells was significantly reduced, in bone marrow of IP3R- the bone marrow, the number of FO B cells was also significantly TKO mice compared with control mice (Fig. 2A, 2B). decreased in IP3R-TKO spleens (Fig. 2C, 2D). However, we did To determine whether expression of CD19-Cre could affect not find significant changes in the number of MZ B cells in IP3R- B cell development, we further examined B cell populations in TKO spleens (Fig. 2C, 2D). FO B cells can freely recirculate bone marrow of Itpr1f/+Itpr2f/+Itpr3f/+CD19-Cre+ and Itpr1f/+ among the peripheral secondary lymphoid tissues. Therefore, we Itpr2f/+Itpr3f/+CD19-Cre2 mice, and found that the percentages of further investigated B cell population in the lymph node, periph- + + total CD19 B220 , pro/pre-B, immature, transitional, and recir- eral blood, and peritoneal cavity of control and IP3R-TKO mice. culating B cells were not significantly changed in Cre+ mice Consistently, the percentage of B220+ B cells was significantly 2 compared with Cre mice (Supplemental Fig. 2A), suggesting that decreased in all peripheral secondary lymphoid tissues of IP3R- expression of CD19-Cre alone did not significantly alter B cell TKO mice compared with control mice (Fig. 2E, 2F). In summary, 2+ development. Furthermore, we observed no changes in the per- these data suggest that IP3R-mediated Ca signaling may play an centages of total CD19+B220+, pro/pre-B, immature, transitional, important role in late B cell development, especially from T2 to and recirculating B cells in bone marrow of Itpr1f/fItpr2f/fItpr3f/+ FO B cell development. + f/f f/+ f/f + CD19-Cre (IP3R1/R2 DKO), Itpr1 Itpr2 Itpr3 CD19-Cre We next investigated whether increased numbers of transitional f/+ f/f f/f + (IP3R1/R3 DKO), and Itpr1 Itpr2 Itpr3 CD19-Cre (IP3R2/R3 B cells in IP3R-TKO mice resulted from altered positive or neg- DKO) mice compared with control mice (Supplemental Fig. 2B). ative selection of immature B cells. It has been shown that acti- 2+ Consistently, we found that BCR-induced Ca signals in IP3R1/ vation of Ras could break B cell tolerance to promote autoreactive R2-DKO, IP3R1/R3-DKO, or IP3R2/R3-DKO B cells were also B cell differentiation and inhibit receptor editing via ERK and comparable with that in control B cells (Supplemental Fig. 2C), PI3K pathways (27). Therefore, we measured ERK1/2 phos- suggesting that different IP3R subtypes could play a redundant phorylation and expression levels of Foxo1, Rag1, and Rag2, all of role in regulating both BCR-mediated Ca2+ release and B cell which were suggested to be regulated by PI3K and essential for development. receptor editing, in bone marrow immature B cells of control and We further examined B cell maturation in the spleen. Once IP3R-TKO mice. We found that ERK1/2 phosphorylation is slightly exiting bone marrow, transitional B cells migrate to the spleen and decreased in IP3R-TKO immature B cells (Supplemental Fig. 3A). undergo further maturation (2). In the spleen, CD93-expressing In addition, we did not find any significant changes of Foxo1, Rag1, transitional B cells can be subdivided into three subsets: transi- and Rag2 mRNA levels between control and IP3R-TKO immature tional type 1 (T1), transitional type 2 (T2), and transitional type 3 B cells (Supplemental Fig. 3B). Taken together, these data suggested (T3) B cells, based on differential expression of CD23 and IgM that increased numbers of transitional B cells in IP3R-TKO mice (2). Among these transitional B cell types, T2 B cells are thought might not be caused by altered positive or negative selection of The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 2. Deletion of IP3Rs partially blocked late conventional B2 B cell development. (A) Surface CD19 and B220 expression of lymphocytes in the + + bone marrow of control and IP3R-TKO mice. Lymphocytes were first gated based on FSC and SSC. CD19 B220 cells were subgated, and surface ex- pression of IgM was examined. (B) Cell numbers of pro/pre-B (B220+IgM2), immature (B220lowIgMint), transitional (B220intIgMhigh), and recirculating high int mature (B220 IgM ) B cells. n = 11 mice per group. Data represent mean 6 SEM. (C) B cell subsets in the spleen of control and IP3R-TKO mice. Immature (B220+CD93+) and mature (B220+CD932) B cells are depicted within the lymphocyte gate. Gated on immature B cells, T1 (CD232IgMhigh), T2 (CD23+IgMhigh), and T3 (CD23+IgMlnt) were distinguished based on differential expression of CD23 and IgM. Gated on mature B cells, FO (CD21int CD23high) and MZ (CD21highCD232) B cells were shown based on CD21 and CD23 expression. (D) Cell numbers of T1, T2, T3, FO, and MZ B cells in + spleen of control and IP3R-TKO mice. n = 4 mice per group. Data represent mean 6 SEM. (E and F) Percentages of B220 cells within the lymphocyte gate by guest on September 27, 2021 in lymph node, peripheral blood, and peritoneal cavity of control and IP3R-TKO mice. n = 5–7 mice per group. Data represent mean 6 SEM. Significance was determined by the two-tailed, unpaired Student t test. *p , 0.05, **p , 0.01, ***p , 0.001 versus control. immature B cells. Because BAFF-R has also been shown to play an anti-IgM, LPS, and anti-CD40. Purified splenic B cells were la- important role in regulating the survival and maturation of transi- beled with CFSE, and cell proliferation was then measured by tional B cells (29), we further checked the expression of Baffr in CFSE dilution. BCR stimulation induced several rounds of cell bone marrow transitional B cells. However, we did not find any division in control B cells, whereas IP3R-TKO B cells did not significant change of Baffr mRNA levels in bone marrow transitional undergo cell division in response to anti-IgM stimulation (Fig. 4A, B cells between control and IP3R-TKO mice (Supplemental Fig. 3C). 4B). By contrast, cell division induced by LPS or anti-CD40 was In addition to conventional B2 B cells, B1 B cells are another unchanged in IP3R-TKO B cells (Fig. 4A, 4B), suggesting that subclass of B cell lymphocytes, which constitute a minor fraction of LPS-induced or anti-CD40–induced cell proliferation is indepen- B cells in the spleen but predominately reside in the peritoneal and dent of IP3Rs, which is also consistent with a previous report that pleural cavities (30). In mice, B1 B cells can be further divided anti-CD40 or LPS did not induce Ca2+ mobilization in B cells into B1a (CD5+) and B1b (CD52) subsets (31). A series of mu- (11). However, we also found that costimulation of anti-IgM tations that interfere with normal BCR signaling has been shown with anti-CD40 could significantly restore the proliferative abil- to reduce B-1 B cell numbers, suggesting a crucial requirement of ity of IP3R-TKO B cells (Fig. 4A, 4B). Furthermore, we examined BCR signals for B1 B cell development (31). Therefore, we next the effects of IP3R deficiency on B cell survival in response to investigated the effect of IP3R deficiency in B1 B cell develop- various mitogenic stimuli. The percentage of viable B cells was ment. Compared with control mice, the proportion of B1a B cells determined by PI exclusion. Survival rate was dramatically in- in the peritoneal cavity was significantly reduced in IP3R-TKO creased in control B cells with BCR stimulation, which was se- mice (Fig. 3A, 3B).Similarly, the proportion of splenic B1a and verely inhibited in IP3R-TKO B cells (Fig. 4C). By contrast, B1b B cells was also decreased in IP3R-TKO mice (Fig. 3C). survival rates of B cells in response to LPS or anti-CD40 were 2+ Taken together, these results suggest that IP3R-mediated Ca comparable between control and IP3R-TKO mice (Fig. 4C), sug- signaling could regulate both conventional B2 B cell and B1 B cell gesting that IP3Rs could also be dispensable for LPS-induced or development in vivo. anti-CD40–induced B cell survival. Furthermore, costimulation of anti-IgM with anti-CD40 could partially restore the survival rate Loss of IP3Rs impairs BCR-mediated cell proliferation of IP3R-TKO B cells (Fig. 4C). Taken together, these results and survival 2+ clearly demonstrate that IP3R-mediated Ca signaling is specif- We next investigated the role of IP3Rs in regulating B cell pro- ically required for BCR-induced B cell proliferation and survival. liferation and survival under various mitogenic stimuli including In contrast, our data also suggest that signaling pathways evoked 6IP3Rs REGULATE MOUSE B CELL DEVELOPMENT AND FUNCTION

FIGURE 3. Loss of IP3Rs resulted in reduced B1 B cell numbers. (A) Surface expression of CD43 and CD5 on B220+-gated lymphocytes isolated from the peritoneal cavities of control and IP3R-TKO mice. Percentages of B1a (CD43+CD5+), B1b (CD43+CD52), and B2 (CD432 CD52) cells in the (B) peritoneal cavity and (C) spleen of control and IP3R-TKO mice. n = 5–6 mice per group. Data represent mean 6 SEM. Significance was deter- mined by the two-tailed, unpaired Student t test. *p , 0.05, **p , 0.01, ***p , 0.001 versus control. Downloaded from

by anti-CD40 could compensate for IP3R deficiency after BCR maintaining basal serum Ig levels. To assess humoral immune stimulation, even though the effects of anti-CD40 on B cell pro- responses, we first examined TD Ab responses by immunizing liferation and survival are not dependent on IP3Rs. mice with NP-CGG, and we monitored the production of NP-specific http://www.jimmunol.org/ We further investigated BCR-induced cell survival and prolif- IgM and IgG1 Abs for the next 3 consecutive wk. The production of eration in different B cell subsets, including transitional, FO, and both NP-specific IgM and NP-specific IgG1 was significantly re- MZ B cells. We found that cell proliferation induced by anti-IgM duced in IP3R-TKO mice at 1 wk postimmunization, but recovered to was all reduced in transitional, FO, and MZ B cells of IP3R-TKO comparable levels within the following 2 wk when compared with mice compared with control mice (Supplemental Fig. 4A–C). In control mice (Fig. 5B). During TD responses, TD Ags will lead to contrast, we found that the percentages of viable PI2 transitional germinal center (GC) formation, where B cells undergo expansion and FO B cells after stimulation of anti-IgM were significantly and affinity maturation (33). Consistently, we also found that the + high + reduced in IP3R-TKO mice compared with control mice, whereas percentage of splenic GC B cells (defined as B220 Fas GL7 ) 2 by guest on September 27, 2021 the percentage of viable PI MZ B cells was comparable between at 1 wk post NP-CGG immunization in IP3R-deficient mice was control and IP3R-TKO mice (Supplemental Fig. 4D). significantly decreased when compared with control mice (Fig. 5C). To further understand the molecular mechanisms underlying the We then analyzed TI Ab responses by immunizing mice with NP- impaired BCR-induced B cell proliferation and survival after IP3R LPS (TI-1 Ag) or NP-Ficoll (TI-2 Ag), and we monitored the pro- deletion, we examined the expression and activation of down- duction of Ag-specific IgM and IgG3 Abs for the next 3 consecutive stream effectors including NFAT, ERK, and JNK in isolated naive wk. We found that the level of NP-specific IgM in IP3R-TKO mice B cells after BCR ligation. First, BCR stimulation in control was slightly higher than that of control mice at 1 wk postim- B cells resulted in apparent NFAT1 dephosphorylation, which was munization of NP-LPS (Fig. 5D), and the level of NP-specific IgM almost totally blocked in IP3R-TKO B cells (Fig. 4D). On the in IP3R-TKO mice was slightly lower than that of control mice at contrary, phosphorylation of ERK and JNK induced by BCR 1 wk postimmunization with NP-Ficoll (Fig. 5E). However, we did stimulation was increased in IP3R-deficient B cells compared with not observe any significant difference in the levels of NP-specific control B cells (Fig. 4D), consistent with a previous report using IgG3 between control and IP3R-TKO within 3 wk postimmunization Mef2c-knockout B cells in which defective cell proliferation was of NP-LPS or NP-Ficoll (Fig. 5D, 5E). Taken together, our results 2+ accompanied by enhanced activation of ERK and JNK (32). Even suggest that loss of IP3R-mediated Ca release in B cells might have though we did not find a significant change of Mef2c expression an acute, but not chronic, influence on humoral immune response. level between control and IP R-TKO splenic B cells (Supplemental 3 Deletion of IP Rs reduces IL-10 production Fig. 4E), we found that expression of Ccnd2, a downstream target of 3 Mef2c, was significantly reduced at both basal levels and in response In addition to producing Abs to protect against infectious Ags, to BCR stimulation (Fig. 4E). Because Mef2c has also been sug- B cells possess a suppressive capability to negatively regulate gested as a target of calcineurin (32), these results implicate that inflammatory responses by secreting anti-inflammatory cytokines 2+ activation of calcineurin after IP3R-mediated Ca release could be such as IL-10 (34). We next investigated whether deletion of IP3Rs a critical step underlying the regulation of BCR-mediated cell could influence the regulatory function of B cells by measuring proliferation. IL-10 production in isolated splenic B cells. It has been shown that BCR stimulation alone is not able to induce IL-10 secretion, but Deletion of IP3Rs affects in vivo immune responses BCR stimulation could potentiate IL-10 secretion when B cells are We next investigated whether deletion of IP3Rs could affect the first preactivated with the agonist of TLRs such as LPS (11, 35, ability of the immune system to produce Abs as a consequence 36). Therefore, we pretreated splenic naive B cells with LPS for of altered B cell development and function. First, we found that 48 h and then applied BCR stimulation with anti-IgM to induce serum titers of IgM, IgG1, IgG2b, IgG3, and IgA were comparable IL-10 secretion. Using quantitative RT-PCR analysis, we found between unimmunized IP3R-TKO and control mice (Fig. 5A), that BCR stimulation could dramatically increase the expression 2+ indicating that IP3R-mediated Ca signaling is not necessary for of IL-10 in LPS-activated control B cells, which was significantly The Journal of Immunology 7 Downloaded from

FIGURE 4. Deletion of IP3Rs impaired BCR-mediated proliferation and survival. (A) Proliferation as assessed by CFSE labeling. Splenic B cells were isolated from control and IP3R-TKO mice, and stimulated for 48 h with vehicle, anti-IgM (10 mg/ml), anti-CD40 (10 mg/ml), LPS (10 mg/ml), and a http://www.jimmunol.org/ combination of anti-IgM (10 mg/ml) with anti-CD40 (10 mg/ml), respectively. The numbers within each histogram represent the percentage of divided cells as measured by CFSE dilution. (B) Percentages of dividing cells in response to different stimuli. n = 4 mice per group. Data represent mean 6 SEM. (C) 2 + Percentages of PI control and IP3R-TKO B220 B cells. Cells were cultured and stimulated as described in (A). n = 4 mice per group. Data represent mean 6 SEM. (D) Western blot analysis of whole-cell lysates of control and IP3R-TKO splenic B cells stimulated with anti-IgM (10 mg/ml) for various durations as depicted. b-Actin was used as loading control. Results represent one of three representative experiments. (E) Quantitative RT-PCR analysis of the expression of cyclin D2 (Ccnd2) in control and IP3R-TKO splenic B cells at basal levels or stimulated for 12 h with anti-IgM (10 mg/ml). n = 3 mice per group. Data represent mean 6 SEM. Significance was determined by the two-tailed, unpaired Student t test. *p , 0.05, **p , 0.01, ***p , 0.001 versus control. by guest on September 27, 2021 inhibited in LPS-activated IP3R-TKO B cells (Fig. 6A). Moreover, suggest that deletion of IP3Rs in B cells resulted in a decreased BCR stimulation also dramatically increased the level of secreted percentage of CD1dhighCD5+ B cells and defective NFAT activa- IL-10 in the medium of LPS-activated control B cells, which was tion, both of which together caused reduced IL-10 expression and also dramatically inhibited in LPS-activated IP3R-TKO B cells secretion. (Fig. 6B). We next investigated the potential cellular and molecular Discussion 2+ mechanisms underlying the regulation of IL-10 production by In this study, we demonstrated that IP3R-mediated Ca release 2+ IP3R-mediated Ca signaling. It has been shown that a regulatory plays an essential role in regulating B cell development and high + B10 subset identified as CD1d CD5 B cell was the major cell function using B cell–specific IP3R knockout mouse models. In type responsible for producing IL-10 (11, 37). Therefore, we first B cells, the strength of BCR signaling has been proposed to guide examined whether deletion of IP3Rs could cause aberrant devel- the positive selection of B cells and negative selection to eliminate opment of B10 cells. Compared with control mice, the percentage autoreactive B cells, thereby regulating the development of B of splenic CD1dhighCD5+ B cells within the B220+ B cells gate in cells. A central response to BCR cross-linking is the elevation 2+ 2+ IP3R-TKO mice was significantly decreased (Fig. 6C), suggesting of intracellular Ca concentration (39), which is mediated by Ca 2+ that IP3R-Ca signaling plays an important role in regulatory release from the ER via IP3Rs and the subsequent SOCE. Mutations B10 cell development or maintenance. Because the calcineurin- in molecules that positively or negatively regulate Ca2+ signaling NFAT signaling pathway has been shown to play a critical role have been reported to regulate B cell development and function. The in regulating BCR-mediated IL-10 production (11, 38), we next absence of positive regulators such as Syk, Btk, BLNK, or PLC-g2 2+ examined whether deletion of IP3Rs could disrupt the calcineurin- results in impaired Ca signaling and blocked B cell development NFAT signaling pathway in B cells. First, calcineurin inhibitors at the early stages in bone marrow (7, 40–43). By contrast, loss of CsA and FK506 could completely abolish the expression of IL-10 negative modulators such as SHP-1 or CD22 leads to enhanced Ca2+ induced by BCR stimulation in both LPS-activated control and increase, hyperresponsive B cells, and autoimmunity (44, 45). All of 2+ IP3R-TKO B cells (Fig. 6D), indicating that IL-10 expression in these studies implicate that Ca signals in B cells may participate in both cell populations relies on the calcineurin-NFAT signaling regulating both development and function. Consistently, the physio- 2+ pathway. Furthermore, we also found that deletion of IP3Rs di- logical role of Ca signals mediated by SOCE in regulating B cell minished the activation of NFAT1 in LPS-activated B cells. In function has been well studied in human patients carrying ORAI1 LPS-activated control cells, BCR stimulation could induce quick and STIM1 mutations (13, 14) or mice with deleted Orai1 and STIM and sustained dephosphorylation of NFAT1. However, the de- (10–12). On the contrary, endogenous Orai1, STIM1, and phosphorylation of NFAT1 was dramatically suppressed in LPS- STIM2 were shown to be dispensable for B cell development (10– 2+ activated IP3R-TKO cells (Fig. 6E). Taken together, these results 12). However, whether endogenous Ca signals can influence B cell 8IP3Rs REGULATE MOUSE B CELL DEVELOPMENT AND FUNCTION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 5. TD and TI Ab responses in control and IP3R-TKO mice. (A) ELISA was used to measure basal Ig titers of IgM, IgG1, IgG2b, IgG3, and IgA in the sera of nonimmunized control and IP3R-TKO mice. n = 8 mice per group. Data represent mean 6 SEM. (B) IgM and IgG1 NP-specific Ab responses of control and IP3R-TKO mice immunized with NP-CGG, as measured by NP-specific ELISA at each time point. n = 4–5 mice per group. Data represent + high + + mean 6 SEM. (C) Percentages of splenic GC B cells (B220 Fas GL7 ) within B220 gate in control and IP3R-TKO mice at baseline (n = 3 mice for both control and IP3R-TKO groups) and 1 wk after NP-CGG immunization (n = 7 mice for both control and IP3R-TKO groups). Data represent mean 6 SEM. (D and E) NP-specific Ab responses of control and IP3R-TKO mice immunized with NP-LPS (D) or NP-Ficoll (E), as measured by NP-specific ELISA at each time point. n = 3–4 mice per group. Data represent mean 6 SEM. Significance was determined by the two-tailed, unpaired Student t test. *p , 0.05, **p , 0.01 versus control. development and the functional difference between Ca2+ release members, RelB and c-Rel, has been demonstrated to regulate late from ER and SOCE remain unclear. Therefore, our study provided B cell development from transitional to mature B cells (47). evidence for the first time, to our knowledge, that endogenous Ca2+ Whether the defect in transitional to mature B cell development in signals mediated by IP3Rs are essential for B cell development. the IP3R-deficient mice was caused by impaired activation of NF-kB Loss of all three IP3R subtypes in B cells could totally abrogate or other transcription factors remains to be investigated. In contrast, 2+ 2+ BCR-induced Ca release and the subsequent SOCE, and impair IP3R-mediated Ca signaling is essential for B1 B cell develop- development of conventional B2, B1, and regulatory B10 cells. It ment. Previous studies have shown that B1 B cell development or is intriguing that the initial intracellular Ca2+ increase mediated maintenance is regulated by the calcineurin-NFAT signaling path- by IP3Rs alone was sufficient to drive normal B cell development, way, given that both calcineurin b1–deficient mice and NFAT2- whereas sustained intracellular Ca2+ increase mediated by both deficient mice exhibited reduced B1 B cell numbers (48, 49). In IP3Rs and SOCE was required for normal B cell function. IP3R-TKO B cells, the activation of NFAT1, another member of 2+ Our results demonstrated that IP3R-mediated Ca signals are NFAT proteins, was defective in response to BCR stimulation, im- specifically important for T2 B cell to FO B cell maturation. plicating that abnormal B1 B cell development in IP3R-TKO mice Deletion of IP3Rs increased transitional B cell numbers and de- might result from defective calcineurin-NFAT signaling. creased mature B cell numbers in the bone marrow. Consistently, We also demonstrated that IP3Rs are required for B cell pro- the numbers of T2 B cells were significantly increased, whereas liferation and survival in response to BCR ligation. Calcineurin FO B cell numbers were decreased in the spleen. In addition, all has been shown to play an important role in B cell proliferation peripheral lymphoid tissues including the lymph node, peripheral (49). In IP3R-deficient B cells, dephosphorylation of NFAT1 in blood, and peritoneal cavity showed reduced proportions of B cells. response to BCR stimulation was dramatically decreased. In ad- A series of transcription factors such as Aiolos, Notch-2, c-Myb, dition, Mef2c, another calcineurin-regulated transcription factor, Pax-5, and NF-kB have been reported to be involved in peripheral was also essential for BCR-mediated B cell proliferation and B cell development (46). Notably, the coordination of NF-kBfamily survival through regulating gene expression of cyclin D2 (32). The Journal of Immunology 9 Downloaded from

high + FIGURE 6. IL-10 production was impaired in IP3R-TKO B cells because of decreased CD1d CD5 regulatory B cell numbers and defective activation of calcineurin-NFAT signaling. (A) Control and IP3R-TKO B cells were cultured with LPS (10 mg/ml) for 48 h (LPS-activated B cells) followed by stimulation with vehicle or anti-IgM for 3 h. Quantitative RT-PCR analysis was used to assess the mRNA levels of IL-10. n = 6 mice per group. Data represent mean 6 SEM. Significance was determined by the two-tailed, unpaired Student t test. **p , 0.01 versus control. (B) ELISA analysis of IL-10 production in LPS-activated control and IP3R-TKO B cells stimulated with vehicle or anti-IgM for 24 h. n = 6 mice per group. Data represent mean 6 SEM.

Significance was determined by the two-tailed, unpaired Student t test. *p , 0.05, **p , 0.01 versus control. (C) Expression of surface CD1d and CD5 on http://www.jimmunol.org/ + high + B220 -gated B cells isolated from control (n = 6) and IP3R-TKO (n = 5) mice. Histograms show the averaged percentages of CD1d /CD5 B cells in control and IP3R-TKO mice. Data represent mean 6 SEM. Significance was determined by the two-tailed, unpaired Student t test. ***p , 0.001 versus control. (D) Quantitative RT-PCR analysis of IL-10 mRNA levels in LPS-activated control and IP3R-TKO B cells treated with vehicle (DMSO), CsA (1 mM), or FK506 (1.25 mM) for 1 h followed by stimulation with anti-IgM for 3 h. n = 4 mice per group. Data represent mean 6 SEM. Significance was determined by the two-tailed, unpaired Student t test. ***p , 0.001 versus DMSO. (E) Western blot analysis of NFAT1 in whole-cell lysates of LPS- activated control and IP3R-TKO B cells stimulated with anti-IgM (10 mg/ml) for various durations as depicted. b-Actin was used as loading control. Results represent one of three representative experiments. by guest on September 27, 2021 IP3R-TKO B cells also showed decreased expression of cyclin D2 question. In addition to TD Ab response, we found that the level of at the mRNA level when stimulated with anti-IgM. In Mef2c- NP-specific IgM in IP3R-TKO mice was slightly higher than that deficient B cells, BCR stimulation resulted in increased phos- of control mice at 1 wk postimmunization of NP-LPS. B1 and MZ phorylation levels of JNK and ERK1/2, which is consistent with B cells have been proposed to be responsible for TI-2 Ab re- what we found in IP3R-TKO B cells. These data together impli- sponses (4, 51). It is likely that the reduced B1 B cell numbers cated that a defective calcineurin-Mef2c pathway might be one of in IP3R-TKO mice contribute to the lower NP-specific IgM pro- the molecular mechanisms accounting for the reduced prolifera- duction at the first wk after immunization. Furthermore, we found tion of IP3R-TKO B cells. In contrast, IP3R-TKO B cells prolif- that the level of NP-specific IgM was slightly increased in IP3R-TKO erated normally in response to LPS and anti-CD40 stimulation mice in response to TI-1 Ags, NP-LPS. Because TI-1 Ag is thought compared with control B cells, suggesting that LPS-induced and to bind both BCR and TLRs to transduce a strong signal for B cell anti-CD40–induced B cell proliferation is independent of intra- activation (52), one possible explanation could be that TLR signaling 2+ cellular Ca signaling, which is also consistent with the fact that in IP3R-TKO mice might compensate B cell activation and function 2+ both LPS and anti-CD40 were not able to induce intracellular Ca in response to TI-1 Ags. Taken together, our data suggest that IP3R- increase in B cells (11). Furthermore, restoration of BCR-induced mediated Ca2+ signaling contributes to Ab response in vivo, which is cell proliferation in IP3R-deficient B cells by costimulation with also partially consistent with a previous study of calcineurin b1– anti-CD40 might be because of the fact that both BCR- and CD40- deficient mice (11). However, the mechanism underlying why loss induced B cell proliferation can share several common molecular of IP3Rs had only a short-term effect in Ab production requires mechanisms, one of which has been identified as cyclin D2 up- further investigation. Because Ab production by B cells is critical regulation (50). for protecting from infection in vivo, deletion of IP3Rs in mice In IP3R-TKO mice, TD Ab response was impaired after deletion may potentially lead to difficulty in fighting infectious diseases, of IP3Rs. It has been shown that FO B cells are the main mediator which would be worthy to be further examined using different mouse responsible for TD Ab response, in which B cells undergo clonal models of infection. expansion by receiving cognate T cell help through cytokine In addition, B cells are known to play a regulatory role in the stimulation and CD40 engagement in GCs (3). Therefore, reduced immune system by secreting inflammatory cytokines, one of which GC B cell numbers and defective proliferation of splenic B cells is IL-10, that can function to prevent inflammatory and auto- might account for the impaired TD Ab response in IP3R-TKO immune pathologies (53). The signaling pathways mediated by mice. However, it remains unclear whether reduced Ab produc- TLRs, combined with BCR and CD40, are critical for B cells to tion and GC formation by deletion of IP3Rs was due to impaired suppress autoimmunity through IL-10 production in vivo (35, 53). B cell development or cell-autonomous defects in mature B cells. In IP3R-TKO B cells that are preactivated by LPS, IL-10 pro- Future experiments using an inducible CreERT2 system instead of duction induced by BCR stimulation was severely impaired. In a constitutive CD19-Cre system would be helpful to address this STIM1/STIM2 double-deficient B cells, IL-10 production is also 10 IP3Rs REGULATE MOUSE B CELL DEVELOPMENT AND FUNCTION reduced, which could result from the defective activation of the 9. Baba, Y., and T. Kurosaki. 2009. Physiological function and molecular basis of STIM1-mediated calcium entry in immune cells. Immunol. Rev. 231: 174–188. calcineurin-NFAT signaling pathway after BCR stimulation (11). 10. Gwack, Y., S. Srikanth, M. Oh-Hora, P. G. Hogan, E. D. Lamperti, M. Yamashita, Consistently, we also found that dephosphorylation of NFAT1 in C. Gelinas, D. S. Neems, Y. Sasaki, S. Feske, et al. 2008. Hair loss and defective response to BCR stimulation was decreased in LPS-activated T- and B-cell function in mice lacking ORAI1. Mol. Cell. Biol. 28: 5209–5222. 11. Matsumoto, M., Y. Fujii, A. Baba, M. Hikida, T. Kurosaki, and Y. Baba. 2011. IP3R-deficient B cells, suggesting that BCR-mediated IL-10 pro- The calcium sensors STIM1 and STIM2 control B cell regulatory function 2+ duction requires both IP3R-mediated Ca release and the subse- through interleukin-10 production. Immunity 34: 703–714. quent SOCE. Intriguingly, we also found that the number of B10 12. McCarl, C. A., S. Khalil, J. Ma, M. Oh-hora, M. Yamashita, J. Roether, T. Kawasaki, A. Jairaman, Y. Sasaki, M. Prakriya, and S. Feske. 2010. Store- cells is decreased in IP3R-defecient mice. Although there is no operated Ca2+ entry through ORAI1 is critical for T cell-mediated autoimmunity specific cell surface marker unique to all B10 cells, they have been and allograft rejection. J. Immunol. 185: 5845–5858. proposed to be enriched within the splenic CD1dhighCD5+ subset 13. McCarl, C. A., C. Picard, S. Khalil, T. Kawasaki, J. Rother, A. Papolos, J. Kutok, C. Hivroz, F. Ledeist, K. Plogmann, et al. 2009. ORAI1 deficiency and lack of (54). BCR signaling is thought to play a role in B10 cell develop- store-operated Ca2+ entry cause immunodeficiency, myopathy, and ectodermal ment, given that human CD19-overexpressing mice showed in- dysplasia. J. Allergy Clin. Immunol. 124: 1311–1318.e7. 14. Picard, C., C. A. McCarl, A. Papolos, S. Khalil, K. Luthy,€ C. Hivroz, F. LeDeist, creased BCR signaling and elevated B10 cell numbers (36), whereas F. Rieux-Laucat, G. Rechavi, A. Rao, et al. 2009. STIM1 mutation associated CD19-deficient mice have impaired BCR signaling and fewer B10 with a syndrome of immunodeficiency and autoimmunity. N. Engl. J. Med. 360: cell numbers (37). Moreover, multiple strains of transgenic mice 1971–1980. 15. Foskett, J. K., C. White, K. H. Cheung, and D. O. Mak. 2007. Inositol with a fixed BCR repertoire have reduced B10 cell numbers (55). trisphosphate receptor Ca2+ release channels. Physiol. Rev. 87: 593–658. 2+ high + IP3R-mediated Ca signaling is critical to maintain CD1d CD5 16. Matsumoto, M., T. Nakagawa, T. Inoue, E. Nagata, K. Tanaka, H. Takano, high + O. Minowa, J. Kuno, S. Sakakibara, M. Yamada, et al. 1996. Ataxia and epileptic B cell numbers. In IP3R-TKO mice, the number of CD1d CD5 seizures in mice lacking type 1 inositol 1,4,5-trisphosphate receptor. Nature 379: B cells was decreased, whereas the numbers of this population is not 168–171. Downloaded from altered by loss of both STIM1 and STIM2, which is also consistent 17. Futatsugi, A., T. Nakamura, M. K. Yamada, E. Ebisui, K. Nakamura, K. Uchida, with the finding that impaired B2 and B1 cell development was only T. Kitaguchi, H. Takahashi-Iwanaga, T. Noda, J. Aruga, and K. Mikoshiba. 2005. IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy found in IP3R-deficient mice and not in STIM1/STIM2 DKO mice. metabolism. Science 309: 2232–2234. The role of regulatory B10 cells has been extensively examined 18. Hisatsune, C., K. Yasumatsu, H. Takahashi-Iwanaga, N. Ogawa, Y. Kuroda, in mouse models of experimental autoimmune encephalomyelitis, R. Yoshida, Y. Ninomiya, and K. Mikoshiba. 2007. Abnormal taste perception in mice lacking the type 3 inositol 1,4,5-trisphosphate receptor. J. Biol. Chem. 282: collagen-induced arthritis, and contact hypersensitivity (55). Further 37225–37231. http://www.jimmunol.org/ studies using these mouse models may help to determine whether 19. Nakazawa, M., K. Uchida, M. Aramaki, K. Kodo, C. Yamagishi, T. Takahashi, K. Mikoshiba, and H. Yamagishi. 2011. Inositol 1,4,5-trisphosphate receptors are reduced production of IL-10 and B10 cell numbers in IP3R-deficient essential for the development of the second heart field. J. Mol. Cell. Cardiol. 51: mice could exacerbate disease severity. 58–66. 2+ 20. Uchida, K., M. Aramaki, M. Nakazawa, C. Yamagishi, S. Makino, K. Fukuda, In summary, we demonstrated that IP3R-mediated Ca release T. Nakamura, T. Takahashi, K. Mikoshiba, and H. Yamagishi. 2010. Gene knock- is important for maintaining normal numbers of conventional B2, outs of inositol 1,4,5-trisphosphate receptors types 1 and 2 result in perturbation B1, and regulatory B10 B cells, as well as regulating the humoral of cardiogenesis. PLoS One 5: e12500. Available at: http://dx.doi.org/10.1371/ immune response in vivo. We also found that IP R-deficient B cells journal.pone.0012500. 3 21. Lin, Q., G. Zhao, X. Fang, X. Peng, H. Tang, H. Wang, R. Jing, J. Liu, W. J. Lederer, exhibited reduced BCR-mediated proliferation and survival, defec- J. Chen, and K. Ouyang. 2016. IP3 receptors regulate vascular smooth muscle tive calcineurin-NFAT signaling, and impaired IL-10 production. contractility and hypertension. JCI Insight 1: e89402. Available at: https:// by guest on September 27, 2021 Therefore, our study revealed a dual role of IP R-mediated Ca2+ insight.jci.org/articles/view/89402. 3 22. Ouyang, K., R. Leandro Gomez-Amaro, D. L. Stachura, H. Tang, X. Peng, signals in B cell development and function. X. Fang, D. Traver, S. M. Evans, and J. Chen. 2014. Loss of IP3R-dependent Ca2+ signalling in thymocytes leads to aberrant development and acute lymphoblastic leukemia. Nat. Commun. 5: 4814. Acknowledgments 23. Sugawara, H., M. Kurosaki, M. Takata, and T. Kurosaki. 1997. Genetic evidence We thank Xing Chang (Shanghai Institutes for Biological Sciences, Chinese for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in Academy of Sciences) for critical suggestions about experimental design, Ju signal transduction through the B-cell antigen receptor. EMBO J. 16: 3078–3088. 24. Cooley, N., K. Ouyang, J. R. McMullen, H. Kiriazis, F. Sheikh, W. Wu, Y. Mu, Chen (University of California, San Diego) and Jennifer Veevers for critical X. J. Du, J. Chen, and E. A. Woodcock. 2013. No contribution of IP3-R(2) to reading of the manuscript, and Xiaodi Chen (Peking University Shenzhen disease phenotype in models of dilated cardiomyopathy or pressure overload Hospital) and Wugen Zhan (Shenzhen Institutes of Advanced Technology, hypertrophy. Circ Heart Fail 6: 318–325. Chinese Academy of Sciences) for technical support of blood collection and 25. Rickert, R. C., J. Roes, and K. Rajewsky. 1997. B lymphocyte-specific, Cre- mediated mutagenesis in mice. Nucleic Acids Res. 25: 1317–1318. flow cytometry. 26. Ouyang, K., C. Wu, and H. Cheng. 2005. Ca(2+)-induced Ca(2+) release in sensory neurons: low gain amplification confers intrinsic stability. J. Biol. Chem. 280: 15898–15902. Disclosures 27. Teodorovic, L. S., C. Babolin, S. L. Rowland, S. A. Greaves, D. P. Baldwin, The authors have no financial conflicts of interest. R. M. Torres, and R. Pelanda. 2014. Activation of Ras overcomes B-cell toler- ance to promote differentiation of autoreactive B cells and production of auto- antibodies. Proc. Natl. Acad. Sci. USA 111: E2797–E2806. References 28. Hobeika, E., S. Thiemann, B. Storch, H. Jumaa, P. J. Nielsen, R. Pelanda, and M. Reth. 2006. Testing gene function early in the B cell lineage in mb1-cre mice. 1. Hardy, R. R., and K. Hayakawa. 2001. B cell development pathways. Annu. Rev. Proc. Natl. Acad. Sci. USA 103: 13789–13794. Immunol. 19: 595–621. 29. Mackay, F., and P. Schneider. 2009. Cracking the BAFF code. Nat. Rev. Immunol. 2. Cariappa, A., C. Chase, H. Liu, P. Russell, and S. Pillai. 2007. Naive recircu- 9: 491–502. lating B cells mature simultaneously in the spleen and bone marrow. Blood 109: 30. Baumgarth, N. 2011. The double life of a B-1 cell: self-reactivity selects for 2339–2345. protective effector functions. Nat. Rev. Immunol. 11: 34–46. 3. Pillai, S., and A. Cariappa. 2009. The follicular versus marginal zone 31. Hardy, R. R. 2006. B-1 B cell development. J. Immunol. 177: 2749–2754. B lymphocyte cell fate decision. Nat. Rev. Immunol. 9: 767–777. 32. Wilker, P. R., M. Kohyama, M. M. Sandau, J. C. Albring, O. Nakagawa, 4. Cerutti, A., M. Cols, and I. Puga. 2013. Marginal zone B cells: virtues of innate- J. J. Schwarz, and K. M. Murphy. 2008. Transcription factor Mef2c is required for like antibody-producing lymphocytes. Nat. Rev. Immunol. 13: 118–132. B cell proliferation and survival after antigen receptor stimulation. Nat. Immunol. 9: 5. Martin, F., and J. F. Kearney. 2001. B1 cells: similarities and differences with 603–612. other B cell subsets. Curr. Opin. Immunol. 13: 195–201. 33. Victora, G. D., and M. C. Nussenzweig. 2012. Germinal centers. Annu. Rev. 6. Rosser, E. C., and C. Mauri. 2015. Regulatory B cells: origin, phenotype, and Immunol. 30: 429–457. function. Immunity 42: 607–612. 34. Mauri, C., and M. R. Ehrenstein. 2008. The ‘short’ history of regulatory B cells. 7. Feske, S. 2007. Calcium signalling in lymphocyte activation and disease. Nat. Trends Immunol. 29: 34–40. Rev. Immunol. 7: 690–702. 35. Lampropoulou, V., K. Hoehlig, T. Roch, P. Neves, E. Caldero´nGo´mez, 8. Baba, Y., M. Matsumoto, and T. Kurosaki. 2014. Calcium signaling in B cells: C.H.Sweenie,Y.Hao,A.A.Freitas,U.Steinhoff,S.M.Anderton,andS.Fillatreau. regulation of cytosolic Ca2+ increase and its sensor molecules, STIM1 and 2008. TLR-activated B cells suppress T cell-mediated autoimmunity. J. Immunol. STIM2. Mol. Immunol. 62: 339–343. 180: 4763–4773. The Journal of Immunology 11

36. Yanaba, K., J. D. Bouaziz, T. Matsushita, T. Tsubata, and T. F. Tedder. 2009. The 46. Casola, S. 2007. Control of peripheral B-cell development. Curr. Opin. Immunol. development and function of regulatory B cells expressing IL-10 (B10 cells) re- 19: 143–149. quires antigen receptor diversity and TLR signals. J. Immunol. 182: 7459–7472. 47. Almaden, J. V., Y. C. Liu, E. Yang, D. C. Otero, H. Birnbaum, J. Davis-Turak, 37. Yanaba, K., J. D. Bouaziz, K. M. Haas, J. C. Poe, M. Fujimoto, and T. F. Tedder. M. Asagiri, M. David, A. W. Goldrath, and A. Hoffmann. 2016. B-cell survival 2008. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls and development controlled by the coordination of NF-kB family members RelB T cell-dependent inflammatory responses. Immunity 28: 639–650. and cRel. Blood 127: 1276–1286. 38. Saraiva, M., and A. O’Garra. 2010. The regulation of IL-10 production by im- 48. Berland, R., and H. H. Wortis. 2003. Normal B-1a cell development requires mune cells. Nat. Rev. Immunol. 10: 170–181. B cell-intrinsic NFATc1 activity. Proc. Natl. Acad. Sci. USA 100: 13459–13464. 39. Braun, J., R. I. Sha’afi, and E. R. Unanue. 1979. Crosslinking by ligands to 49. Winslow, M. M., E. M. Gallo, J. R. Neilson, and G. R. Crabtree. 2006. The surface immunoglobulin triggers mobilization of intracellular 45Ca2+ in calcineurin phosphatase complex modulates immunogenic B cell responses. B lymphocytes. J. Cell Biol. 82: 755–766. Immunity 24: 141–152. 40. Conley, M. E., O. Parolini, J. Rohrer, and D. Campana. 1994. X-linked agam- 50. Lam, E. W., J. Glassford, L. Banerji, N. S. Thomas, P. Sicinski, and G. G. Klaus. maglobulinemia: new approaches to old questions based on the identification of 2000. Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes the defective gene. Immunol. Rev. 138: 5–21. activated via the antigen receptor and CD40. J. Biol. Chem. 275: 3479–3484. 41. Kurosaki, T. 2002. Regulation of B-cell signal transduction by adaptor proteins. 51. Yammani, R. D., and K. M. Haas. 2013. Primate B-1 cells generate antigen- Nat. Rev. Immunol. 2: 354–363. specific B cell responses to T cell-independent type 2 antigens. J. Immunol. 190: 42. Minegishi, Y., J. Rohrer, E. Coustan-Smith, H. M. Lederman, R. Pappu, 3100–3108. D. Campana, A. C. Chan, and M. E. Conley. 1999. An essential role for BLNK in 52. Hou, B., P. Saudan, G. Ott, M. L. Wheeler, M. Ji, L. Kuzmich, L. M. Lee, human B cell development. Science 286: 1954–1957. R. L. Coffman, M. F. Bachmann, and A. L. DeFranco. 2011. Selective utilization 43. Yu, P., R. Constien, N. Dear, M. Katan, P. Hanke, T. D. Bunney, S. Kunder, of Toll-like receptor and MyD88 signaling in B cells for enhancement of the L. Quintanilla-Martinez, U. Huffstadt, A. Schro¨der, et al. 2005. Autoimmunity antiviral germinal center response. Immunity 34: 375–384. and inflammation due to a gain-of-function mutation in phospholipase C gamma 53. Fillatreau, S., C. H. Sweenie, M. J. McGeachy, D. Gray, and S. M. Anderton. 2 that specifically increases external Ca2+ entry. Immunity 22: 451–465. 2002. B cells regulate autoimmunity by provision of IL-10. Nat. Immunol. 3: 44. O’Keefe, T. L., G. T. Williams, F. D. Batista, and M. S. Neuberger. 1999. De- 944–950. ficiency in CD22, a B cell-specific inhibitory receptor, is sufficient to predispose 54. Matsushita, T., K. Yanaba, J. D. Bouaziz, M. Fujimoto, and T. F. Tedder. 2008. to development of high affinity autoantibodies. J. Exp. Med. 189: 1307–1313. Regulatory B cells inhibit EAE initiation in mice while other B cells promote Downloaded from 45. Shultz, L. D., T. V. Rajan, and D. L. Greiner. 1997. Severe defects in immunity disease progression. J. Clin. Invest. 118: 3420–3430. and hematopoiesis caused by SHP-1 protein-tyrosine-phosphatase deficiency. 55. Lykken, J. M., K. M. Candando, and T. F. Tedder. 2015. Regulatory B10 cell Trends Biotechnol. 15: 302–307. development and function. Int. Immunol. 27: 471–477. http://www.jimmunol.org/ by guest on September 27, 2021 Supplementary Figures and legends

2+ Supplementary Figure 1. Characterization of IP3R expression and Ca release in B cells of control and IP3R-TKO mice. (A) and (B) Lymphocytes were isolated from the bone marrow of

+ + control and IP3R-TKO mice. CD19 B220 cells were subgated and examined for surface expression of IgM to sort pro-/pre-B (B220+IgM-), immature (B220lowIgMint), transitional (B220intIgMhigh), and

high int recirculating mature (B220 IgM ) B cells, respectively. The mRNA level of each IP3R subtype was measured by quantitative RT-PCR analysis. (A) IP3R mRNA levels are presented as the ratio to

Gapdh. Data shown were averaged from four independent experiments. Data represent mean  SEM.

*p < 0.05, **p < 0.01 as the significance was determined by the 2-tailed, unpaired Student’s t test. (B) qRT-PCR analysis of IP3Rs in IP3R-TKO B cells. n = 4 mice per group. Data is normalized to corresponding Gapdh, and is expressed as fold change versus control. Data represent mean  SEM. Significance was determined by the 2-tailed, unpaired Student’s t test. *p < 0.05, **p < 0.01, ***p <

0.001. (C) - (F) Splenic B cells were isolated from control and IP3R-TKO mice by negative selection of CD43+ cells with anti-CD43 magnetic beads, loaded with Ca2+ indicator Fluo4, and imaged using confocal microscopy. (C) Sequential confocal images, before (t0) and after (t1) IP3 uncaging.

2+ Intracellular Ca mobilization was induced in selected cells (red box) by photolysis of caged IP3 with the 405nm ultraviolet (UV) light. Bar indicates 20 m. (D) Traces of averaged Ca2+ fluorescence during IP3 uncaging. Data represent one of three independent experiments. (E) TG 2+ induced Ca mobilization in control and IP3R-TKO splenic B cells. ER stores were depleted by TG

2+ 2+ (2M) in Ca -free (zero [Ca ]o) buffer with subsequent reintroduction of regular buffer with normal extracellular Ca2+ concentration. Traces of averaged Ca2+ fluorescence are depicted. Data represent one of three independent experiments. (F) The amplitude of changes of fluorescence

2+ (F/F0) induced by TG in control and IP3R-TKO splenic B cells in the absence of extracellular Ca . n=3 mice per group. Data represent mean ± SEM. Significance was determined by the 2-tailed, unpaired Student’s t test. (G) Relative mRNA levels of UPR chaperone-encoding genes in control and IP3R-TKO splenic B cells. n=3 mice per group. Data are presented as the ratio to Gapdh and represent mean ± SEM. Significance was determined by the 2-tailed, unpaired Student’s t test.

+ Supplementary Figure 2. B cells in CD19-Cre mice and B cell-specific IP3R double knockout mice. (A) Expression of CD19-Cre did not alter B cell development in bone marrow. Percentages of

B220+CD19+ B cells within lymphocytes and percentages of pro-/pre-B (B220+IgM-), immature

(B220lowIgMint), transitional (B220intIgMhigh), and recirculating mature (B220highIgMint) B cells within B220+CD19+ B cells from bone marrow of Itpr1f/+Itpr2f/+Itpr3f/+CD19-Cre- (n = 5) and

Itpr1f/+Itpr2f/+Itpr3f/+CD19-Cre+ (n = 7) mice. Data represent mean  SEM. Significance was determined by the 2-tailed, unpaired Student’s t test. (B) Percentages of B220+CD19+ B cells within lymphocytes and percentages of pro-/pre-B, immature, transitional, and recirculating mature B cells within B220+CD19+ B cells from bone marrow of control (n = 8), Itpr1f/fItpr2f/fItpr3f/+CD19-Cre+

f/f f/+ f/f + f/+ f/f (IP3R1/R2 DKO, n = 5), Itpr1 Itpr2 Itpr3 CD19-Cre (IP3R1/R3 DKO, n = 4), Itpr1 Itpr2

f/f + Itpr3 CD19-Cre (IP3R2/R3 DKO, n = 9) mice. Data represent mean  SEM. Significance was determined by the 2-tailed, unpaired Student’s t test. (C) BCR-induced Ca2+ release in control,

2+ IP3R1/R2 DKO, IP3R1/R3 DKO and IP3R2/R3 DKO splenic B cells. Ca release was elicited by

2+ 2+ 2+ anti-IgM (10g/ml) in Ca -free (zero [Ca ]o) buffer and monitored by Ca indicator Fluo4. Data represent one of three independent experiments.

Supplementary Figure 3. Characterization of critical molecules involved in B cell development.

(A) Flow cytometry analysis of basal ERK1/2 phosphorylation levels in control and IP3R-TKO bone marrow immature B cells (left). Cells were treated with sodium pervanadate before ERK1/2 phosphorylation analysis. Bar graph (right) represents mean fluorescence intensity (MFI) of phosphorylated-ERK1/2 (p-ERK) relative to total ERK1/2. n = 3 mice per group. Data represent mean  SEM. Significance was determined by the 2-tailed, unpaired Student’s t test. **p < 0.01. (B)

Expression of Foxo1, Rag1, and Rag2 in control and IP3R-TKO bone marrow immature B cells identified by qRT-PCR analysis. n=3 mice per group. Data are presented as the ratio to Gapdh and represent mean ± SEM. Significance was determined by the 2-tailed, unpaired Student’s t test. (C)

Relative Baffr mRNA levels in bone marrow transitional B cells from control and IP3R-TKO mice. n=3 mice per group. Data are normalized to Gapdh levels and represent mean ± SEM. Significance was determined by the 2-tailed, unpaired Student’s t test.

Supplementary Figure 4. BCR-mediated cell proliferation and survival in transitional, FO, and

MZ B cells from control and IP3R-TKO mice. (A) Gating strategies for proliferation and survival

analysis in B cell subsets. Splenic B cells were isolated from control and IP3R-TKO mice, labeled with CFSE and stimulated for 48 hr with vehicle or anti-IgM (10g/ml), stained with PI and fluorescence conjugated anti-B220, anti-CD93, anti-CD21 and anti-CD23 antibodies. Top: Viable

PI-negative (PI-) cells were shown within B220+ gate. Middle: Surface expression of B220 and

CD93 on gated PI-B220+ cells. Transitional (B220+CD93+) and mature (B220+CD93-) B cells are shown. Bottom: Surface expression of CD23 and CD21 on gated mature B cells. FO (CD23+CD21+) and MZ (CD23-CD21+) B cells are shown. Relative cell numbers for indicated gates are depicted. (B) BCR-mediated cell proliferation in control and IP3R-TKO transitional (T-B), FO, and MZ B cells.

The numbers within each histogram represent the percentage of divided cells as measured by CFSE dilution based on PI- B cell subsets. (C) Percentages of dividing cells in response to anti-IgM. n = 3 mice per group. Data represent mean  SEM. Significance was determined by the 2-tailed, unpaired

Student’s t test. *p < 0.05, **p < 0.01. (D) BCR-mediated cell survival in control and IP3R-TKO transitional, FO, and MZ B cells. Cell survival was presented by percentages of PI- total, transitional, mature, FO, and MZ B cells within B220+ B cells. n=3-5 mice per group. Data represent mean ±

SEM. Significance was determined by the 2-tailed, unpaired Student’s t test. ***p < 0.001. (E)

Relative Mef2c mRNA levels in splenic B cells of control and IP3R-TKO mice. n = 3 mice per group.

Data are normalized to Gapdh levels and represent mean ± SEM. Significance was determined by the 2-tailed, unpaired Student’s t test.