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CD36 Is Differentially Expressed on B Cell Subsets during Development and in Responses to Antigen

This information is current as Woong-Jai Won, Martin F. Bachmann and John F. Kearney of September 25, 2021. J Immunol 2008; 180:230-237; ; doi: 10.4049/jimmunol.180.1.230 http://www.jimmunol.org/content/180/1/230 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD36 Is Differentially Expressed on B Cell Subsets during Development and in Responses to Antigen1

Woong-Jai Won,*† Martin F. Bachmann,‡ and John F. Kearney2*†

Of a number of mAbs made by immunization with sort-puriﬁed marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag .challenge, we found that CD36 was expressed on IgM؉ plasma cells, but down-regulated on isotype-switched plasma cells in vivo Although development, localization, and phenotype of MZ B cells in CD36؊/؊ mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-speciﬁc IgM and IgG levels in CD36؊/؊ mice were slightly reduced

compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC Downloaded from IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2. The Journal of Immunology, 2008, 180: 230–237.

ature B cells in mammals are divided into three major loids (17, 21–27). A role for CD36 has also been suggested in the 3

B cell subsets, marginal zone (MZ), follicular (FO), pathogenesis of Alzheimer’s disease and atherosclerosis (18, 25, http://www.jimmunol.org/ M and B1 B cells, depending on surface phenotypes, lo- 28–30). Recent studies showed that CD36 enhances TLR2 signal- cation, and immunological functions (1–4). Although MZ and B1 ing to induce cytokine secretion by macrophages, although the B cells represent subpopulations of naive mature B cells, they play exact mechanism was not established (31, 32). an important role in early innate-like and primary Ab responses (1, Because CD36 is expressed uniquely on freshly isolated mature 2, 5). In particular, they respond quickly to T-independent (TI) MZ B cells and is induced to high levels on other mature B cell Ags, resulting in a rapid production of large numbers of short-lived subsets after stimulation in vitro, we investigated the role of CD36 plasma cells (6, 7). with respect to B cell development and function and examined a Previously, our laboratory identified two surface markers, CD9 possible cooperation between CD36 and TLR2 in Ab responses. and FCRL5 (Fc receptor-like 5), which help distinguish MZ B and by guest on September 25, 2021 B1 cells from FO B cells in mice (8, 9). Recently, we and others Materials and Methods showed by different approaches that CD36 is expressed on resting Mice MZ B cells, but not the majority of FO B cells (10, 11). CD36Ϫ/Ϫ mice were obtained from Dr. Maria Febbraio at the Lerner Re- CD36 is a Type BI scavenger receptor family member, which is search Institute (Cleveland, OH). T15 H chain knock-in and M167Id Ig expressed by a variety of cell types including endothelium, eryth- transgenic mice were obtained from Dr. Jim Kenny at National Institutes of Health (Bethesda, MD), and BALB/cJ, C57BL/6J, and TLR2Ϫ/Ϫ mice rocytes, platelets, adipocytes, dendritic cells, neutrophils, mono- Ϫ Ϫ were purchased from The Jackson Laboratories (33). CD36 / mice were cytes/macrophages, microglia, and muscle cells (12–20). CD36 backcrossed at least to the sixth generation to C57BL/6 mice. All mice binds multiple ligands including native and modified low density were bred, and maintained in our animal facilities at the University of lipoproteins, oxidized phosphoryl choline (PC) epitope, anionic Alabama at Birmingham (UAB). All studies and procedures were approved phospholipids, collagens, thrombospondin-1, and fibrillar ␤-amy- by the UAB Institutional Animal Care and Use Committee (IACUC). Flow cytometry All Abs were purchased from BD Biosciences unless specified. Spleen, *Division of Developmental and Clinical Immunology and †Department of Microbi- peritoneal cavity (PEC), peripheral blood, and bone marrow (BM) cells ology, University of Alabama at Birmingham, Bikminghan AL 35294; and ‡Cytos were prepared as single cell suspensions after RBC lysis, and stained with Biotechnology AG, Wagistr 25, Schlieren-Zurich, Switzerland FITC conjugated anti-mouse CD1d, CD19, CD23, B220, IgM (SBA), IgG1 Received for publication August 7, 2007. Accepted for publication October 3, 2007. (SBA), TLR2 (eBioscience), peanut agglutinin (Jackson Immunoresearch Laboratories); PE conjugated anti-mouse CD5, CD23, CD38, syndecan1, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance IgD (SBA); biotin conjugated anti-mouse syndecan1; allophycocyanin- with 18 U.S.C. Section 1734 solely to indicate this fact. conjugated AA4.1 (eBioscience), Mac1. Alexa 647 and Alexa 488 conju- 1 gated CD9 and CD36, PE conjugated CD21, and biotinylated CD36 (MZ1) This work was supported in part by National Institutes of Health Grants CA13148 were made in our laboratory, according to the manufacturer’s instructions and AI14782. (Invitrogen Life Technologies and Pierce). FITC conjugated PC-Dextran 2 Address correspondence and reprint requests to Dr. John F. Kearney, Division of and PE conjugated M167 Id mAb were provided by Dr. Louis Rezanka at Developmental and Clinical Immunology, Department of Microbiology, SHEL 410, the National Institute on Aging (Baltimore, MD). Biotinylated mAbs were 1825 University Boulevard, University of Alabama, Birmingham, AL 35294-2182. incubated with streptavidin-allophycocyanin as a secondary reagent. Cells E-mail address: [email protected] were analyzed using FACSCalibur flow cytometer and plotted with Win- 3 Abbreviations used in this paper: MZ, marginal zone; FO, follicular; PEC, perito- MDI (Scripps Institute) software. neal cavity; BM, bone marrow; DC, dendritic cell; PLC, plasma cell; GC, germinal center; WT, wild type; KO, knock-out; TG, transgenic; PC, phosphoryl choline; PEC, Cell sorting and semi-quantitative RT-PCR peritoneal cavity. To examine mRNA transcripts by mouse B cell subsets, total spleen B cells Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 obtained after RBC lysis were stained with FITC-conjugated anti-CD23 www.jimmunol.org The Journal of Immunology 231 Downloaded from http://www.jimmunol.org/

FIGURE 2. Rapid induction of CD36 on B cells. A, Rapid induction of CD36 on both MZ and FO B cells. FACS-sorted MZ and FO B cells were incubated with B cell activating agents, LPS (10 ␮g/ml), or anti-CD40 (1 by guest on September 25, 2021 ␮g/ml), for 1–3 days, and CD36 was detected by flow cytometry. B, In- duction of CD36 on PEC B1 cells. FACS-sorted PEC B1a (CD5ϩB220lowMac1ϩ) or B1b (CD5ϪB220lowMac1ϩ) cells were incubated with LPS for 1–3 days. Gray-filled histogram indicates isotype-con- FIGURE 1. Unique expression of CD36 by MZ B cells. A, Surface trol staining. staining of mature B cells with anti-CD36 mAb (MZ1 clone). Spleen cells from BALB/cJ mice were stained with FITC ␣-CD23, PE ␣-CD21, biotinylated ␣-CD36. PEC-exudate cells were stained with FITC ␣-B220, PE ␣ ␣ -CD5, and biotinylated -CD36, then developed with SA-allophycocya- In vitro mitogenic stimulation nin. Gray filled histograms indicate isotype-control staining. B, Expression of CD36 mRNA transcripts by splenic mature B cells. mRNA levels of The sorted MZ and FO B cells were cultured for 1 to 3 days with LPS CD36 from MZ and FO B cells were detected by semi-quantitative RT- (Sigma-Aldrich), anti-CD40 (BD Biosciences), or synthetic TLR2 ligands PCR. As a positive control, ␤-actin PCR was amplified with the same such as FSL1 and Pam3Cys-SKKKK (EMC microcollections GmbH). CD36 expression was determined by flow cytometry using our Alexa 647- number of cycles in parallel. C, Immunohistological staining of CD36 in conjugated anti-mouse CD36 mAb (MZ1). mouse spleen section. Frozen spleen sections from BALB/cJ mouse were stained with MOMA1, then developed with 7-amino-4-methyl coumarin- Cytokine measurement 3-acetic acid goat-anti-rat IgG (Blue). Sections were blocked with normal rat serum, then stained with Alexa488 MZ1 (Green) or tetramethyl rhoda- Sorted cells were stimulated with various activation agents such as LPS, mine iso-thiocyanate IgM (Red). Each staining histogram and immunohis- Pam3Cys-SKKKK, and FSL-1, and supernatants from cultures were col- ␣ tologic photograph is a representative of at least six mice. lected at day 2. Levels of various cytokines such as TNF- , IL-2, IL-4, IL-6, IL-10, IL-12 (p40 and p70), and IFN-␥, were measured using BD cytokine-bead assay kits with a FACSArray flow cytometer and calculated using the manufacturer’s software (BD Biosciences). and PE-conjugated anti-CD21, and sorted using a MoFlo sorter or Immunohistochemistry FACSAria sorter (BD Biosciences). mRNA and cDNA of each B cell subset were prepared according to the manufacturer’s protocol (Invitrogen Portions of mouse spleen and small intestines were embedded in OCT Life Technologies). RT-PCR of serially diluted cDNA were conducted by (Optimal Cutting Temperature) compound (Sakura Finetek). The frozen the following conditions: CD36 5Ј primer (GGC ACC ACT GTG TAC OCT-embedded tissues were cut to a thickness of 4 ␮m, put on the glass AGA CAG), CD36 3Ј primer (GGA AAG GAG GCT GCG TCT GTG C), slides, fixed in acetone, and stored at Ϫ80°C. Slides were first blocked with Taq polymerase (1 unit/reaction, Invitrogen Life Technologies), OP#4 10% horse serum and stained with Alexa 488-conjugated anti-mouse buffer (Stratagene), preheated at 94°C for 3 min, (94°C 1 min, 54°C 1 min, CD36; tetra-methyl rhodamine iso-thiocyanate-conjugated goat anti-mouse 72°C 1 min for 35 cycles), 72°C 10 min, and then soaked at 4°C. RT-PCR IgM (SBA); supernatant of MOMA1 hybridoma. Anti-rat IgG 7-amino-4- of mouse TLR1, 2, and 6 was followed as previously described at other methyl coumarin-3-acetic acid (Jackson Immunoresearch Laboratories) laboratories (34). was added as a secondary reagent for MOMA1 staining. 232 CD36 EXPRESSION BY B CELL SUBSETS

FIGURE 3. Differential expression of CD36 by activated B cells and plasma cells in vivo.A, Expression of CD36 by natural plasma cells. Spleen cells from un- immunized C57BL/6J mice were stained with PE Syn- decan1, Alexa-647 MZ1, and either FITC PC-Dextran or FITC IgM. B, Expression of CD36 by PC-speciﬁc plasma cells induced by Streptococcus pneumoniae R36A strain. C57BL/6J mice were injected i.v. with 108 R36A and the expression of CD36 was examined at day 3. *ND (not detected). C, Expression of CD36 by ger- Downloaded from minal center B cells. Spleen cells at day 7 after SRBC injection were stained. Germinal center cells were de- ﬁned, as previously described (9, 46). D, Expression of CD36 by isotype-switched plasma cells. Top panel shows surface staining and lower panel shows an intra- cytoplasmic staining of spleen cells of SRBC immunized mice at day 7. http://www.jimmunol.org/ by guest on September 25, 2021

Bacteria preparation and immunization BH8, 32-17-1, and 59.6C5.5 were used as standards for anti-PC IgM, IgG, and IgG3 isotype determination, respectively. Nonencapsulated Streptococcus pneumoniae R36A strain was plated on blood agar plates overnight. Separate colonies were picked, transferred, Statistical analysis and grown in Todd Hewitt Broth plus 0.5% yeast extract (BD Biosciences) until OD reaches 0.5 at 420 nm. The cells were harvested and washed with Concentrations of serum Ig of different isotypes and anti-PC IgM and IgG PBS three times. The cells were heat-inactivated at 60°C for 1 h and pep- Ab were expressed as the arithmetic mean of individual serum samples sin-treated (7). To induce anti-PC Ab response, 1 ϫ 108 heat-killed Strep- from four to ten mice Ϯ SD. Concentrations of IgM and cytokine secreted tococcus pneumoniae R36A strain were injected i.v. or i.p. into CD36Ϫ/Ϫ in sorted B cell culture supernatants were expressed as the arithmetic mean and control littermate mice. To induce soluble TI Ag responses, TNP-LPS of triplicate cultures from pooled spleens from three mice Ϯ SD. Levels of (TI-1 Ag), and TNP-Ficoll (TI-2 Ag) (Biosearch Technologies) were significance of the differences between groups were determined by Stu- tested, except that sera titers were measured by ELISA using alkaline phos- dent’s t test. A value of p Ͻ 0.05 was considered statistically significant. phatase-conjugated Abs, as described by others (35). In response to SRBC, C57BL/6J mice were injected i.v. with 200 ␮l of packed SRBC (Vector Laboratories), as previously described in our laboratory (8). Results CD36 is a specific marker of mouse MZ B cells Dendritic cell (DC)-B cell coculture Of three mAbs previously generated by immunizing a rat with BM-derived DC were generated as previously described in our laboratory sort-purified MZ B cells in our laboratory (38), we found that one (36). DCs were primed with R36A at the ratio of 1:10 (DC:R36A) in 5% reacted with mouse CD36 by using ␣-virus expression cloning (10, complete RPMI 1640 medium at 37°C for 4 h. Spleen B cells were isolated from CD36ϩ/Ϫ or CD36Ϫ/Ϫ M167 Ig transgenic (TG) mice by magnetic 39). As shown in Fig. 1A, CD36 is expressed on most MZ B cells, sorting with anti-mouse CD43 beads, and incubated with primed DC at the but on only very small numbers of freshly isolated FO B cells and ratio of 106 :2ϫ 105 cells (B cells:DCs) for 3–8 days. Generation of B1 cells in spleen and peritoneal cavity. CD36 mRNA transcripts ϩ ϩ M167Id plasma cells (PLC) in the coculture was calculated as Id PLC are also exclusively expressed in MZ B cells, but not in FO B ϫ percentage total live cell numbers collected for same interval by cells (Fig. 1B). The expression of CD36 was higher on MZ B cells FACSCalibur flow cytometer. outside of the metallophilic macrophage layer in the MZ (Fig. 1C). ELISA Although CD36 is widely expressed in other cell types such as Sera were prepared from the blood of immunized mice and the concentra- macrophages, endothelial cells, platelets, and erythrocytes, it is tions of anti-PC of either IgM or IgG isotypes were determined by ELISA, clear that CD36 is differentially expressed on freshly isolated ma- as previously described in our laboratory (37). Purified hybridoma Abs of ture B cell subsets. The Journal of Immunology 233

CD36 is induced rapidly on mature B cells by TLR and CD40 stimulation Although CD36 is not expressed by the majority of freshly isolated FO B and B1 cells, as shown in Fig. 2A, CD36 is rapidly induced on FO B cells after stimulation with LPS and anti-CD40, peaking at day 3 where it is expressed at a level comparable to freshly isolated MZ B cells. In contrast, only a portion of PEC B1 cells were induced to express CD36 following LPS stimulation by day 3 (Fig. 2B). These results show that CD36 is induced rapidly on mature spleen B cells followed by multiple activation agents, but not on most B1 cells. CD36 is expressed on IgMϩ plasma cells but down-regulated on IgG isotype-switched plasma cells in vivo We next immunized C57BL/6 mice with heat-killed S. pneumoniae (R36A) and SRBC as particulate TI and T-dependent Ags, respectively, and examined the expression of CD36 by plasma cells induced in each response. In un-immunized mice, natural IgMϩ PLC did not express CD36 (Fig. 3A). In response to R36A, Downloaded from all syndecan1-positive PC-binding cells in the spleen expressed CD36 (Fig. 3B, top panel). The majority of the total IgMϩ PLC induced by R36A also expressed CD36, but in a biphasic manner (Fig. 3B, middle panel). In contrast, most IgG1ϩ plasma cells induced by SRBC immunization expressed CD36 at much lower ϩ levels, although IgM plasma cells in response to SRBC expressed http://www.jimmunol.org/ CD36 at a level comparable to IgMϩ plasma cells induced by R36A (Fig. 3D). Most germinal center (GC) B cells induced by SRBC weakly expressed the CD36 except for a small population (Fig. 3C). These results show that CD36 is expressed at high levels on short-lived IgMϩ plasma cells compared with isotype-switched plasma cells, which appear to slowly lose expression as they ter- minally differentiate.

Lack of CD36 has minimal effects on mature B cell development by guest on September 25, 2021 during transitional B cell stages in BM and spleen Examination of the development and the localization of FO and MZ B cells in the spleens of CD36Ϫ/Ϫ mice revealed only a slight reduction in the total numbers of mature B cells and MZ precursors in CD36Ϫ/Ϫ mice. Splenic and PEC B1 cell compartments were comparable between wild-type (WT) and knock-out (KO) mice (data not shown). However, spleen and BM transitional B cells were slightly increased compared with WT mice (Fig. 4), suggesting that there may be a minor block in the transition of immature B cells into mature B cell compartments. Other cell compartments Ϫ Ϫ FIGURE 4. B cell development in CD36 / mice. To examine a pos- in various tissues such as peripheral blood, mesenteric lymph sible role of CD36 in B cell development, B cell compartments of WT node, and Peyer’s patches, also appeared normal (data not shown). CD36ϩ/Ϫ mice were compared with that of CD36Ϫ/Ϫ mice. A, FACS plot Absence of CD36 affects plasma cell generation and humoral of transitional and mature B cell compartment in spleens and BM of CD36Ϫ/Ϫ mice. The numbers in FACS plots indicate the values of a rep- Ab response to particulate TI-2 Ags resentative individual mouse. B, Comparison of total cell numbers of each Ϫ Ϫ Resting serum Ig levels of CD36 / mice showed small differ- B cell subset in CD36Ϫ/Ϫ mouse spleen. †, MZP (marginal zone precursor) ences in several IgG isotypes (IgG1, IgG2a, IgG2b, and IgG2c), indicates a population of CD21highCD23highCD1dhigh. TR (transitional), SP .p Ͻ 0.05 ,ءء ,p Ͻ 0.01 ,ء .(but not IgM and IgG3 isotypes, compared with WT mice (Fig. 5A). (spleen), BM (bone marrow Because MZ B cells are important for bacterial TI-2 Ab response, we examined conventional Ab response of CD36Ϫ/Ϫ mice with TNP-LPS and TNP-Ficoll as TI-1 and TI-2 soluble Ags, respec- signiﬁcant. We also examined the production of PC-binding PLC tively. Both anti-TNP titers to both Ags were comparable between at day 3 after R36A challenge. As shown in Fig. 5C, the frequency CD36WT and CD36KO mouse groups (data not shown). Next, we and actual number of PC-speciﬁc PLC in CD36Ϫ/Ϫ mice were challenged CD36Ϫ/Ϫ mice with heat-killed S. pneumoniae as a ϳ2-fold lower than in wild-type (WT) mice, correlating with se- particulate Tl-2 Ag and examined the generation of PC-binding rum anti-PC levels. To examine a secondary response, R36A-im- plasma cells and anti-PC titers. As shown in Fig. 5B, total anti-PC munized mice were rechallenged at day 14 with the same dose of IgM levels were reduced to ϳ65% of the WT group at day 7 ( p Ͻ bacteria, and anti-PC titers were compared. Both anti-PC IgM and 0.05), which is the usual peak of the anti-PC IgM titer (Fig. 5B). anti-PC IgG titers in CD36Ϫ/Ϫ mice were consistently lower (Fig. Similarly, CD36Ϫ/Ϫ mice also showed lower anti-PC IgG levels 5D). We also found that a majority of anti-PC IgG isotypes was compared with WT mice, although the p value is not statistically IgG3 (data not shown), which also showed a reduction to the same 234 CD36 EXPRESSION BY B CELL SUBSETS

FIGURE 5. Anti-PC Ab response and plasma cell generation of CD36Ϫ/Ϫ mice after TI-2 bacterial challenge. A, Serum Ig levels of different isotypes in un-immunized CD36Ϫ/Ϫ mice. All mice were 8–12 p Ͻ 0.05. B, Anti-PC ,ء .wk-old females IgM and IgG titers of CD36Ϫ/Ϫ mice. CD36ϩ/Ϫ and CD36Ϫ/Ϫ mice were immunized i.p. with 108 R36A, and anti-PC titers were measured at different time points. Levels of anti-PC IgM and IgG were calculated by anti-PC IgM (BH8) and IgG1

p Ͻ 0.05. Downloaded from ,ء .mAbs as standards (32-17-1) The graph shows the results from the total pool of mice used in three independent experiments. The sample number at day 28 is three per group. C, PC-specific plasma cell generation of CD36Ϫ/Ϫ mice. Each mouse group was injected i.p. with 108 R36A, and the number of PC-specific plasma cells was http://www.jimmunol.org/ compared between the WT and KO groups at day 4. The graphs are representative of the plots of three mice and the numbers inside contour plots indicate the percentage of a p Ͻ 0.05. D, Anti-PC ,ء .representative mouse secondary responses of CD36Ϫ/Ϫ mice. The upper panel shows anti-PC IgM responses of CD36ϩ/Ϫ and CD36Ϫ/Ϫ mice and the lower panel shows anti-PC IgG responses. Each by guest on September 25, 2021 mouse group was rechallenged i.p. with the same dose of R36A at 14 days after the first injection.

degree. This result shows that lack of CD36 has marginal effects cells showed a significant decrease in plasma cell generation as on anti-PC IgM and IgG levels in response to the bacterial TI Ag well as anti-PC IgM production ( p Ͻ 0.05), compared with associated with S. pneumoniae. the WT counterpart, irrespective of whether WT or CD36Ϫ/Ϫ DCs were used. Neither CD36Ϫ/Ϫ macrophages, nor supernatants from Defective anti-PC Ab response is due to the absence of CD36 CD36Ϫ/Ϫ DC culture, showed any difference in their ability to on B cells induce M167Idϩ plasma cell generation (data not shown). These To examine the function of CD36 in B cells, we sorted MZ and FO results suggest that the mild defects in plasma cell generation of B cells from CD36Ϫ/Ϫ and WT mice and compared several in vitro Ag-specific plasma cells were regulated by the expression of CD36 activities, such as proliferation, plasma cell differentiation, cyto- on B cells. kine production by mitogen stimulation, including TLR ligands, anti-CD40, IL-4, and anti-IgM or in combination. All of these CD36 cooperates with TLR2 in the anti-PC IgG Ab response responses were comparable between CD36Ϫ/Ϫ and WT B cells Recent studies showed that CD36 enhances TLR2 signaling in the (data not shown). To check whether the defective anti-PC response cytokine response of activated macrophages (32). TLR2 forms observed in CD36Ϫ/Ϫ mice is intrinsic to B cells, we studied Ag- dimers with TLR1, TLR6, or TLR2 itself, which give different specific plasma cell generation ex vivo in response to Ag-pulsed specificities to ligands (40). We tested the mRNA expression of DC (36). We prepared DC derived from CD36ϩ/Ϫ or CD36Ϫ/Ϫ TLR2 and other dimer partners in mouse B cell subsets. RNA BM, primed them with heat-killed R36A, and coincubated them transcripts of TLR1, 2, and 6 were detected in both MZ and FO B with B cells from either CD36ϩ/Ϫ or CD36Ϫ/Ϫ M167 Ig TG cells, but MZ B cells express ϳ5 times more mRNA of each than mouse spleen cells. As shown in Fig. 6, CD36Ϫ/Ϫ M167 TG B FO B cells (Fig. 7A). TLR2 protein level in MZ B cells is slightly The Journal of Immunology 235

FIGURE 6. Anti-PC Ab production and plasma cell generation is modulated by CD36 on B cells. A, Anti-PC Ab responses in DC-B cell coculture system. Spleen B cells from M167 Id TG mice with either CD36ϩ/Ϫ or CD36Ϫ/Ϫ background and DC derived from CD36ϩ/Ϫ or CD36Ϫ/Ϫ mouse BM were cocultured for 3–8 days, and the generation of PC-speciﬁc plasma cells were measured by ﬂow cytometry. B, Anti-PC IgM titer of coculture supernatants. Anti-PC IgM titers in the culture supernatants were measured by ELISA, as described in Materials and Methods. The graph shows a representative of two independent experiments, both showing the .p Ͻ 0.05 ,ءء ;p Ͻ 0.01 ,ء .same results Downloaded from

higher than that of FO B cells, which correlated with RNA mes- mice ( p Ͻ 0.05), suggesting that CD36 and TLR2 cooperate in the sage level, although the expression level was low (Fig. 7B). anti-PC IgG response to S. pneumoniae (Fig. 8). Ϫ/Ϫ After activation of B cells from CD36 mice with chemically http://www.jimmunol.org/ synthesized TLR2 ligands, proliferation, plasma cell generation, Discussion and cytokine production of CD36Ϫ/Ϫ B cells were similar to WT We previously showed that CD36 is expressed by freshly isolated B cells (Fig. 7C, data not shown). However, as expected, TLR2Ϫ/Ϫ MZ B cells, but only by a very few FO and B1 cells (10). The B cells showed severe defects in proliferation (Fig. 7C), cytokine differential expression of CD36 by B cell subsets provides another expression, and plasma cell generation (data not shown). This re- useful marker for discrimination of MZ B cells along with CD9 sult confirms that B cells are strongly dependent on TLR2 in in and FCRL5 (8, 9). However, because CD36 is rapidly induced in vitro responses to these ligands, but not on CD36 expression. FO B cells by stimulation by TLR ligands and ␣-CD40, it may To test whether CD36 and TLR2 cooperate in the anti-PC Ab represent an early activation marker of B cells, similar to CD25 response in vivo, we compared the anti-PC humoral response to S. and CD69. Of interest, PEC B1a cells expressed CD36 much less by guest on September 25, 2021 pneumoniae in CD36 TLR2 doubly deficient and singly deficient than B1b cells following LPS stimulation. In addition to the minor mice with WT mice. As shown in Fig. 8, anti-PC IgM levels of populations of FO B cells that express CD36, it was also expressed doubly deficient mice were similar to those of TLR2 singly defi- at a slightly lower level on most MZ precursors and a subset of cient mice compared with WT or CD36 singly deficient mice. transitional B cells, suggesting that these cells may be activated. However, anti-PC IgG titers of CD36 TLR2 double KO mice were CD36 expression along with other MZ B cell markers such as CD9 much lower than those from either TLR2 or CD36 singly deficient and FcRL5 may be indicative of activation signals involved in the

FIGURE 7. Expression of TLR2 by B cells and function of TLR2 in B cell activation. A, mRNA transcript levels of TLR1, 2, and 6 by MZ and FO B cells. Semi-quantitative RT- PCR of each TLR1, 2, and 6 was performed as described (34). ␤-actin was used as a positive Loading was less compared with ,ء .control other wells. B, TLR2 surface expression by spleen B cell subsets. Spleen cells from C57BL/6J mice were stained with FITC TLR2 (clone 6C2), CD21-PE, or biotinylated CD23, then developed with SA-allophycocyanin. †, MZP(CD23highCD21high). C, Proliferation of CD36Ϫ/Ϫ and TLR2Ϫ/Ϫ B cells induced by TLR2 ligands. Sorted CD36Ϫ/Ϫ and TLR2Ϫ/Ϫ MZ B cells along with WT MZ B cells were stimulated with LPS and synthetic TLR2 ligands for 2 days and labeled with 1␮Ci [3H]thymidine in 96-well microplate. The in- corporation of radioactive thymidine is pre- sented by a gamma scintillation counter as a proliferation index. Pam3 (Pam3Cys-SKKKK). 236 CD36 EXPRESSION BY B CELL SUBSETS

generation, IgM secretion, and cytokine secretion in vitro, similar to other studies (13). MZ B cells and B1 cells are responsible for TI Ab response (3, 7, 43). We examined TI Ab responses with conventional soluble Ags such as TNP-LPS and TNP-Ficoll, but anti-TNP titers between CD36Ϫ/Ϫ and WT mouse groups were comparable. Corco- ran et al. (13) also reported Ab responses of CD36Ϫ/Ϫ mice, but their studies targeted the anti-Leishmania Ab response, which also showed minor differences between WT and KO mice. Our studies targeted Streptococcus pneumoniae, a Gram-positive bacterium that expresses TLR2 ligands. Different results between anti-PC, conventional TI Ab responses, and anti-Leishimania response may be due to the difference in Ag structure and the degree of cooperation with other TLR ligands on bacterial surfaces. It is of interest that TLR2 and CD36 cooperate to fully induce anti-PC IgG response at early time points. This result suggests that they may physically interact with each other on B cells to directly regulate Ab production or on other cells, such as macrophages or

DCs, to indirectly regulate secretion of cytokines, thus affecting Downloaded from FIGURE 8. TLR2 and CD36 cooperate in anti-PC Ab responses. To test Ϫ Ϫ IgG switching. However, the mechanism for cooperation remains for cooperation between CD36 and TLR2 in anti-PC responses, TLR2 / and CD36Ϫ/Ϫ mice were bred, and four different genotypes of mice were unknown at this point, and further studies will be needed (44). challenged with 108 heat-killed R36A strain i.p. Tested mouse numbers of Anti-PC IgG levels are lower than anti-PC IgM levels, but both each group are five, three, four, and five (WT, CD36KO, TLR2KO, and isotypes have been shown to protect against S. pneumoniae (45). DKO), respectively. The graph shows a representative of two independent Collectively, we showed that after R36A challenge, both anti-PC Ϫ/Ϫ /p Ͻ 0.05. IgM and anti-PC IgG levels in CD36 mice were lower than http://www.jimmunol.org ,ء .experiments, both showing the same results CD36WT mice, which suggest that both responses have a syner- gistic effect in anti-PC production to S. pneumoniae. positive selection of mature B cell precursors from transitional B We showed that the reduction in PC-specific plasma cell (PLC) cells. generation in vitro by CD36Ϫ/Ϫ B cells is B cell intrinsic. Culture One interesting observation is the differential expression of of M167Idϩ MZ and FO B cells with DCs also showed similar CD36 on IgMϩ PLC from germinal center B cells and Ig-isotype results, although only M167Idϩ MZB cells gave strong Ab pro- switched PLC derived from SRBC immunization. Additionally, duction, and as shown previously, Idϩ FO B cells did not (data not only some IgMϩ plasma cells expressed CD36. However, all PC- shown) (36). Given the wide range of CD36 ligands, including its binding PLC express CD36, perhaps indicating the Ag-driven in- function as a receptor for fatty acids, the role of CD36 on B cells by guest on September 25, 2021 duction of CD36 on B cells in early immune responses. Although may not have been revealed in the experiments we have conducted. there was a strong induction of CD36 on FO B cells by in vitro Nevertheless, its differential expression on B cell surfaces makes it stimulation, only a small fraction of GC B cells expressed CD36. another B cell subset marker and its rapid up-regulation after ac- Low expression of CD36 on GC B cells indicates that it may play tivation may make it useful as a B cell activation marker. a minor role in T-dependent Ab responses. Previously, Konig et al. (41) showed that CD36 expression is strictly regulated by Oct2 in Acknowledgments B cells. Thus, it will also be of interest to check Oct2 expression We acknowledge Lisa Jia and Xiaoying Liu for technical assistance, Jer- by B cell subsets. Collectively, our results suggest that CD36 is emy Foote for testing J558L Ig TG mice, Dr. Ute Saunders for help in the induced at an early stage of B cell activation and its induction in dendritic cell culture system, Dr. Maria Febbraio for providing CD36Ϫ/Ϫ vivo on B cells results from Ag-specific activation. mice, Dr. Francis Lund for help with cytokine measurement of B cell In CD36Ϫ/Ϫ mice, the localization and compartment of mature culture, and Dr. Inho Park and Dr. Nicholas Kin for discussion and reading B cells are minimally affected, while the number of mature B cells the manuscript. is slightly reduced and TR B cell compartments is increased. Al- though the unique expression of CD36 by MZ B cells predicted a Disclosures specific role in MZ B cell development, CD36 appears to affect The authors have no financial conflict of interest. both MZ and FO B cell compartments. This minor difference in B cell development may be due to a small blockage at the stage References 1. Martin, F., and J. F. Kearney. 2001. B1 cells: similarities and differences with between transitional and mature B cells. We also tested Ig TG other B cell subsets. Curr. Opin. Immunol. 13: 195–201. mouse model system such as M167 (anti-PC) Ig TG, J558 (anti- 2. Martin, F., and J. F. Kearney. 2002. Marginal-zone B cells. Nat. Rev. Immunol. ␣-1,3 dextran) Ig TG, and T15 (anti-PC) Ig H chain knock-in mice 2: 323–335. Ϫ/Ϫ 3. Oliver, A. M., F. Martin, G. L. Gartland, R. H. Carter, and J. F. Kearney. 1997. crossed with a CD36 background. 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