Human B Lymphocytes and Autocrine

CS1 (CRACC, CD319) Induces Proliferation and Autocrine Cytokine Expression on Human B Lymphocytes

This information is current as Jae Kyung Lee, Stephen O. Mathew, Swapnil V. Vaidya, of September 28, 2021. Pappanaicken R. Kumaresan and Porunelloor A. Mathew J Immunol 2007; 179:4672-4678; ; doi: 10.4049/jimmunol.179.7.4672 http://www.jimmunol.org/content/179/7/4672 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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CS1 (CRACC, CD319) Induces Proliferation and Autocrine Cytokine Expression on Human B Lymphocytes1

Jae Kyung Lee,* Stephen O. Mathew,† Swapnil V. Vaidya,‡ Pappanaicken R. Kumaresan,§ and Porunelloor A. Mathew2†

CS1 (CRACC, CD319), a member of the CD2 family of cell surface receptors, is implicated in the activation of NK cell- mediated cytotoxicity. Previous studies showed that CS1 is also expressed on activated B cells. However, the functional role of CS1 in human B-lymphocytes is not known. Two isoforms of CS1, CS1-L and CS1-S, are expressed in human NK cells that differentially regulate NK cell function. CS1-L contains immunoreceptor tyrosine-based switch motifs in its cytoplasmic domain whereas CS1-S lacks immunoreceptor tyrosine-based switch motifs. In this study, we show that human B lymphocytes express only the CS1-L isoform, and its expression is up-regulated upon B cell activation with various stimulators.

Moreover, anti-CS1 mAb strongly enhanced proliferation of both freshly isolated as well as activated B cells. The enhanced Downloaded from proliferation effects of CS1 were most prominent on B cells activated by anti-CD40 mAbs and/or hrIL-4. The effects of CS1 on B cell proliferation were shown on both naive and memory B cells. Human cytokine microarray and quantitative real-time PCR results indicated that CS1 activation enhanced mRNA transcripts of ﬂt3 ligand, lymphotoxin A, TNF, and IL-14. Neutralizing Abs against lymphotoxin A, TNF-␣, and/or ﬂt3 ligand abolished the ability of CS1 on the B cell proliferation. These results suggest that activation of B lymphocytes, through surface CS1, may be mediated through secretion of autocrine cytokines and CS1 may play a role in the regulation of B lymphocyte proliferation during immune responses. The Journal http://www.jimmunol.org/ of Immunology, 2007, 179: 4672–4678.

he proliferation and differentiation of lymphocytes are liferation and Ig synthesis by activated human B lymphocytes (10). regulated by receptors localized on the cell surface. Es- SLAM has several isoforms and among them soluble secreted T pecially, activation of B cells is initiated by the recogni- form of SLAM and membrane-bound form of SLAM had B cell tion of specific Ag by cell surface Ig, and it is pivotal in deter- growth promoting effects even in the absence of other stimuli and mining the Ag specificity of the response. Cytokines derived from had more potent effect in the presence of polyclonal B cell stimuli Th2 such as IL-4, IL-5, and IL-13 and membrane-bound costimu- and cytokines (10). by guest on September 28, 2021 latory molecules expressed by activated CD4ϩ T cells are required CS1 (CRACC, CD319) is a novel member of the CD2 family for subsequent proliferation, Ig production, Ig isotype switching, (11–13) and is expressed on activated B lymphocytes, NK cells, and differentiation of activated B cells (1, 2). Regulation of B cell CD8ϩ T lymphocytes, and mature dendritic cells (11, 12). We proliferation and differentiation can also be mediated by mono- have previously showed that it is a self ligand, and homophilic cytes through release of cytokines such as IL-8 and IL-10 (3, 4). interaction of CS1 regulates NK cell cytolytic activity (14). The The B cell coreceptor, CD19, has recently been shown to regulate cytoplasmic domain of CS1 contains two immunoreceptor ty- positive selection and maturation in B lymphopoiesis (5). The rosine-based switch motifs (ITSM) observed in some of the CD2 CD40 on B cells interact with CD40L (CD154) on activated CD4ϩ family members (11, 12) that provide docking sites to recruit small T cells to provide an essential signal for T cell-dependent B cell SH2 domain-containing adapter proteins, including Src homology activation (6–9). One of the CD2 family receptors, signaling lym- 2 domain protein 1A/SLAM-associated protein (SAP) and EWS- phocyte activation molecule (SLAM, CD150)3, could induce pro- activated transcript-2 (EAT-2) (15). A recent study identified that CS1 recruits EAT-2 and activates the PI3K and phospholipase C␥ signaling pathways in NK cells (16). In addition, human NK cells *Department of Physiology, University of Texas Southwestern Medical Center at express two splice variant forms of CS1, CS1-S that lack the ITSM Dallas, Dallas, TX 75390; †Department of Molecular Biology and Immunology and Institute for Cancer Research, University of North Texas Health Science Center at in the cytoplasmic domain and CS1-L, which contain two Fort Worth, Fort Worth, TX 76107; ‡Department of Internal Medicine, University cytoplasmic ITSM motifs. Only CS1-L was able to mediate of Arkansas for Medical Sciences, Little Rock, AR 72205; and §Department of cytotoxicity in human NK cells (17). Internal Medicine, Division of Hematology and Oncology, University of Califor- nia Davis Cancer Center, University of California Davis Medical Center at Sac- In this study, we showed that human B lymphocytes express ramento, CA 95817 only the CS1-L isoform. Contrary to the previous studies in which Received for publication October 13, 2006. Accepted for publication July 30, 2007. the expression of CS1 was shown only in activated B cells, our The costs of publication of this article were defrayed in part by the payment of page results demonstrate that CS1 is expressed on both naive and acti- charges. This article must therefore be hereby marked advertisement in accordance vated B cells. The expression levels of CS1 were up-regulated with 18 U.S.C. Section 1734 solely to indicate this fact. upon activation with various stimulators including anti-CD40 1 This work was supported by National Institutes of Health Grant CA85753. 2 Address correspondence and reprint requests to Dr. Porunelloor Mathew, Depart- ment of Molecular Biology and Immunology, University of North Texas Health Sci- protein; EAT-2, EWS-activated transcript-2; hr, human recombinant; RT, reverse ence Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107- transcriptase; LTA, lymphotoxin-␣; flt3L, flt3 ligand; TNFSF13B, TNF superfamily 2699. E-mail address: [email protected] factor 13B; NHL, non-Hodgkin’s lymphoma. 3 Abbreviations used in this paper: SLAM, signaling lymphocytic activation molecule; ITSM, immunoreceptor tyrosine-based switch motifs; SAP, SLAM-associated Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 4673

mAb, IL-4, and anti-␮ mAb. Our results also demonstrated the Proliferation assay functions of CS1 on human B-lymphocytes. The data indicated Purified B cells were cultured in the presence of increasing concentrations that CS1 increased proliferation of freshly isolated as well as ac- of anti-CS1 mAb. B cells were also activated with anti-␮ mAb (10 ␮g/ml) tivated B cells. Effects of CS1 on B cell proliferation were most or anti-CD40 mAb (10 ␮g/ml) in the presence or absence of hrIL-4 (5 prominent when B cells were activated with anti-CD40 mAb ng/ml), hrIL-2, hrIL-10 or hrIL-12 (100 U/ml) and proliferation was mea- and/or IL-4. Interestingly, cytokine microarray and real-time PCR sured by MTS/PMS (Promega) incorporation during the last4hofa4-day culture. The proliferation was also measured by [3H]-thymidine incorpo- data showed that CS1 increased mRNA transcripts of autocrine ration assay during the last 18 h of a 5-day culture. Neutralizing Abs cytokines, which are mostly involved in B cell growth. against cytokines lymphotoxin-␣ (LTA; 0.5 ␮g/ml), TNF-␣ (0.5 ␮g/ml), and flt3 ligand (flt3L; 1 ␮g/ml) were also used. Cell proliferation is expressed as mean cpm Ϯ SD for triplicate wells. Materials and Methods Reagents ELISA Purified human recombinant (hr) IL-4, IL-2, IL-10, and IL-12 were pur- Quantitation of IgG in cell-free supernatants was performed by an ELISA chased from BD Biosciences. The purified anti-CD40 mAbs and anti-␮ specific for human IgG (Rockland). Ninety-six-well plates were coated mAb were purchased from Beckman Coulter and BD Biosciences, respec- with isotype-specific capture Abs at 1 ␮g/well in 0.1 ml of 0.5 M sodium tively. Anti-CS1 mAb (1G10 mAb) was generated and purified as de- carbonate (pH 9.6) for overnight at room temperature. The plates were scribed earlier (14). washed five times with a wash solution containing 50 mM Tris (pH 8.0), 0.1 M NaCl, and 0.05% Tween 20, and incubated for 30 min at room B cell isolation and culture conditions temperature with a postcoat solution (1% BSA in 50 mM Tris (pH 8.0) and 0.15 M NaCl) to block nonspecific binding. Subsequently, cell-free super- B cells were obtained by density gradient centrifugation over Histopaque- natants and standards were added in duplicate to the plate wells (100 ␮l/ Downloaded from 1077 (Sigma-Aldrich). For depletion of T cells, NK cells, monocytes, gran- well). Dilution of the standards was made in a blocking solution to avoid ulocytes, dendritic cells, basophiles, platelets, and early erythroid cells binding of serum components to the wells. All plates were incubated for 1 h from PBMC, these cells were magnetically labeled using a mixture of at room temperature, washed five times with wash solution, and incubated hapten-conjugated CD2, CD4, CD11b, CD16, CD36, and anti-IgE Abs for1hatroom temperature with 100 ␮l/well optimal concentration of (Miltenyi Biotec). Cells were labeled for 10 min at 6–12°C with hapten Ab isotype-specific (anti-human IgG) Abs conjugated with HRP. After wash- mixture and FcR blocking reagent (Miltenyi Biotec). After removing the ing three times, the enzyme substrate hydrogen peroxide plus 3,3Ј,5,5Ј-

unbound Abs by washing with cold PBS plus 1% BSA, the cells were tetramethylbenzidine was added for 20 min at room temperature in the http://www.jimmunol.org/ ␮ incubated with anti-hapten microbead (Miltenyi Biotec) for 15 min at dark, and the reaction was stopped with 50 lof2MH2SO4. Plates were 6–12°C, and then magnetically labeled cells were depleted by retaining read in a microplate reader and isotype concentrations were extrapolated them on a magnetic column (MACS; Miltenyi Biotec). For isolation of from a reference curve. A revelation program incorporated with the mi- memory B cells, the negatively isolated B cells were incubated with CD27 croplate reader calculated the Ig concentration. microbeads for 15 min at 4–8°C. The cells were washed with cold PBS plus 1% BSA, and then the magnetically labeled cells were depleted by Microarray experiments and analysis retaining them on a magnetic column while the negatively selected cells were the naive CD19ϩ CD27Ϫ B cells. Total cellular RNA was isolated using the RNeasy mini kit (Qiagen) ac- Puriﬁed total B cells, naive, and memory B cells were cultured in round- cording to the manufacturer’s instruction. Human common cytokine array bottom 96-well plates in 0.2 ml RPMI 1640 medium supplemented with was obtained from SuperArray Bioscience Corporation and used according

␮ by guest on September 28, 2021 10% FBS at a concentration of 105 cells/well and incubated at 37°C in a to manufacturer’s instructions. In brief, 2 g of total RNA was used for generating the biotin-16-UTP-labeled cRNA (Roche). The arrays were ex- humidified atmosphere containing 5% CO2. Cultures were performed in triplicate when proliferation of B cells was studied and in quadruplets when posed to an Alpha Innotech FluorChem Image system for image correction. Ig synthesis was studied. The images were then analyzed using GEArray Expression Analysis Suite (SuperArray Bioscience). The level of gene expression was determined as the average density and mean has been adjusted to value of 100 and min- Reverse transcriptase (RT)-PCR imum positive value was 10. Background correction was set on minimum Total RNA was isolated with the RNAstat 60 reagent according to the value, which is a method in which the lowest average density spot on the manufacturer’s protocol (Tel-Test) and first-strand cDNA was synthesized array is found and the average across that spot is used as the background from 5 ␮g of total RNA using superscript II (Invitrogen Life Technologies) correction value. Normalization was performed using GAPDH with com- RT and random primers in a volume of 20 ␮l. PCR was performed using parable results. Comparison of the two arrays was performed using a fold Ͼ 2 ␮l of cDNA mixture, under standard conditions for 35 cycles. PCR was (ratiometric analysis (x/y)). Genes showing a fold change of 2.5 were performed using primers that were described before (17). Following am- selected for further evaluation. plification, portions of the PCR were electrophoresed through a 1.2% agarose gel. Quantitative real-time PCR Total RNA was reverse transcribed to cDNA using Omniscript RT (Qia- Flow cytometry gen) and random primers (New England Biolabs). The RT2 PCR primer sets from SuperArray (SuperArray Bioscience) were used for each gene, B cells were cultured as described above and harvested and washed. Cells which were designed to analyze the relative expression of a specific gene were incubated with anti-CS1 mAb followed by PE-conjugated mouse IgG. based on the sequence from GenBank. Primers were LTA (PPH00337A), For double staining, FITC-conjugated CD19 mAbs were used. For staining TNF (PPH00341A), (IFN-␥) (PPH00380A), IL-14 (PPH00806A), TNF memory B cells, CD27 mAb was used. FITC- and PE-conjugated mAbs 5 superfamily factor 13B (TNFSF13B) (PPH01180A), and FLT3LG with irrelevant specificity were used as negative controls. A total of 10 (PPH06324A). RT-PCRs using 2 ␮g of total RNA were performed under cells with light scatter characteristics of lymphocytes of each sample were conditions recommended by the manufacturer. PCRs were conducted in the analyzed using FACScan flow cytometry (Corixa EPICS XL-MCL). The Cepheid SmartCycler (Cepheid) in a 25-␮l reaction mixture containing mean fluorescence intensity in the binding assay represents the average of 12.5 ␮lofRT2 Real-Time SYBR Green PCR Master Mixture (SuperArray three independent experiments with similar results. ␮ 2 ␮ Bioscience), 1.0 lofRT PCR primer set, and 11.5 l of RNase-free H2O containing a diluted template cDNA. Samples were preincubated for 15 Western blotting min at 95°C, then subjected to 40 cycles of amplification at 95°C for 30 s ϫ 6 for denaturing, and at 55°C for 30 s for annealing-extension. Data were Approximately 2 10 cells were incubated with or without anti-CD40 displayed by SmartCycler Software 2.0 (Cepheid). The relative mRNA unit mAb (10 ␮g/ml) for 24 min at 37°C. Cells were then lysed with 1% Non- ␮ for a given gene measured from a single reverse-transcription reaction was idet P-40, 10 mM Tris (pH 7.4), 150 mM NaCl, 100 g/ml PMSF, and divided by the value obtained for ␤-actin. protease inhibitor mixture (Sigma-Aldrich) for 30 min on ice. The lysate was separated by 8–10% SDS-PAGE. Membranes were probed with 1G10 Statistical analysis mAb and anti-actin (Santa Cruz Biotechnology). Western blots were performed according to the manufacturer’s chemiluminiscence detection sys- Statistical analysis was done using Student’s t test for two samples with tem instructions (Kirkegaard & Perry Laboratories). equal variance. A p value of 0.05 or less was considered significant. 4674 EXPRESSION AND FUNCTION OF CS1 ON HUMAN B-LYMPHOCYTES

FIGURE 1. Expression of CS1 on human B cells. A, mRNA expression of CS1 on B cells. Fresh isolated human B cells of a healthy donor were Downloaded from cultured in medium for 0 h, 72 h, and 72 h with the stimulation of anti- CD40 mAbs (10 ␮g/ml). RT-PCR was performed using CS1 primers and products were run on a 1.2% agarose gel and stained with ethidium bro- mide. Molecular mass standards are indicated on the left. B, Surface expression of CS1 on human B cells. Negatively selected human B cells were incubated in medium or activated with anti-CD40 mAbs for 24 h http://www.jimmunol.org/ and stained with FITC-conjugated anti-CD19 mAbs and 1G10 mAbs followed by PE-conjugated secondary Abs. C, Protein expression of CS1 on human B cells. Negatively selected human B cells were incubated in medium or activated with anti-CD40 mAbs for 24 h. The lysate were separated by 7.5% SDS-PAGE. Membranes were probed with FIGURE 3. Effect of 1G10 mAb on proliferation of freshly isolated and 1G10 mAb and anti-actin mAb. activated human B cells. A, Negatively selected human B cells were cultured in the presence of increasing concentration (␮g/ml) of 1G10 mAb for 4 days. IL-4 (5 ng/ml) and IL-2 (100U/ml) were treated on B cells as Results positive controls. Proliferation was measured by MTS incorporation during by guest on September 28, 2021 Human B lymphocytes express only CS1-L isoform last4hofa4-day culture. B, Freshly isolated human B cells were cultured The expression of CS1 on human B lymphocytes was studied by in medium in the presence of cAb (open) or anti-CS1 (1G10mAb) (ﬁlled) ␮ RT-PCR. In an earlier study, we found that there are two isoforms or anti-CD40 mAb (10 g/ml) in the absence (open) or presence (ﬁlled) of ␮ of CS1 expressed on human NK cells: CS1-L containing two 1G10 mAb (20 g/ml) as indicated for 4 days. hrIL-2 (100 U/ml), IL-4 (5 ng/ml), IL-10, or IL-12 (100 U/ml) were also used to stimulate B cells. Proliferation was measured by [3H]-thymidine incorporation assay during last 18 h of a 5-day culture. Cell proliferation is expressed as mean cpm Ϯ .p Ͻ 0.05 ,ء .SD for triplicate wells from separate experiments

ITSMs in the cytoplasmic domain and CS1-S, which does not contain ITSMs (17). Interestingly, the RT-PCR result indicates human B cells only expressed the long isoform of CS1. The stimulation of isolated B cells with anti-CD40 mAb for 72 h did not alter the pattern of mRNA expression (Fig. 1A). Next, we studied the surface expression of CS1 on B cells by performing double immunofluorescence using mAbs specific for CD19 and CS1. Freshly isolated human B cells were incubated in medium or with anti-CD40 mAb for 24 h. The purity of human B cells from PBMCs was Ͼ90% for each experiment. Results showed that Ͼ95% of freshly isolated B cells expressed CS1 on the surface. Moreover, surface expression, as well as protein lev- FIGURE 2. Effects of anti-CD40 mAb-activated B cells on CS1 expres- els, has been up-regulated after incubation with anti-CD40 mAbs sion. Negatively selected human B cells were cultured in medium, anti- for 24 h (Fig. 1, B and C). CD40 mAb (10 ␮g/ml), hrIL-4 (5 ng/ml), or anti-␮ mAb (10 ␮g/ml) for the indicated time points. Thereafter, the cell were harvested, washed, and Expression of CS1 is up-regulated upon B cell activation stained with mAb CD19-FITC and anti-CS1 mAb, followed by PE-conjugated secondary Ab. Open and filled histograms represent staining with We next studied the effects of various other stimulators of CS1 control Ab and mAb 1G10, respectively. CS1 expression on CD19 positive expression on B cells. Purified human B cells were incubated with cells was analyzed using a FACScan flow cytometer. Numbers in the his- stimulators including anti-CD40 mAb, IL-4, and anti-␮ mAb. The togram indicate mean fluorescence intensity values. The data shown are levels of CS1 on B cells that were incubated in medium have not representative of three independent experiments. changed throughout 72 h. However, anti-CD40 mAbs, IL-4, and The Journal of Immunology 4675

FIGURE 5. Effects of 1G10 mAb on Ig synthesis by puriﬁed B cells. Negatively selected total B cells were cultured for 12 days in the conditions of medium alone, 1G10 mAb (20 ␮g/ml), anti-CD40 mAbs (10 ␮g/ml), or combination of the anti-CD40 mAbs and 1G10 mAb. Ig levels in the cultured supernatants were measured by ELISA. The data represent mean Ϯ SEM of quadruplicate cultures in four experiments. Downloaded from

1G10 mAb induces proliferation of both naive and memory B FIGURE 4. Effects of 1G10 mAb on proliferation of naive and memory cells B cells. A, Negatively selected naive B cells (ﬁlled) and positively selected The effect of 1G10 mAb was studied on naive and memory B cells. memory B cells (open) were cultured in medium or anti-CD40 mAb (10 CS1 induced greater proliferation of memory B cells as compared ␮g/ml) and/or IL-4 (5 ng/ml) and/or 1G10 mAb (20 ␮g/ml) for 4 days.

with naive B cells. This effect was augmented when incubated with http://www.jimmunol.org/ 3 Proliferation was measured by [ H]-thymidine incorporation assay during anti-CD40 mAbs and IL-4 (Fig. 4A). However, the CS1-stimulated last the 18 h of a 5-day culture. Cell proliferation is expressed as mean proliferating B cells did not seem to show any change in the mem- cpm Ϯ SD for triplicate wells. B, Negatively selected B cells were cultured ϩ in medium with and without 1G10 mAb (20 ␮g/ml) for 5 days. Thereafter, ory phenotype as there was no change in the expression of CD27 the cells were harvested, washed, and stained with CD27 mAb followed by B cells after CS1 stimulation for 5 days (Fig. 4B). PE-conjugated secondary Ab. Open and filled histograms represent staining with control Ab and CD27 mAb, respectively. CD27 expression on the CS1 stimulation did not induce Ig production B cells was analyzed using a Coulter FC500 flow cytometer. We next studied the effects of CS1 on Ig-synthesis using purified B cells cultured in the presence or absence of 1G10 mAb. Anti-

CD40 mAb was used as a positive stimulator. Neither 1G10 mAb by guest on September 28, 2021 alone nor combined with anti-CD40 mAb failed to induce IgG anti-␮ Abs stimulation increased the surface expressions of CS1 synthesis by puriﬁed B cells, although the effect of anti-CD40 during a culture period of 24 h and the high expression levels were mAb in IgG production was quite obvious as a positive control sustained for up to 72 h (Fig. 2). The most rapid effect was ob- (Fig. 5). This suggests that CS1 may not be involved in regulating served in response to anti-CD40 mAbs, which induced high levels IgG production. We also tested production of IgM and IgE, and of CS1 expression on B cells already during a culture period of 6 h found no induction by 1G10 mAb (data not shown). (data not shown). Optimal enhancement of CS1 expression by anti- ␮ mAb was observed after 24 h, whereas IL-4 effect was observed Expressions of common cytokine genes in activated human after 48 h. This suggests that CS1 is expressed on B cells consti- B-lymphocytes upon CS1 stimulation tutively and its expression is up-regulated upon activation. To determine the mechanism of CS1 induction of B cell proliferation, we performed cytokine microarray for the transcriptional 1G10 mAb induces proliferation of freshly isolated as well as activated B cells Table I. Upregulated genes induced by CS1 on activated human B Generation of mAb against CS1 (1G10 mAb) as well as the func- cells tional role of CS1 on human NK cells have been previously described (17). In this study, we used 1G10 mAb to study the effect Fold Increase of CS1 on the proliferation of human B cells. 1G10 mAb directly Unigene No. Gene Description (CS1/Control) induced B cell proliferation of freshly isolated B cells in a dose- Hs.519702 Cytoplasmic FMR1 interacting 3.09 dependent manner. 1G10-induced proliferation was in the similar protein 2 range as that induced by IL-2 or IL-4 (Fig. 3A). Optimal effects by Hs.434053 Family with sequence similarity 3, 2.73 1G10 mAb were observed even at a concentration of 2.2 ␮g/ml. member 3 Hs.428 Fms-related tyrosine kinase 3 2.52 1G10 mAb also enhanced proliferation of activated B cells. CS1 ligand stimulation had the most synergistic effect on B cell proliferation Hs.856 INF-␥ 3.28 induced by anti-CD40 mAbs and IL-4 (Fig. 3B), and to a lesser Hs.1121150 IL-14 (Taxilin) 2.84 extent by anti-␮ mAb (data not shown). Strong B cell growth Hs.459095 IL-16 3.34 Hs.512234 IL-6 4.11 promoting effects of CS1 was seen when B cells were incubated Hs.624 IL-8 4.57 with CD40 mAb alone. However, other cytokines such as IL-2, Hs.36 Lymphotoxin A 2.40 IL-10, and IL-12 did not show enhanced proliferation effects, sug- Hs.241570 TNF 2.85 gesting that the effect of CS1 is dependent on CD40 or CD40 Hs.478275 TNF, superfamily, member 10 3.14 combined with IL-4 cytokines (Fig. 3B). Hs.525157 TNF superfamily, member 13B 3.14 4676 EXPRESSION AND FUNCTION OF CS1 ON HUMAN B-LYMPHOCYTES

FIGURE 7. Effect of neutralizing Abs against cytokines abolishes CS1- induced B cell proliferation. Negatively selected human B cells were cultured in medium or anti-CD40 mAb (10 ␮g/ml) in the presence (filled) or absence (open) of 1G10 mAb (20 ␮g/ml) for 4 days. Neutralizing Abs Downloaded from against LTA (0.5 ␮g/ml), TNF-␣ (0.5 ␮g/ml), and flt3L (1 ␮g/ml) alone and combination (comb) of all three were also used. Proliferation was measured by [3H]-thymidine incorporation assay during the last 18 h of a 5-day culture. Cell proliferation is expressed as mean cpm Ϯ SD for trip- .p Ͻ 0.05 ,ء .licate wells from three separate experiments http://www.jimmunol.org/ the mRNA of six cytokines (flt3L, IFN-␥, IL-14, LTA, TNF, and TNFSF13B) were measured by real-time quantitative PCR. The mRNA level of LTA was increased ϳ3-fold, and the transcripts for flt3L, IL-14, and TNF also showed significant increases, ranging from 1.5- to 2.5-fold (Fig. 6). Neutralizing Abs against LTA, TNF-␣, and/or flt3L abolished the ability of CS1 on the B cell proliferation (Fig. 7). This suggests that CS1 may induce produc- FIGURE 6. 1G10 mAb increased mRNA expression of cytokines on activated B cells. A, Human cytokine microarray. Negatively isolated hu- tion of autocrine cytokines, which stimulate B cell proliferation. by guest on September 28, 2021 man B cells were cultured in CD40 mAb (10 ␮g/ml) and IL-4 (5 ng/ml) in the presence (right panel) or absence (left panel) of 1G10 mAbs (20 ␮g/ml) Discussion for 4 days. Total RNAs were isolated and biotinylated cRNAs were used In this study, we have showed that only one isoform of CS1 as probes and hybridization was performed as described in Materials and (CS1-L) is expressed on human B lymphocytes and ligation of Methods. The arrays were exposed to an Alpha Innotech FluorChem Image surface CS1 by the mAb 1G10-induced proliferation as well as system for image correction. Normalization was performed using GAPDH expression of autocrine genes in B cells. B cell activation requires with comparable results. Comparison of the two arrays was performed two signals; the first signal is delivered through its Ag receptor. using a fold (ratiometric analysis (x/y)) analysis. Genes showing a fold The second signal is delivered by a helper T cell by the interaction change of Ͼ2.4 were selected for further evaluation. B, Real-time quantitative PCR showing high expression of LTA, TNF, IL-14, and flt3L genes between CD40L on the T cell and CD40 on the B cell. CD40 in CS1-treated CD40/IL-4-activated human B cells. Freshly isolated human signaling activates different mediators and pathways that are re- B cells were incubated with CD40 (10 ␮g/ml) and IL-4 (5 ng/ml) in the lated to cell growth and differentiation (18). These signaling path- presence (filled) or absence (open) of 1G10 mAb for 4 days. Real-time ways lead to the activation of transcription factors (19, 20). PCR of each transcript was performed. Results are presented as relative Through these signaling pathways, CD40 signals can induce pro- ,p Ͻ 0.01; liferation in B cells (20–25), induction of isotype switching (26 ,ءء ;p Ͻ 0.05 ,ء .units of each transcript compared with ␤-actin -p Ͻ 0.001 (compared with CS1-treated B cells). 27), differentiation to Ab-secreting plasma cells (28, 29), and gen ,ءءء eration of memory (30), and stimulate the up-regulation of cell surface proteins such as LFA-1 (31), ICAM-1 (31, 32), CD23 (33), levels of autocrine cytokines upon CS1 stimulation. The human and B7 (34). This, along with the fact that CS1 is homophilic, led common cytokine microarray contained 114 known cytokine us to believe that CS1 might be one of the cell adhesion molecules genes. Purified human B cells were stimulated with anti-CD40 regulating B cell proliferation and activation. mAb and IL-4 for 4 days in the presence or absence of 1G10 mAb. There are many studies that indicate a role for members of the Total RNAs were isolated from each sample and used for gener- CD2 family of cell surface molecules in activation of lymphocytes. ating biotinylated cRNA probe. The results showed that 19 cyto- Meuer et al. (35) showed that CD2 engagement induces T cell kine genes (18.3%) were expressed in B cells that are activated proliferation in the absence of TCR signaling (36), and its natural with CD40/IL-4. Thirty-five genes (30.8%) were expressed upon ligand, CD58 (37), on APCs reduce the concentration of Ag re- CS1 stimulation. The data indicated that 12 genes with Ͼ2.4-fold quired for T cell activation (38). Anti-CD2 mAb also could induce increase could be differentially expressed between 1G10 mAb proliferation of CD2 positive human B progenitor cell lines (39, stimulated and unstimulated B cells. Seven of these 12 genes were 40). Moreover, blocking of CD2 on the surface of T cells inhibits highly expressed in 1G10 mAb stimulated human B cells (Table I). the capacity of these cells to mediate productive T-B cell interac- To confirm the differential transcriptional activities of human tions (6). Recombinant soluble secreted form of SLAM and murine cytokines between 1G10 mAb stimulated and unstimulated B cells, L cells transfected with cDNA encoding membrane-bound form of The Journal of Immunology 4677

SLAM induced proliferation and IgM, IgG, and IgA production by mAb/IL-4 induced rapid TNF-␣ gene transcription. B cell prolif- unfractionated B cells. However, only IgM production by sorted eration induced by CD40/IL-4 stimulation was inhibited by anti- sIgDϩ B cells was observed, indicating that SLAM acts as a B cell TNF-␣ Ab which suggests that TNF-␣ is a required autocrine B growth and differentiation-promoting molecule, but not as an Ig cell growth factor (57). isotype switch factor (10). CS1 also increased flt3L mRNA expression significantly. Dur- Our data showed that 1G10 mAb alone increased naive and ing B cell development, interaction between B cell progenitors and memory B cell proliferation and also enhanced proliferation of stromal cells are quite important. flt3L is a potent hemopoietic activated B cells. At present, we do not know the mechanism by cytokine that affects the growth and differentiation of progenitor which CS1 induces B cell proliferation. One could envisage two and stem cells both in vivo and in vitro (58). It has been shown that probable pathways for CS1 induction of B cell proliferation: first, flt3L synergies with IL-7 in the proliferation of committed B220ϩ CS1 signaling itself could induce B cell proliferation by activating pro-B cells and may contribute to the maintenance of an earlier growth-related signaling pathways; or second, CS1 may induce pro-B cell population, implicating an important role for flt3L in the cytokine productions to promote proliferation. differentiation and proliferation of early B cell progenitors in vitro CS1 contains ITSMs in its cytoplasmic domain and ITSMs can (59). A recent study using flt3L and IL-7R␣-deficient mice showed recruit small adaptor molecules (41). The SAP family adaptors that signaling through the cytokine tyrosine kinase receptor flt3 bind to the ITSMs in the cytoplasmic tail of SLAM (CD150) (42), and IL-7R ␣ are indispensable for fetal and adult B cell develop- 2B4 (CD244) (43–46), Ly-9 (CD229), and CD84 (47) via its SH2 ment (60). domain. There are three members of SAP family adaptors, which It is very interesting that all cytokines enhanced by 1G10 mAb include SAP, EAT-2, and EAT-2-related transducer (48). In the stimulation are related to growth-promoting activity. Thus, CS1 Downloaded from previous study, EAT-2 was expressed in highly purified cell-sorted may accelerate proliferation of CD40/IL-4 activated human B cells B lymphocytes and macrophages in mice (49) and it has been by inducing prolonged secretions of autocrine cytokines. shown that EAT-2 binds to CD84, CD150, CD244, and CD229 through its SH2 domain (50). However, recent studies showed that Disclosures neither SAP nor EAT-2 is expressed in human B-lymphocytes (16, The authors have no financial conflict of interest.

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