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DifferentiationProduction during Plasma Cell Galectin-1 Promotes Immunoglobulin

Shie-Liang Hsieh and Kuo-I LinChih-Ming Tsai, Yi-Kai Chiu, Tsui-Ling Hsu, I-Ying Lin,

http://www.jimmunol.org/content/181/7/4570doi: 10.4049/jimmunol.181.7.4570

2008; 181:4570-4579; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2008 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Galectin-1 Promotes Immunoglobulin Production duringPlasma Cell Differentiation1

Chih-Ming Tsai,*† Yi-Kai Chiu,*† Tsui-Ling Hsu,† I-Ying Lin,† Shie-Liang Hsieh,*†

and Kuo-I Lin2†

Galectin-1, a �-galactoside-binding soluble lectin, has been implicated in regulating immune system homeostasis. We investigatedthe function of galectin-1 in plasma cell differentiation and found that it is induced in primary murine and human differentiatingB cells. B lymphocyte-induced maturation protein-1 (Blimp-1), a master regulator for plasma cell differentiation, was necessaryand sufficient to induce galectin-1 expression. Notably, ectopic expression of galectin-1 in mature B cells increased Ig �-chaintranscript levels as well as the overall level of Ig production. This function of galectin-1 was dependent on binding to cell surfaceglycosylated counter receptors, as a galectin-1 mutant deficient in �-galactoside binding showed diminished ability to promote Igproduction. Extracellular galectin-1 bound more significantly to mature B cells than to plasma cells. Lastly, we found that thesugar compound N-acetyllactosamine blocked the binding of galectin-1 to murine splenic B cells and inhibited their differentiation.Taken together, these data are the first to demonstrate a role for galectin-1 in promoting Ig production during plasma celldifferentiation. The Journal of Immunology, 2008, 181: 4570–4579.

G alectins are a family of lectins having characteristicamino acid sequences for carbohydrate recognition andan affinity for �-galactosides. Galectins play important

roles in regulating immune cell homeostasis, in host-pathogen in-teractions, and in tumorigenesis (1–3). Thus far, 15 mammaliangalectins have been identified and sequenced (3, 4). Galectins arelocalized to the cytoplasm, nucleus, and the extracellular environ-ment (5). Secretion of galectins occurs via a nonclassical secretionpathway that requires association of galectins with glycosylatedcounter-receptors (6, 7).

Galectin-1 modulates cells of the immune system in a number ofways. Galectin-1 produced by thymic epithelial cells causes apo-ptosis in human thymocytes (8). In peripheral blood, galectin-1causes apoptosis of activated T cells by cooperating with TCRengagement, but it supports the survival of naive T cells (9–11).Galectin-1 has also been proposed to shift the T cell polarizationreaction from Th1 to Th2 by triggering apoptosis in Th1 cells (12,13). Galectin-1 also promotes surface exposure of phosphatidyl-serine (PS)3 without accompanying apoptosis in human T cell lines(14). In addition to its role in regulating many aspects of T cell

function, galectin-1 appears to have a role in stromal cells. Galec-tin-1 is expressed in bone marrow stromal cells (15, 16), and se-creted galectin-1 anchors to integrins to interact with pre-BCR andacts as a survival signal for pre-B cells during development (15,17). In late-stage B cell activation and maturation, soluble galec-tin-1 produced by activated B cells resulting from Trypanosomacruzi infection in mice causes T cell apoptosis and affects IFN-�production (18). Intracellular galectin-1 may associate with the Bcell-specific Oct-1-associated coactivator, OCA-B, to negativelyregulate BCR signaling (19). Whether there is a role for galectin-1in plasma cell differentiation, however, remains elusive.

Plasma cell differentiation is regulated by a master regulator,termed B lymphocyte-induced maturation protein-1 (Blimp-1) (20,21). B cell-specific deletion of Prdm1, the gene encoding Blimp-1,in mice showed that in response to either thymus-dependent orthymus-independent Ags, short-lived plasma cells, postgerminalcenter plasma cells, and plasma cells in a memory response areabsent (22). Blimp-1 is a transcriptional repressor (23). Our pre-vious microarray study revealed that galectin-1 is up-regulatedupon ectopic expression of Blimp-1 in mature human B cell lines(24). Herein, we investigated the importance of Blimp-1-depen-dent induction of galectin-1 during plasma cell differentiation. Wefound that the effect of galectin-1 on promoting Ig production ap-pears to be mediated through an extracellular receptor(s) and todepend on the binding of �-galactosides before terminal differen-tiation of B cells. Our results may broaden current knowledge oflectins in modulating B cell function.

Materials and MethodsCell lines, mouse strains, and reagents

BCL-1 cells were maintained in RPMI 1640 (Invitrogen) containing 10%FBS (Invitrogen) and 50 �M 2-ME (Invitrogen); CESS and MOLT-4 cellswere grown in RPMI 1640 containing 10% FBS. All the abovementionedcell lines were grown in medium containing penicillin/streptomycin (100U/ml; Invitrogen). IL-2 (20 ng/ml; eBioscience) and IL-5 (20 ng/ml;eBioscience) were used to stimulate BCL-1 cells. Splenic B cells werepurified using B220 microbeads (Miltenyi Biotec) from 12–16-wk-oldC57BL/6 mice (purchased from BioLASCO Taiwan), Prdm1f/f

CD19Cre�/�, or littermate control Prdm1f/fCD19�/� mice, as describedpreviously (22). Purified splenic B cells (purity �95%) were cultured as

*Institute and Department of Microbiology and Immunology, National Yang-MingUniversity, Taipei, Taiwan; and †Genomics Research Center, Academia Sinica,Taipei, Taiwan

Received for publication March 19, 2008. Accepted for publication July 26, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Academia Sinica (to K.L.) and by National HealthResearch Institutes Grant NHRI-EX95, 96, 97-9509NC (to K.L.).2 Address correspondence and reprint requests to Dr. Kuo-I Lin, Genomics ResearchCenter, Academia Sinica, 128 Academia Road, Section 2, Nankang District, Taipei115, Taiwan. E-mail address: kuoilin@gate.sinica.edu.tw3 Abbreviations used in this paper: PS, phosphatidylserine; Blimp-1, B lympho-cyte-induced maturation protein-1; Gal-1, galectin-1; GlcNAc, N-acetylglu-cosamine; KO, knockout; LacNAc, N-acetyllactosamine; �m, membrane form of�-chain transcript; �s, secreted form of �-chain transcript; PPIA, peptidylprolylisomerase A; QPCR, quantitative PCR; 7-AAD, 7-aminoactinomycin D; YFP,yellow fluorescent protein.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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described (25) and were stimulated with LPS (2 �g/ml; Sigma-Aldrich),IL-2 (20 ng/ml) � IL-5 (20 ng/ml), IL-4 (10 ng/ml; PeproTech) � CD40L(1 ng/ml; PeproTech) or LPS � anti-IgM (5 �g/ml; Sigma-Aldrich). Re-combinant galectin-1, with endotoxin level �1.0 EU/�g (determined byLimulus amebocyte lysate method), was purchased from R&D Systems.Human PBMC were isolated from healthy donors and grown as described(25). CD19� human B cells, at a density of 2 � 106 cells/ml, were stim-ulated with IL-21 (500 ng/ml; BioSource) � anti-CD40 (1 �g/ml; R&DSystems) � IL-2 (100 U/ml; PeproTech) � anti-IgM (5 �g/ml; JacksonImmunoResearch Laboratories). 293T and 3T3 cells were maintained inDMEM supplemented with 10% FBS and penicillin/streptomycin (100U/ml). 293F cells were maintained in FreeStyle 293 expression medium(Invitrogen). Lactose and sucrose were purchased from Sigma-Aldrich.Sugar compounds, N-acetyllactosamine (LacNAc), and Lac-di-NAc (pro-vided by the Consortium of Functional Glycomics) were dissolved in dis-tilled water.

Plasmids

pGC-yellow fluorescent protein (YFP), pGC-HABlimp-YFP, pFUGW, andpFUW-BlimpGFP (a gift from Dr. Kathryn Calame, Columbia University,New York, NY) were described previously (26, 27). Full-length humangalectin-1 was amplified by RT-PCR from H929 multiple myeloma cells.The amplified product was cloned into a pCMV2-FLAG vector (Sigma-Aldrich) at BamHI/NotI sites to generate N-terminally FLAG-tagged ga-lectin-1. Mutated human galectin-1, W69G, was generated by mutagenesisusing the following primers with the mutated bases underlined: 5�-ACGGCGGGGCCGGGGGGACCGAG-3� and 5�-CTCGGTCCCCCCGGCCCCGCCGT-3�. The FLAG-tagged galectin-1 and FLAG-tagged W69GPCR products were then blunt-end digested and subcloned into vectorpGC-YFP at the NotI site to generate pGC-NGal-1-YFP and pGC-W69G-YFP, respectively. To generate a galectin-1-hIgGFcm (Gal-1-Fc) or W69G-hIgGFcm (W69G-Fc) fusion gene, primers 5�-ATTAGGCCCAGCCGGCCGCTTGTGGTCTGGTCGCCAGCAACC-3� and 5�-GGATGCGGCCGCGCGTCAAAGGCCACACATTTGATCTTG-3� were used to PCR

amplify from pCMV2-FLAG galectin-1 or pCMV2-FLAG-W69G vector.The amplified product was ligated in-frame into the pSecTagFcm vector (akind gift from Dr. H.-H. Lin, Chang-Gung University, Taiwan) at the Sfi/NotI sites.

RNA isolation, real-time quantitative PCR (QPCR),and RT-PCR

Total RNA isolation, cDNA synthesis, and subsequent QPCR analysis inan ABI Prism 7000 sequence detection system (Applied Biosystems) orRT-QPCR analysis was according to published protocols (25). The Taq-Man primer sets used here were: Prdm1 (assay ID: Mm 00476128_ml),L32 (assay ID: Mm 00777741_sH), Lgals1 (assay ID: Mm 00777741_sH),peptidylprolyl isomerase A (PPIA) (assay ID: Hs99999904_ml), andLGALS1 (assay ID: Hs 00169327_ml), all purchased from Applied Bio-systems. The primers used for SYBR Green detection were: GAPDH, 5�-TTAGCACCCCTGGCCAAGG-3� and 5�-CTTACTCCTTGGAGGCCAT-3�; �s(secreted form of �-chain transcript), 5�-TCTGCCTTCACCACAGAAG-3�and 5�-TAGCATGGTCAATAGCAGG-3�; �m (membrane form of �-chaintranscript), 5�-AGGACAGCAGAGACCAAGAGAT-3� and 5�-GCCAGACATTGCTTCAGAT-3�; and total Ig �-chain mRNA, 5�-ATCTGCATGTGCCCATTC-3� and 5�-TTAGGATGTCTGTGGAGG-3�.

Western blotting

Cells were lysed in 2� SDS loading buffer as reported (22). The proce-dures for performing immunoblotting essentially followed published pro-tocols (25, 28). Abs specific to Blimp-1 or �-tubulin were as described(29). The anti-galectin-1 Ab was purchased from Santa Cruz Biotechnol-ogy (1/500 dilution was used). The immunoreactive proteins were detectedby an enhanced chemiluminescence system (Amersham Biosciences) ac-cording to the manufacturer’s protocol. Chemiluminescent signals werecaptured using a CCD camera (Fujifilm LAS-3000).

Table I. Galectin-1-Fc-binding glycans detected on printed glycan arraya

Glycan No. Name Avg SD % CV Intensity Index

52 Gal�1–4GlcNAc�1–2Man�1–3(Gal�1–4GlcNAc�1–2Man�1–6)Man�1–4GlcNAc�1–4GlcNAc�-Gly

53,927 7,245 13 16.38

35 �3OSO3�Gal�1–4�6OSO3�GlcNAc�–Sp8 36,711 12,066 33 11.1533 �3OSO3�Gal�1–3GlcNAc�–Sp8 25,781 4,462 17 7.831 AGP 24,747 4,274 17 7.5236 �3OSO3�Gal�1–4GlcNAc�–Sp0 22,808 5,924 26 6.93105 Gal�1–3Gal�1–4GlcNAc�–Sp8 16,499 3,094 19 5.01143 Gal�1–4GlcNAc�1–3(Gal�1–4GlcNAc�1–6)GalNAc�–Sp8 16,477 1,783 11 5.0137 �3OSO3�Gal�1–4GlcNAc�–Sp8 15,958 14,766 93 4.856 Transferrin 14,848 8,243 56 4.5124 (Gal�1–4GlcNAc�)2–3,6-GalNAc�–Sp8 13,782 3,937 29 4.1930 �3OSO3�Gal�1–4�6OSO3�Glc�–Sp8 13,639 1,672 12 4.1426 �3OSO3��6OSO3�Gal�1–4�6OSO3�GlcNAc�–Sp0 12,912 4,118 32 3.9229 �3OSO3�Gal�1–4(6OSO3)Glc�–Sp0 12,750 2,274 18 3.873 AGP-B 12,314 3,553 29 3.74147 Gal�1–4GlcNAc�1–3Gal�1–4GlcNAc�–Sp0 10,462 645 6 3.184 Ceruloplasmine 10,384 5,955 57 3.152 AGP-A 8,939 1,559 17 2.72149 Gal�1–4GlcNAc�1–3Gal�1–4Glc�–Sp8 8,789 1,032 12 2.67132 Gal�1–3GlcNAc�1–3Gal�1–4Glc�–Sp10 7,562 1,337 18 2.30227 Neu5Ac�2–3Gal�1–4�6OSO3�GlcNAc�–Sp8 7,542 652 9 2.29173 GlcNAc�1–4GlcNAc�1–4GlcNAc�–Sp8 7,471 188 3 2.27116 Gal�1–3(Fuc�1–4)GlcNAc�1–3Gal�1–4GlcNAc�–Sp0 7,387 866 12 2.2470 Fuc�1–2Gal�1–4GlcNAc�1–3Gal�1–4GlcNAc�1–3Gal�1–4GlcNAc�–Sp0 6,978 4,136 59 2.12100 Gal�1–3(Gal�1–4)Gal�1–4GlcNAc�–Sp8 6,960 1,785 26 2.11144 Gal�1–4GlcNAc�1–3GalNAc�–Sp8 6,931 1,185 17 2.1169 Fuc�1–2Gal�1–4GlcNAc�1–3Gal�1–4GlcNAc–Sp0 6,874 2,988 43 2.09110 Gal�1–4Gal�1–4GlcNAc�–Sp8 6,850 1,581 23 2.08236 Neu5Ac�2–3Gal�1–4GlcNAc�–Sp0 6,737 9,020 134 2.05131 Gal�1–3GlcNAc�1–3Gal�1–4GlcNAc�–Sp0 6,664 3,330 50 2.02153 Gal�1–4GlcNAc�–Sp8 6,653 5,452 82 2.02

a The carbohydrate-binding profile of Gal-1-Fc was determined by covalent printed array (version 2.1) in Core H, Consortium for Functional Glycomics. The average signalintensity (Avg) represents the values of relative fluorescence units and SDs among glycan replicates that were analyzed. % CV is calculated as follows: SD/Avg � 100%. Themean of the average signal intensity detected from all glycans was calculated and set as a baseline. Glycans with the average signal intensity that were �2-fold of the baseline(shown in Intensity Index) were selected to show in this table.

Disaccharide units that represent LacNAc (Gal�1–4GlcNAc) or lactose (Gal�1–4Glc) are shown in boldface type. Abbreviations: Gal, galactose; GalNAc, N-acetylgalac-tosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; Man, mannose; Fuc, fucose; Neu5Ac, N-acetylneuraminic acid.

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Production of Gal-1-Fc and W69G-Fc fusion protein

Gal-1-Fc or W69G-Fc fusion proteins were overexpressed using the Free-Style 293 expression system as reported (30). Briefly, 30 �g pSecTagga-lectin-1-hIgGFcm or pSecTagW69G-hIgGFcm was used to transfect 3 �107 293F cells with 293fectin. At 48 and 72 h posttransfection, culturesupernatants were collected for further purification. The cultured mediumwas passed through a protein A column (Amersham Biosciences), and thebound proteins were then eluted with 0.1 M glycine buffer (pH 3.0). Thepurity of the recombinant protein was analyzed by 8% SDS-PAGE andvisualized by Coomassie blue staining.

Flow cytometric analysis and recombinant protein bindingassay

Cell surface marker staining was performed as described (28). Fluores-cence intensity was analyzed by FACSCanto (BD Biosciences) and FCSExpress 3.0 software. The Abs used in this study (all from BD Pharmingen)were: PE-conjugated anti-mouse CD138/syndecan-1 (clone 281-2), allo-phycocyanin-conjugated anti-mouse CD45R/B220 (clone RA3-6B2),FITC-conjugated anti-human IgD (clone IA6-2), and APC-conjugated anti-human CD38 (clone HB7). Apoptotic and dead cells stained by annexin Vand 7-aminoactinomycin D (7-AAD), respectively, were determined as de-scribed (29). To detect the binding of galectin-1, 10 �g of Gal-1-Fc orW69G-Fc was added to the binding buffer (PBS plus 2% FBS in 100 �l)with 1 � 105 cells for 60 min, followed by the addition of FITC-conjugatedanti-human IgG (Fc-specific; Sigma-Aldrich) for 30 min. To detect the

blocking of Gal-1-Fc binding by sugar compounds, various concentrationsof sugar compounds were incubated together with 10 �g Gal-1-Fc beforethe addition of secondary Ab. For the B cell proliferation assay, mousesplenic B cells (2 � 107 cells/ml in PBS) were stained with 1 �M CFSE(Sigma-Aldrich) for 8 min at 25°C. Cells were then washed twice withcomplete medium and plated as previously described (22).

ELISA and ELISPOT

Cell supernatants were harvested for ELISA to determine the amount ofIgM or IgG as described (25). For galectin-1 ELISA, supernatants wereserially diluted in PBS containing 1% BSA into 96-well plates coated withgoat anti-mouse galectin-1 (100 �g/ml) (R&D Systems). Captured galec-tin-1 was further incubated with biotinylated anti-mouse galectin-1 (50�g/ml) (R&D Systems) and streptavidin-HRP conjugate (100 �g/ml)(R&D Systems). ELISPOT analysis for detecting IgM-secreting cells iso-lated by YFP-positive sorting essentially followed a reported procedure(31). Photomicrographs of the ELISPOT assay were read and analyzed bythe AID EliSpot Reader System (AID Autoimmun Diagnostika).

Retrovirus generation and transduction

The preparation of retroviral vectors and transduction of virus followeda described protocol (24, 32). We transduced BCL-1 and CESS celllines at a multiplicity of infection of 2–5 and primary splenic B cells ata multiplicity of infection of 10 –15. For cells transduced with retroviralvector-expressing YFP or lentiviral vector expressing GFP, YFP, or

FIGURE 1. Induction of galectin-1 during plasma cell differentiation. A–C, pFUGW or pFUW-BlimpGFP was used to transduce CESS cells. After 2days of transduction, transduced GFP� cells were sorted by flow cytometry. Sorted GFP� cells, at a density of 2 � 105 cells/ml, were further cultured for2 days. The amounts of secreted IgG in the supernatants were determined by ELISA. B and C, Sorted GFP� cells were immediately subjected to RT-QPCRanalysis (B) or immunoblot analysis for galectin-1 expression (C). PPIA mRNA and �-tubulin were used as the internal controls for real-time QPCR andimmunoblotting, respectively. D, RT-QPCR analysis of galectin-1 and Blimp-1 expression in mouse splenic B cells stimulated with LPS at the indicatedtimes. Ribosomal protein L32 mRNA was used as the internal control for normalization. Results are representative of three independent experiments. E,The amounts of secreted galectin-1 in the culture supernatants from D were determined on day 6 by ELISA and normalized to cell counts in each condition.F, RT-QPCR showing induction of galectin-1 mRNA by various stimuli in murine splenic B cell culture for 3 days. Relative galectin-1 levels werenormalized to the levels of internal control L32 mRNA and calculated by comparing galectin-1 levels at day 0. G, Splenic B cells isolated fromPrdm1f/fCD19Cre�/� (KO) and Prdm1f/fCD19�/� (control) mice were stimulated with LPS. Cultured supernatants harvested at the indicated time pointswere used to determine galectin-1 levels by ELISA. Error bars in A, B, and E–G represent SD of data means from three independent experiments. H,Peripheral CD19� B cells purified from three donors were treated with IL-21 � anti-CD40 combined with IL-2 � anti-IgM for 6 days. Cells were thenharvested for RT-QPCR to analyze the levels of galectin-1 and Blimp-1 mRNAs. PPIA mRNA was used as the internal control. Results were normalizedto sham treatment samples from the same time points as the experimental samples.

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GFP, positive cells were sorted on FACSAria (BD Biosciences) foradditional experiments.

Glycan array

The carbohydrate-binding profile of galectin-1 was determined by the CoreH, Consortium for Functional Glycomics, using a printed glycan microar-ray. Briefly, Gal-1-Fc (200 �g/ml) in binding buffer (1% BSA, 150 mMNaCl, 2 mM CaCl2, 2 mM MgCl2, 0.05% (w/v) Tween 20, and 20 mMTris-HCl (pH 7.4)) was applied onto covalent printed glycan array (version2.1) slides and incubated for 1 h at room temperature. A secondary incu-bation was conducted using Alexa Fluor 488-conjugated goat anti-humanIgG (10 �g/ml in binding buffer). Slides were scanned, and the averagesignal intensity (shown as “Avg” in Table I) was calculated. The commonfeatures of glycans with stronger binding are depicted in Fig. 6A. Theaverage signal intensity detected from all glycans was calculated and set asthe baseline.

Statistics

Data were analyzed statistically using the Student’s t test. p values of �0.5(two-sided tests) were considered statistically significant.

ResultsBlimp-1-dependent induction of galectin-1 during plasma celldifferentiation

A previous microarray study of changes in gene expression uponintroduction of Blimp-1 into human B cell lines demonstrated thatgalectin-1 expression is induced under these circumstances (24).We therefore sought to examine a possible causal relationship be-

tween Blimp-1 and galectin-1 expression. The lentiviral vector ex-pressing either GFP alone or GFP-tagged Blimp-1 (27) was used totransduce CESS cells, an EBV-transformed human lymphoblas-toid B cell line. GFP� CESS cells ectopically expressing Blimp-1(GFP-Blimp-1) showed a 10-fold increase in IgG production com-pared with GFP� CESS cells (GFP) (Fig. 1A). We confirmed thatGFP-Blimp-1 cells had increased levels of galectin-1 mRNA (Fig.1B) and protein (Fig. 1C).

We next examined expression of galectin-1 in response to stim-ulation by various cytokines and mitogens. We found that induc-tion of galectin-1 mRNA was concomitant with induction ofBlimp-1 mRNA following T cell-independent LPS stimulation inprimary mouse splenic B cells (Fig. 1D) and that galectin-1 wassecreted into the culture medium in this context (Fig. 1E). Two Tcell stimuli, IL-2 � IL-5 and IL-4 � CD40L, also induced galec-tin-1 mRNA expression (Fig. 1F). Splenic B cells in which theBlimp-1 gene Prdm1 is deleted show severe impairment of IgMsecretion and induction of CD138 when stimulated with LPS invitro (22). We examined the levels of secreted galectin-1 fromPrdm1f/fCD19Cre�/� cells, Blimp-1 knockout (KO) splenic Bcells, and from Prdm1f/fCD19�/� (control) splenic B cells in re-sponse to LPS stimulation. We found that the levels of secretedgalectin-1 were dramatically diminished in Prdm1-deficient B cellculture (Fig. 1G). Thus, galectin-1 is induced during plasma celldifferentiation in a Blimp-1-dependent manner.

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FIGURE 2. Secreted galectin-1 promotes Ig production. A, Expression of FLAG-tagged galectin-1 (pGC-NGal1-YPF) and FLAG-tagged W69G (pGC-W96G-YPF) in 3T3 cells; 95% of the cells were YFP�. Actin was used as an internal control. B, Ectopic expression of galectin-1 promotes IgM productionin BCL-1 cells. BCL-1 cells transduced with pGC-YFP or pGC-NGal1-YFP for 1 day were sorted by flow cytometry. YFP� cells, at a density of 5 � 105

cells/ml, were further cultured for 2 days. The amounts of secreted IgM in the supernatants were determined by ELISA. C, Splenic B cells stimulated withLPS � anti-IgM overnight were transduced with pGC-YFP, pGC-NGal1-YFP, or pGC-W69G-YFP, and 1 day after transduction YFP� cells were sortedby flow cytometry to determine the number of IgM-secreting cells by ELISPOT. D, Splenic B cells stimulated with a suboptimal dose of LPS (0.5 �g/ml)overnight were transduced with retrovirus as described in C. YFP� cells were sorted on day 3, and the numbers of IgM-secreting cells were analyzed byELISPOT. E, BCL-1 cells were transduced with pGC-YFP or pGC-NGal1-YFP for 1 day, and then YFP� cells were sorted by flow cytometry. YFP� cellswere further cultured with either lactose (3 mM) or sucrose (3 mM) for another 2 days. The amounts of secreted IgM in the supernatants were determinedby ELISA. F, The sorted YFP� BCL-1 cells transduced with pGC-YFP or pGC-NGal1-YFP were subjected to RNA isolation and RT-QPCR to determinetotal �-chain transcripts using SYBR Green. BCL-1 cells stimulated with IL-2 � IL-5 or sham treatment were used as controls. G, cDNAs from F weresubjected to real-time QPCR to analyze the amounts of �m mRNA and �s mRNA using SYBR Green. GAPDH was used for normalization in F and G.Diagrams in F and G indicate the primers designed specifically to detect �m (gray arrow line), �s (white arrow line), and total Ig � (black arrow line)mRNA. Error bars represent the SD of data means from three independent experiments: �, p � 0.05; ��, p � 0.01; ���, p � 0.001.

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IL-21 � anti-CD40 combined with IL-2 � anti-IgM treatmentinduces human peripheral B cells to form Ab-secreting cells (33).We took advantage of this fact to examine the correlation betweengalectin-1 and Blimp-1 mRNA expression during human periph-eral B cell differentiation. Indeed, human peripheral B cellsshowed significant induction of IgG production (data not shown),induction of CD38 plasma cell surface marker (see Fig. 5D), andinduction of Blimp-1 mRNA (Fig. 1H, right panel) under this cul-ture condition on day 6 (33). As expected, we observed that theinduction of Blimp-1 mRNA was accompanied by induction ofgalectin-1 mRNA on day 6 (Fig. 1H, left panel).

Extracellular galectin-1 promotes Ig production

To directly test the function of galectin-1 during plasma cell dif-ferentiation, we transduced B cells with a bicistronic retroviruscarrying the cDNAs encoding FLAG-tagged galectin-1 and YFP(pGC-NGal1-YFP). Expression of FLAG-tagged galectin-1 was

confirmed by immunoblotting using an Ab to FLAG (Fig. 2A).Notably, ectopic expression of galectin-1 in BCL-1 cells, a mouseB cell lymphoma line capable of producing IgM in response tocytokines, led to increased IgM secretion compared with controlpGC-YFP-expressing cells (Fig. 2B). Similar results were obtainedfor splenic B cells ectopically expressing galectin-1 that were stim-ulated with LPS � anti-IgM, a treatment that causes B cell pro-liferation but not differentiation (Fig. 2C). Furthermore, the ectopicexpression of galectin-1 enhanced the formation of IgM-secretingcells induced by suboptimal doses of LPS in splenic B cells (Fig.2D). A similar effect of ectopic expression of galectin-1 on pro-moting Ig production was also found in CESS cells (data notshown). These data suggest that galectin-1 induced during plasmacell differentiation may have a role in promoting Ig production.

Galectin-1 has been suggested to function in a variety of cellularcompartments (34). In most cases, the intracellular function of ga-lectin-1 is independent of its carbohydrate-binding ability (2, 34).

FIGURE 3. Exogenously added recombinant galectin-1 increases IgM production. A, MOLT-4 T cells were incubated with the indicated concentrationsof recombinant galectin-1 (rGalectin-1) or with solvent only (mock) for 4 h, followed by annexin V and 7-AAD staining. B, Splenic B cells were treatedwith the indicated concentrations of recombinant galectin-1 or solvent (mock) for 3 days followed by analysis of the IgM level in culture supernatants byELISA. C, Splenic B cells were treated with 5 �M recombinant galectin-1 plus either 10 mM or 3 mM of either lactose or sucrose for 3 days followedby ELISA to determine the IgM level in supernatants (��, p � 0.01). D, Splenic B cells treated with the indicated concentrations of recombinant galectin-1or solvent (mock) for 3 days followed by annexin V and 7-AAD staining. E, Trypan blue exclusion assay to measure cell viability demonstrates reducedspontaneous cell death in splenic B cells treated with the indicated concentrations of recombinant galectin-1 or solvent (mock) for 3 days. Error barsrepresent SD of data means from three independent experiments.

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To further address which cellular localization of galectin-1 pro-motes Ig production, we generated a mutant galectin-1, W69G (7,35), a �-galactoside binding and export mutant containing a pointmutation that changes Trp at residue position 69 to Gly (W69G).The W69G mutant, which remained intracellular, allowed us toexamine whether extracellular galectin-1 is responsible for pro-moting Ig production. We found that BCL-1 cells and splenic Bcells expressing W69G galectin-1 failed to increase Ig productioncompared with cells expressing wild-type galectin-1 (Fig. 2B–D),although the protein levels of intracellular FLAG-tagged wild typeand W69G galectin-1 in transduced cells were comparable (Fig.2A). Furthermore, exogenously added lactose, a known galectin-1ligand, blocked the enhanced IgM production by pGC-NGal1-YFPtransduction in BCL-1 cells; in contrast, sucrose, which does notbind galectin-1, failed to influence IgM production (Fig. 2E).These data suggest that galectin-1 enhances Ig secretion in a car-bohydrate- and an extracellular-dependent manner.

To further investigate the molecular mechanism by which ga-lectin-1 promotes Ig secretion, we examined the levels of total H(�)-chain transcripts and the ratio of the secreted form (�s) tomembrane form (�m) of �-chain transcripts in pGC-NGal1-YFP-or pGC-YFP-transduced BCL-1 cells. The levels of each form ofmRNA in sham and IL-2 � IL-5-treated cells were included asnegative and positive controls, respectively. We found that pGC-NGal1-YFP-transduced BCL-1 cells showed a 2.0-fold increase inthe total amount of �-chain transcripts compared with that in pGC-YFP-transduced cells (Fig. 2F). This extent of increase of the�-chain transcript was similar to that of BCL-1 cells stimulatedwith IL-2 � IL-5 (1.7-fold), suggesting that galectin-1 up-regu-lates �-chain mRNA expression. Changes in the ratio of �m to �smRNA upon ectopic expression of galectin-1 were also monitored.We found that ectopic expression of galectin-1 caused only a 1.5-fold increase in the ratio of �m to �s mRNA expression, in con-trast to the dramatic change in the ratio (10-fold) upon IL-2 � IL-5stimulation of BCL-1 cells (Fig. 2G), suggesting that galectin-1does not dramatically influence splicing of the �-chain mRNA.These results suggest that galectin-1 promotes �-chain mRNAtranscription during plasma cell differentiation.

To further establish the role of extracellular galectin-1 in Igsecretion, we tested whether exogenously added recombinant ga-lectin-1 could enhance IgM production. The purified recombinantgalectin-1 was found to be endotoxin free (data not shown), and ithad biological function because it increased the cell surface expo-sure of PS in a dose-dependent manner, as determined by annexin

V staining, in MOLT-4 T cells as reported (Fig. 3A) (14). Wefound that, similar to the effect of ectopic galectin-1 expression,exogenously added recombinant galectin-1 increased IgM produc-tion in splenic B cells (Fig. 3B). The effect of recombinant galec-tin-1 on promoting Ig production was blocked by the addition oflactose in a dose-dependent manner (Fig. 3C). However, unlike itsability to induce PS exposure in the T cell lineage, exogenouslyadded recombinant galectin-1 did not increase the percentageof annexin V-positive or trypan blue-stained splenic B cells (Fig.3, D and E). In fact, incubation with exogenous recombinant ga-lectin-1 reduced spontaneous cell death of splenic B cells, as de-termined by trypan blue staining (Fig. 3E). This effect could be dueto the activity of recombinant galectin-1 to activate B cells. Col-lectively, these data demonstrate that extracellular galectin-1 canpromote Ig production, but it does not induce PS exposure in Bcells.

Exogenous galectin-1 cannot rescue CD138 expression and IgMproduction in Prdm1-deficient B cells

Because galectin-1 works downstream of Blimp-1 and is capableof promoting Ig production, we sought to determine whether ectopicexpression of galectin-1 could rescue the defect of Ig secretion and ofCD138 induction in Prdm1-deficient B cells in response to LPS. Asshown in Fig. 4, in contrast to the effect of reintroduction of Blimp-1,we found that the percentage of CD138� cells and the amounts ofIgM-secreting cells from infected populations remained diminishedwhen introducing galectin-1 to LPS-stimulated Prdm1-deficient Bcells, indicating that ectopic expression of galectin-1 could not com-pensate for Blimp-1 deficiency.

Galectin-1 binds to the surface of mature B cells

Because we observed that galectin-1 is induced and has a role inpromoting Ig production, we wished to examine the pattern ofgalectin-1 binding during plasma cell differentiation. Plasmids en-coding recombinant galectin-1-hIgGFcm (Gal-1-Fc) or W69G-hIgGFcm (W69G-Fc) were generated, and the proteins were puri-fied with a protein A column (30). The fusion protein containedfull-length galectin-1 or W69G in the N-terminal and Fcm portionof human IgG in the C-terminal, which allows it to be detected byanti-human IgG. Fcm used here prevented binding to cell surfaceFc receptors as described (30); W69G-Fc was a control to excludethe possibility of nonspecific binding by Gal-1-Fc. The expressionof purified recombinant fusion protein was monitored (Fig. 5A).We used Gal-1-Fc to monitor the binding of galectin-1 to B cells

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1000FIGURE 4. Ectopic expression ofgalectin-1 cannot rescue Blimp-1 defi-ciency. A, Splenic B cells isolated fromPrdm1f/fCD19Cre�/� (KO) and Prdm1f/f

CD19�/� (control) mice were stimulatedwith LPS (2 �g/ml) and transduced withpGC-YFP, pGC-NGal1-YFP, or pGC-HABlimp-1-YFP. Two days after retro-viral infection, transduced cells weresorted by flow cytometry, and YFP�

cells were further subjected to ELISPOTanalysis determining the number of IgM-secreting cells. B, Three days after virustransduction the levels of CD138 on thecell surface of transduced cells describedin A were analyzed by flow cytometry.Results are a representative of two inde-pendent experiments.

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and found that Gal-1-Fc bound to purified mouse splenic B cells(Fig. 5B). This binding was dependent on galactoside recognition,as neither incubation with W69G-Fc alone (Fig. 5B, top) nor withGal-1-Fc in the presence of lactose in the binding reaction (Fig.5B, bottom) showed binding. Unexpectedly, following LPS-medi-ated differentiation for 3 days, we found reduced binding of ga-lectin-1 to B220lowCD138� plasma cells compared with theB220highCD138� undifferentiated populations (Fig. 5C). We alsoexamined whether galectin-1 bound to human B cells when stim-ulated to differentiate by IL-2 � IL-21 and anti-CD40 � anti-IgM.Likewise, we observed that Gal-1-Fc bound to unstimulated ma-ture B cells (Fig. 5D, top), but after 6 days of stimulation almostno Gal-1-Fc binding to CD38highIgD� plasma cells was observed(Fig. 5D, bottom). Therefore, our data suggest that galectin-1 bindsbetter to mature B cells than to plasma cells.

LacNAc blocks plasma cell differentiation in vitro

To further understand which glycan ligands are recognized by ga-lectin-1, we screened Gal-1-Fc for carbohydrate ligands by the

glycan microarray screening service available at the Consortium ofFunctional Glycomics. As expected, we found that Gal-1-Fc hadthe highest affinity for sugar compounds containing multipleGal�1–4 N-acetylglucosamine (GlcNAc) (LacNAc), Gal�1–4Glc(lactose), or Gal�1–3GlcNAc units (Fig. 6A) (36). Glycans with anaverage signal intensity for Gal-1-Fc binding �2-fold that of thebaseline (an intensity index of �2.00) are shown in Table I. �-2,6-Sialylation of the galactose residue or fucosylation of the N-acetyl-glucosamine abolished Gal-1-Fc binding (data not shown).

Based on the sugar array profile (Fig. 6A and Table I) and thefact that galectins recognize multiple LacNAc units present on thebranches of N- or O-linked glycans (37, 38), we asked if LacNAccould block the binding of galectin-1 to B cells. We used lactoseas a positive control in this experiment. Sucrose and N-acetylgalac-tosamine (GalNAc)�1–4GlcNAc (Lac-di-NAc) were used as neg-ative controls. Notably, we found that the binding of Gal-1-Fc tomurine splenic B cells was efficiently blocked by LacNAc in adose-dependent manner (Fig. 6B). In fact, LacNAc was a morepotent inhibitor than lactose in blocking galectin-1 binding to B

FIGURE 5. Extracellular galectin-1 binds to mature B cells. A, SDS-PAGE analysis of expression of Gal-1-Fc and W69G-Fc fusion proteins andsubsequent staining of the gel by Coomassie blue. The expected molecular mass of the fusion proteins is 43.5 kDa. B, Purified mouse splenic B cells wereincubated with 10 �g Gal-1-Fc or W69G-Fc or preincubated with 100 mM lactose for 10 min before Gal-1-Fc binding (bottom). Following the additionof FITC-conjugated Fc-specific anti-human IgG, binding was analyzed by flow cytometry. C, Purified splenic B cells were stimulated with LPS for 3 days,and cells were harvested for Gal-1-Fc binding as described above. The cells were also stained with the surface markers B220 and CD138. D, Humanperipheral B cells before (day 0, top) and after (day 6, bottom) treatment with a combination of IL-21, anti-CD40, IL-2, and anti-IgM for 6 days wereincubated with Gal-1-Fc in a binding assay and subsequently stained with the surface markers CD38 and IgD. Results were analyzed by flow cytometry.PC indicates plasma cells. Gray histograms in B–D represent stained with FITC-conjugated anti-human IgG only. Results from B–D are representative ofthree independent experiments.

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cells, as LacNAc at 1 mM greatly reduced Gal-1-Fc binding,whereas a dose of 100 mM lactose was required to achieve a sim-ilar blockade (Fig. 6B). Sucrose at 100 mM and Lac-di-NAc at 1mM did not affect Gal-1-Fc binding (Fig. 6B). We next determinedwhether LacNAc could block murine splenic B cell differentiation.As shown in Fig. 6C, LacNAc at 10 mM and 1 mM significantlyreduced LPS-mediated induction of IgM in splenic B cells,whereas Lac-di-NAc did not affect IgM production. Because B cellstimulation followed by differentiation requires cell division (39),we labeled purified splenic B cells with the cell division trackingdye CFSE and stained cells for CD138 expression after 3 days inculture with LPS plus various doses of LacNAc or Lac-di-NAc tomonitor the effects of sugar compounds on B cell proliferation andinduction of CD138 following LPS stimulation. Results in Fig. 6D

show that LacNAc at higher doses (10 mM and 1 mM) interferedmore obviously with the induction of CD138high. The effect ofLacNAc was not completely due to its ability to alter LPS-inducedcell proliferation because reduced frequencies of CD138� cellswere observed at concentrations of LacNAc (1 mM and 0.1 mM)that did not affect CFSE dye dilution (Fig. 6D). Taken together,these data show a correlation between abrogation of galectin-1binding to mature B cells by LacNAc and the effect of LacNAc onthe inhibition of plasma cell differentiation.

DiscussionGalectins have a variety of roles in regulating immune cell ho-meostasis; however, the role of galectin-1 in plasma cell differen-tiation in particular has been unclear. We demonstrate here for the

FIGURE 6. LacNAc binds Gal-1-Fc and can block LPS-stimulated splenic B cell differentiation. A, Glycan binding profile of Gal-1-Fc. RecombinantGal-1-Fc was examined for carbohydrate binding properties using a glycan microarray by the Core H of Consortium for Functional Glycomics. Thecommon features of the glycans that showed the strongest binding to Gal-1-Fc are depicted. Symbol nomenclature is shown in the inset. See the “Avg”column in Table I for the values of relative fluorescence units. B, Mouse splenic B cells were either incubated with 10 �g Gal-1-Fc (open histograms) aloneor with 10 �g Gal-1-Fc and various concentrations of lactose (1 mM, light gray histogram; 10 mM, dark gray histogram; or 100 mM, black histogram),sucrose (100 mM, dark gray histogram), or LacNAc or Lac-di-NAc (0.01 mM, light gray histograms; 0.1 mM, dark gray histograms; or 1 mM, blackhistograms), followed by the addition of FITC-conjugated anti-human IgG for the detection of galectin-1 binding. Open histograms filled with diagonalslashed lines represent cells stained with FITC-conjugated anti-human IgG only. C and D, LacNAc blocks LPS-mediated IgM production and CD138induction in splenic B cells. C, Splenic B cells were incubated with the indicated concentrations of sugar compounds and LPS (0.25 �g/ml). Cell culturesupernatants were harvested after 3 days of treatment for ELISA to determine IgM levels. Data shown are means SD of triplicate samples fromrepresentative of two independent experiments (���, p � 0.001). D, Flow cytometric analysis of cell surface CD138 expression in CFSE-labeled cells.Concentrations of sugar compounds are indicated on the top of the dot plots. The percentage of CD138high plasma cells in each population is shown. Datashown are representative of two independent experiments.

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first time that, during plasma cell differentiation, induction of ga-lectin-1 is Blimp-1 dependent. Galectin-1 increases the levels of�-chain transcripts and Ig production during plasma cell differen-tiation. Because exported galectin-1 was responsible for this ac-tivity and did not bind to cells with plasma cell surface markers,the data suggest that the effect of extracellular galectin-1 is topromote Ig production by B cells after their activation, rather thanto induce Ig secretion by plasma cells. Our findings are summa-rized in Fig. 7.

Galectin-1 functions in various cellular compartments. The in-tracellular activities of galectin-1 appear to be independent of itslectin activity (2). We showed that a galectin-1 mutant, W69G, isimpaired in its ability to promote Ig production, that exogenouslyadded lactose blocks the induction of Ig caused by ectopic expres-sion of galectin-1, and that exogenously added recombinant ga-lectin-1 increases Ig production, indicating that binding to cell sur-face counter-receptors is required for galectin-1 function duringplasma cell differentiation. Additionally, blocking the binding ofgalectin-1 to murine splenic B cells by LacNAc abrogated LPS-stimulated plasma cell differentiation (Fig. 6); this also supportsthe notion that galectin-1 requires carbohydrate recognition for itsactivity during plasma cell differentiation. Numerous galectin-1receptors, including CD45, CD7, CD43, CD2, CD3, CD4, CD7,integrins, fibronectin, laminin, and pre-BCR have been reported(4). Our finding that galectin-1 preferentially binds to mature Bcells (Fig. 5) suggests that these cells contain potential galectin-1receptors. One potential receptor is CD45; it has been implicatedas the galectin-1 receptor in Burkitt’s lymphoma B cells (40), andits expression is down-regulated in plasma cells (41). Alterna-tively, galectin-1 may preferentially bind to mature B cells becauseonly they retain certain glycosylation modification patterns thatgalectin-1 recognizes. In support of the latter hypothesis, a recentpublication indicated that Th1, Th2, and Th17 cells have differ-ential surface glycosylation patterns, revealed by plant lectin bind-ing, thus rendering them differentially susceptible to galectin-1-mediated cell death (13). Nevertheless, further work is needed toelucidate the key surface glycoconjugates that mediate the effect ofgalectin-1 during plasma cell differentiation. Additionally, our re-sults cannot exclude the possibilities that other galectins might beinvolved in modulating Ig production because several galectins

bind LacNAc with differential affinity (37, 38). However, the roleand mechanism of regulation of the expression of other galectinsduring B cell activation/differentiation remain unclear. Galectin-3is induced during B cell activation by IL-4 or anti-CD40, but itsinduction seems to favor the memory B cell fate (42). Therefore,it remains to be investigated whether other galectin family mem-bers participate in modulating B cell activation/differentiation.

Are there other possible functions for galectin-1 secreted byactivating/differentiating B cells in addition to promoting Ig pro-duction? Galectin-1 can induce phenotypic and functional matu-ration in human monocyte-derived dendritic cells (43). Also, sev-eral lines of study have indicated that galectin-1 is involved inregulating apoptosis in activated T cells (10, 44). Thus, galectin-1may act as a surveillance factor by which activated B cells elim-inate or change the properties of activated T cells during immuneresponses in secondary lymphoid tissues. Unlike its known role inincreasing the number of annexin V-positive T cells, we found thatgalectin-1 had no significant role in regulating apoptosis in matureB cells: neither ectopic expression of galectin-1 by retroviral vec-tor (data not shown) nor exogenously added recombinant galec-tin-1 increased cell death or annexin V-positive cells in B cells inthis study (Fig. 3, C and D). It has been shown that in early stagesof B cell development, the galectin-1/pre-BCR complex functionsin pre-B cell proliferation (15), which also suggests that galectin-1does not cause B cell death.

The mechanism underlying the ability of galectin-1 to promoteIg production remains unclear. Intracellular galectin-1 has beenimplicated in regulating pre-mRNA splicing in a complex withgalectin-3 (45–47). Blimp-1 also has a possible role in regulatingthe switch from �m to �s (22). Our results suggest that it is un-likely that the role of Blimp-1 in affecting splicing of �m mRNAto �s mRNA is through the action of galectin-1 because we did notfind significant change in the ratio of �s/�m transcripts followingectopic expression of galectin-1 (Fig. 2G). We found, however,that total levels of �-chain mRNA were elevated (Fig. 2F), sug-gesting that galectin-1 may trigger a signaling pathway(s) to in-crease �-chain expression. One clue in this regard is that the effectof galectin-1 in promoting Ig production is not by up-regulatingthe expression of Blimp-1 or the activated form of XBP-1 (XBP-1s) because we did not find changes in the levels of Blimp-1 orXBP-1s mRNA in BCL-1 cells ectopically expressing galectin-1(data not shown). Thus, galectin-1 may signal through the uniden-tified counter-receptors that function independently of triggeringBlimp-1 induction during B cell activation/differentiation. Al-though galectin-1 enhanced Ig production 2-fold, we found thatexogenous galectin-1 could not rescue the deficiency of Blimp-1 insplenic B cell culture (Fig. 4). This observation is similar to aprevious finding that ectopic expression of XBP-1s cannot com-pensate for a Prdm1 deficiency (22). These data suggest that mul-tiple Blimp-1 targets are required to coordinately regulate Ig pro-duction. In conclusion, we have established a previously undefinedrole and mechanism for Blimp-1-dependent induction of galectin-1in promoting Ig production during plasma cell differentiation.

AcknowledgmentsThe authors thank Drs. Chi-Huey Wong and Tse Wen Chang for discus-sions and critical reading of the manuscript, Hui-Kai Kuo and Wen-WenChen for technical assistance, Consortium of Functional Glycomics (GrantGM62116) for the gift of the sugar compounds and performing the Gal-1-Fc glycan array, and Dr. Rachel Ettinger (National Institutes of Health,Bethesda, MD) for human peripheral B cell culture advice.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 7. Proposed mode of action of Blimp-1-dependent inductionof galectin-1 during plasma cell differentiation. During B cell activation/differentiation, Blimp-1 causes up-regulation of galectin-1, which is thensecreted to the extracellular environment to bind with its counter-recep-tor(s) on less differentiated B cells. The conjugation of glycosylated surfacereceptors by galectin-1 promotes Ig production. Plasma cells gradually losethe ability to bind galectin-1 as differentiation proceeds. In addition toactivated B cells, other cell types, such as follicular dendritic cells andendothelial cells (48), can express galectin-1, which might also contributeto promoting the production of Ab-secreting cells during immune re-sponses in lymphoid follicles.

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