Galectin-8 provides costimulatory and proliferative signals to T lymphocytes

Galectin-8 provides costimulatory andproliferative signals to T lymphocytes

Marıa Virginia Tribulatti,* Valentina Cattaneo,* Ulf Hellman,† Juan Mucci,* andOscar Campetella*,1

*Instituto de Investigaciones Biotecnologicas-Instituto Tecnologico de Chascomus, CONICET-Universidad Nacional de SanMartın, Buenos Aires, Argentina; and †The Ludwig Institute for Cancer Research, Uppsala University, Sweden

RECEIVED SEPTEMBER 5, 2008; REVISED FEBRUARY 23, 2009; ACCEPTED MARCH 23, 2009. DOI: 10.1189/jlb.0908529

ABSTRACTGalectin (Gal) constitute a family of carbohydrate-rec-ognizing molecules ubiquitously expressed in mam-mals. In the immune system, they regulate many pro-cesses such as inflammation, adhesion, and apoptosis.Here, we report the expression in the spleen of the twosame Gal-8 splice variants described previously in thethymus. Gal-8 was found to induce two separate bio-logical activities on T lymphocytes: a robust naive CD4�

T cell proliferation in the absence of antigen and nota-bly, a costimulatory signal that synergized the cognateOVA peptide in DO11.10 mice transgenic for TCROVA.The antigen-independent proliferation induced by Gal-8displayed increased expression of pro- and anti-inflam-matory cytokines, thus suggesting the polyclonal ex-pansion of Th1 and Th2 clones. The costimulatory effecton antigen-specific T cell activation was evidencedwhen the Gal and the peptide were assayed at dosessuboptimal to induce T cell proliferation. By mass spec-tra analysis, several integrins and leukocyte surfacemarkers, including CD45 isoforms, as well as othermolecules specific to macrophages, neutrophils, andplatelets, were identified as putative Gal-8 counter-re-ceptors. Gal-8 triggered pZAP70 and pERK1/2. More-over, pretreatment with specific inhibitors of CD45phosphatase or ERK1/2 prevented its antigen-depen-dent and -independent T cell-proliferative activities.This seems to be associated with the agonistic bindingto CD45, which lowers the activation threshold of theTCR signaling pathway. Taken together, our findingssupport a distinctive role for locally produced Gal-8 asan enhancer of otherwise borderline immune re-sponses and also suggest that Gal-8 might fuel the re-activity at inflammatory foci. J. Leukoc. Biol. 86:371–380; 2009.

IntroductionProtein glycosylation is becoming a rapidly growing study fieldin cell and tissue physiology as a result of the huge complexity

involved that allows a fine regulation of several processes [1].In fact, the interaction between different cell types and theirenvironment relies in part on their differential glycosylationprofiling. Together with many surface molecules able to recog-nize glycosylated binders, some soluble mediators such as Galare involved in homeostasis and in pathological events [2, 3].Gal constitute a family of �-galactoside binding mammalianlectins that contains conserved carbohydrate recognition do-mains. They are classified in three groups based on their struc-ture: prototype such as Gal-1; chimera type with Gal-3 as itsonly representative; and tandem repeat type, where Gal-8 isincluded. As they are involved in a growing number of differ-ent biological processes, their structure and biochemistry arecurrently receiving strong attention.

Regarding their activity on the immune system, severalmembers of this protein family including Gal-1, Gal-3, Gal-8,and Gal-9 [2, 4 –7] are expressed by different cell popula-tions, where acting mostly in an autocrine/paracrine man-ner, they are involved in several phenomena such as regula-tion, adhesion, differentiation, and apoptosis [8 –10]. Someof them are also associated with tumor evasion and progres-sion [3]. The activity of some Gal, particularly Gal-1 andGal-3, in the immune system has been analyzed in detail,but in contrast, scarce information about the effects ofGal-8 is still available. Gal-8 belongs to the tandem repeat-type group, which contains two carbohydrate recognitiondomains joined by a linker peptide, whose variable lengthdefines different isoforms in mice, rats, and humans [7, 11–13]. Gal-8 is widely expressed in several tissues under nor-mal and altered conditions [2]. Through the interactionwith integrins [13, 14], Gal-8 induces an adhesive pheno-type in several cells such as Jurkat T cells [15, 16] and neu-trophils [17]. Moreover, Gal-8-induced spreading of Jurkatcells depends on the interaction of this Gal with specific �1integrins, thus suggesting a modulating property in the cell-driven immune response [15]. We have shown recently thattwo isoforms of Gal-8 differing in the length of their linker

1. Correspondence: Instituto de Investigaciones Biotecnologicas, Av GeneralPaz 5445, Predio INTI, Edificio 24, B1650WBA San Martin, Buenos Aires,Argentina. E-mail: [email protected]

Abbreviations: Gal-8�Galectin-8, Mac-1�macrophage antigen-1,MMP�matrix metallopeptidase, MPCR�multiplex PCR, NIHnu/nu�NIHnude, p�phospho, PTPase�phospho-tyrosine phosphatase,TDG�thiodigalactoside

Article

0741-5400/09/0086-371 © Society for Leukocyte Biology Volume 86, August 2009 Journal of Leukocyte Biology 371

peptide are present in the thymus, where both are able toinduce apoptosis of the CD4highCD8high thymocytes througha mechanism involving the caspase activation pathway [7].The aim of the present work was to analyze the expressionof Gal-8 in the spleen and explore its functions on periph-eral lymphocytes.

MATERIALS AND METHODS

Mice, cell lines, and cell purificationC57BL/6J and C.Cg-Tg (DO11.10) 10Dlo/J (DO11.10) breeding pairswere obtained from The Jackson Laboratories (Bar Harbor, ME, USA)and bred in our animal facilities. The Committee of Ethics of the Insti-tuto de Investigaciones Biotecnologicas (Buenos Aires, Argentina) ap-proved all animal experiments. Dr. Anne I. Sperling (University of Chi-cago, Chicago, IL, USA) generously provided B6.CD43null mice, back-crossed eight times to C57BL/6J mice. Athymic NIHnu/nu or euthymicNIH�/� mice were obtained from the animal facility of the NationalUniversity of La Plata (Buenos Aires, Argentina). The human Jurkat Tcell line was obtained from American Type Culture Collection (Manas-sas, VA, USA). For mouse splenocyte purification, spleens from 4- to8-week-old animals were removed and disrupted against a stainless steelmesh in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA). The cellsuspension was washed and incubated with RBC lysis buffer (SigmaChemical Co., St. Louis, MO, USA) and washed again with medium. ForT cell purification, C57BL/6J mouse splenocytes were treated with anti-CD19-coated beads (Dako, Carpinteria, CA, USA) following the manu-facturer’s protocol for cell depletion. Purity was confirmed by flow cy-tometry with PE-labeled anti-CD19 (Becton Dickinson, San Jose, CA,USA). CD4 and CD8 T cells were purified from splenocytes fromC57BL/6J mice by using the miniMACS columns and anti-CD4- or anti-CD8-coupled paramagnetic particles from Miltenyi Biotec (Germany).Cells assayed by FACS for purity indicated no contaminants in theeluted population. Cell lines and mouse primary cells were cultured at37°C in 5% CO2 in RPMI-1640 in the presence of 10% FBS (Invitro-gen), 2 mM glutamine, and gentamycin (complete medium).

Antibodies and reagentsAnti-mouse Gal-8 rabbit antibodies affinity-purified through a Gal-8-Sepha-rose column were obtained as described earlier [7]. Dr. Yehiel Zick (TheWeizmann Institute of Science, Israel) kindly provided a mouse anti-ratGal-8 mAb [18]. Fluorescent mAb against mouse CD19 was from BectonDickinson. The anti-p44/42 MAPK (ERK1/2), pp44/42 MAPK (Thr202/Tyr204), ZAP70, and pZAP70 antibodies and the U0126 ERK1/2 phosphory-lation inhibitor were from Cell Signaling Technology (Beverly, MA, USA).The CD45 PTPase inhibitor N-(9, 10-dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide [19] was from Calbiochem (La Jolla, CA, USA).TDG was from Sigma Chemical Co.

Expression of mouse rGal-8Both recombinant isoforms of Gal-8 cloned from mouse thymic cells(Gal-8L and Gal-8S) were expressed in Escherichia coli and purified by twosteps of affinity chromatography as described previously [7]. Lectin activityof these molecules was tested by hemagglutination assays [7].

RT-PCRSplenocytes were solubilized in Trizol reagent (Invitrogen), and total RNAwas extracted following the manufacturer’s instructions. mRNA was puri-fied using the PolyATract mRNA isolation kit from Promega (Madison, WI,USA) and used as template for the retrotranscriptase Superscript II (In-vitrogen) to obtain cDNA. Controls without the retrotranscriptase were in-cluded. Gal-8 transcripts were detected with a set of primers (forward 5�-AAAGCTAGCATGTTGTCCTTAAATAACCTA-3�, reverse 5�-AAAAGATCTC-

CGGATAAGCCATTTTGTA-3�), which amplifies the entire codingsequence of Gal-8 using mouse splenocyte cDNA as template.

Western blotSplenocyte lysates from C57BL/6J male mice were seeded onto a lactosyl-Sepharose column (Sigma Chemical Co.) and eluted with lactose as de-scribed [7]. Eluates were solved in a 10% SDS-PAGE and transferred topolyvinylidene difluoride membranes. Blots were probed with affinity-puri-fied anti-mouse Gal-8 antibodies 1/100, followed by HRP-labeled goat IgGanti-rabbit IgG 1/5000, and developed by chemiluminescence (both fromPierce, Rockford, IL, USA). In a similar set of assays, blots were probedwith a mouse mAb anti-rat Gal-8 followed by HRP-labeled anti-mouse IgGsecondary antibodies (Dako).

Gal-8 binding assaysMouse C57BL/6J splenocytes were treated with Gal-8, 0.1 �M, washed withcold PBS or PBS plus 100 mM lactose, blocked with anti-FcRs (BectonDickinson), and incubated with affinity-purified rabbit anti-Gal-8 antibodies[7] with sodium azide in an ice bath, followed by incubation with 1/1000FITC-conjugated anti-rabbit IgG antibodies (Molecular Probes, Invitrogen,Eugene, Oregon, USA). Cells were washed, fixed, and analyzed by flow cy-tometry. Two controls were included: one where cells were first incubatedwith a nonrelated rabbit IgG and another one where cells were processedwithout incubation with Gal-8. A FACSCalibur cytometer (Becton Dickin-son) and WinMdi software were used throughout this work.

Proliferation and costimulation assaysSplenocytes (5�105) from C57BL/6J male mice were cultured for 48 h in96-multiwell plates in a final volume of 0.2 ml RPMI-1640 complete me-dium. An aliquot of 50 �l containing 1 �Ci [3H]-methyl thymidine (NewEngland Nuclear, Boston, MA, USA) was added to each well for the last16 h before cell harvesting. TDG or CD45 PTPase inhibitor was alwaysadded 20 min before Gal-8. U0126 inhibitor (10 �M) was added 1 h beforethe assay. No significant differences with basal cpm were found in TDG,PTPase, or U0126 inhibitors, only added cultures. An aliquot of 5 �g/mlLPS or Con A (Sigma Chemical Co.) was added as proliferation controlswhen indicated. For costimulatory assays, splenocytes (1.5�105 cells) weretaken from 4- to 8-week-old female DO11.10 mice and cultured in a finalvolume of 0.1 ml in the presence of 0.4–1 �g/ml cognate OVA323–339 pep-tide (Sigma-Genosys, The Woodlands, TX, USA) for 48 h, adding [3H]-methyl thymidine 16 h before harvesting. For short assays, 24 h cultureswere carried out with the addition of labeled thymidine 6 h before collec-tion. The peptide dose was determined in preliminary experiments (notshown) so as to induce low cell proliferation under these conditions. Allassays were performed in quadruplicate.

Affinity chromatography and mass spectrometrySplenocytes from C75BL/6J mice were lysed with 1% Triton X-100 in thepresence of a cocktail of protease inhibitors and passed through a Gal-8affinity column made by coupling purified rGal-8L to N-hydroxysciccinim-ide-activated HiTrap columns (GE Healthcare, Uppsala, Sweden). Gal-8binders were eluted with 100 mM lactose in PBS and concentrated by Ami-con Centriprep YM-3 (Millipore, Bedford, MA, USA). After alkylation withiodoacetamide, samples were resuspended in cracking buffer and solved by10% SDS-PAGE. After Coomassie blue staining, discrete bands were cutout, in-gel digested overnight with trypsin, and subjected to peptide massfingerprinting on an Ultraflex TOF/TOF (Bruker Daltonics, Bremen, Ger-many) MALDI mass spectrometer.

RT-MPCRFor RT-MPCR assays, the MPCR Kit for Mouse Th1/Th2 Cytokines Set-2(Maxims Biotech, Rockville, MD, USA) was used. This kit contains specificprimers for IFN-�, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, and GAPDH as aninternal control. cDNA from splenocytes cultured for 4–24 h in the pres-

372 Journal of Leukocyte Biology Volume 86, August 2009 www.jleukbio.org

ence of Gal-8 or Gal-8 plus TDG was used as template for these assays. Gal-8-treated splenocytes were washed with 200 mM lactose before harvestingto detach them from the plate.

pp44/42 MAPK (PERK1/2)Jurkat cells (3�106) were cultured in complete medium for 1 h in thepresence of Gal-8, alone or in combination with TDG or CD45 PTPase in-hibitor. Cells were washed with cold PBS plus lactose 100 mM before har-vesting to detach them from the plate bottom and then washed with coldPBS. Western blots were carried out following the manufacturer’s protocol(Cell Signaling Technology), with the addition of 2 mM sodium orthovana-date to the lysis buffer as the only modification.

pZAP70Splenocytes were obtained from DO11.10 female mice, and 5 � 106 nucle-ated cells were cultured for 1 h in a final volume of 2 ml RPMI completemedium with the addition of 0.1 �M Gal-8, 1 �g/ml OVA323–339 peptide,and 2 �M CD45 PTPase inhibitor as indicated and processed as forPERK1/2.

Statistical analysisStudent’s t-test was used.

RESULTS

Expression of Gal-8 in the mouse spleenGal-8 transcripts were detected by RT-PCR using mousesplenocyte cDNA as template (Fig. 1A), and the main ampli-con (�1 kbp band) was cloned. A control without the retro-transcriptase rendered no signal. Noticeably, the two sameisoforms (Gal-8S and Gal-8L), found previously in thymo-cytes [7], were identified in mouse splenocytes. Nine inde-pendent clones were sequenced that resulted in eight corre-sponding to Gal-8S and one to Gal8-L. To confirm the ac-tual presence of the protein, mouse spleen homogenateswere loaded onto lactosyl-Sepharose affinity columns, andthe fraction eluted with lactose was probed by Western blotusing affinity-purified anti-Gal-8 rabbit polyclonal antibodiesand a mouse anti-rat Gal-8 mAb [18] (Fig. 1B).

Gal-8 induces splenocyte proliferationAs Gal-8 is secreted to the extracellular space where it exertsits function as a paracrine/autocrine effector [13], we exam-ined the ability of soluble Gal-8 to bind mouse splenocytes. Asshown for Gal-8L in Figure 2A, both rGal-8 isoforms bound toalmost all cell populations. More importantly, both of themwere competed out effectively by lactose, thus supporting theirspecific interaction with the splenocyte surface glycans.

Several Gal promote apoptosis in mature human and mu-rine T cells upon binding. In the case of Gal-8, its proapop-totic effect has been demonstrated in several tumor cell lines[18] as well as in the CD4highCD8high thymocyte subpopula-tion [7]. Nevertheless, this effect was not observed by the pro-pidium iodide assay in mouse splenocytes cultured in the pres-ence of 0.5–2 �M Gal-8 (either isoform) for 16 h (not shown).Furthermore, we found a decreased percentage of hypoploidcells in Gal-8-treated splenocytes as compared with controls(not shown), thus suggesting that Gal-8 might actually have anopposite role in this system. These findings prompted us to

search for a possible role of Gal-8 in splenocyte activation. In-deed, when splenocytes were cultured for 48 h in the presenceof 0.5–2 �M Gal-8 (the S or the L isoform), strong prolifera-tion indexes (five- to 90-fold increase) were observed (shownfor Gal-8L in Fig. 2B). This effect was dose-dependent, reach-ing its maximum effect at 2 �M of the rGal. Splenocyte prolif-eration was prevented by the addition of TDG, which blocksthe interaction of Gal-8 with carbohydrates. This findingstresses the requirement of the interaction of the carbohy-drate-recognition domains with surface glycans to induce pro-liferation and rules out the possible involvement of bacterialcontaminants such as LPS in this phenomenon. Besides, theaddition of sucrose as a nonrelated sugar did not prevent theproliferative effect of Gal-8 (not shown). As similar findingswere obtained with both Gal-8 isoforms, further assays wereperformed with the Gal-8L recombinant protein.

CD4 T cells are the main targets for Gal-8-inducedproliferationTo narrow down the population of Gal-8-responsive cells inthe proliferation studies, assays similar to those performed be-fore were carried out on splenocytes collected from athymicnude mice. As shown in Figure 3A, splenocytes from nudemice failed to proliferate with Gal-8 or Con A, although as ex-pected, responded well to LPS. On the other hand, additionof 2 �M Gal-8 led to cpm values twofold higher in B cell-de-

Figure 1. Expression of Gal-8 in mouse spleen. (A) RT-PCR. cDNAfrom splenocytes and thymocytes were used as template in a RT-PCRassay with specific primers for mouse Gal-8 open-reading frame, whichresults in 1 kb-length transcripts. (B) Western blots. Whole homoge-nates from spleens or thymuses (as positive control) were affinity-puri-fied by lactosyl-Sepharose, and the eluted fractions were precipitatedwith trichloroacetic acid and subjected to 10% SDS-PAGE. Blots werereacted with affinity-purified rabbit anti-mouse Gal-8 antibodies ormouse anti-rat Gal-8 mAb. A lower molecular weight band in the mon-oclonal-developed Western blot corresponds to mouse Igs.

Tribulatti et al. Galectin-8 induces T cell proliferation

www.jleukbio.org Volume 86, August 2009 Journal of Leukocyte Biology 373

pleted suspensions (content of T cells, �97%) than in nonde-pleted ones (Fig. 3B). Taken together, these results stronglysupport T cells as the principal targets for Gal-8-induced pro-liferation. To explore these observations further, purifiedCD4� and CD8� lymphocytes were tested separately in prolif-eration assays in response to the Gal-8 stimulus. CD4� cellsstrongly proliferated; meanwhile, the CD8� population wascomparatively unresponsive (Fig. 3C). These findings indicatethe strong effect exerted by Gal-8 and support an antigen-in-dependent stimulating effect on CD4� T cell populations thatmight be involved in fueling inflammatory processes.

Gal-8 induces expression of IL-4, IFN-�, and IL-2Some Gal are known to alter the outcome of the immune re-sponse; Gal-1, for example, shifts the balance from Th1- to-

ward a Th2-polarized immune response [20]. To deepen ourunderstanding of Gal-8-mediated T cell activation, we won-dered which cytokines were expressed in proliferating cells. Toundertake this task, a RT-MPCR assay was carried out usingcDNA from splenocytes after 24 h of treatment with Gal-8 astemplate. Enhanced transcription of IL-4 as well as of IFN-�and IL-2 was observed in Gal-8-treated cells (Fig. 4). On theother hand, IL-10, IL-5, IL-13, which were undetectable under

Figure 2. Gal-8 induces naıve T cell proliferation. (A) Gal-8 binding tosplenocyte surface. Splenocytes were treated with 0.1 �M Gal-8L andbinding tested with rabbit anti-Gal-8 antibodies. Control, Anti-Gal-8antibodies in the absence of Gal-8 addition; Lac, cells washed with 100mM lactose after Gal-8 treatment; Normal IgG, antibodies purifiedfrom naive rabbits. (B) Splenocytes were cultured with the indicatedGal-8L amounts and proliferation quantified. TDG was added at 30mM 20 min before Gal-8 addition. No significant differences withbasal cpm were found in cultures where only TDG was added. *, P �0.01; **, P � 0.001. Results are representative of more than five inde-pendent assays.

Figure 3. Gal-8 induces CD4� T cell proliferation. (A) Splenocytesfrom NIHnu/nu mice or their euthymic counterparts were assayed. (B)Splenocytes from C57BL/6J mice depleted of B cells by anti-CD19-coated bead treatment (�2.5% residual B cells) were tested. (C) Gal-8-induced proliferation on purified T cells. Splenocytes from C57BL/6Jwere subjected to magnetic cell sorting using anti-CD4 or anti-CD8microbeads (Miltenyi Biotec), following the manufacturer’s instruc-tions for magnetic labeling and separation. Each group (5�105 cells)was incubated with the indicated amounts of Gal-8. (A and B) cpmcorresponding to Gal-8 plus TDG were discounted. Results are repre-sentative of three to five independent assays. LPS and Con A wereadded at 5 �g/ml as controls.


the conditions of this assay, and IL-12 showed no significantvariations in their transcription rates upon Gal-8 treatment.Identical results were obtained for every cytokine tested whensplenocytes were incubated in the presence of Gal-8 for ashorter period of 4 h (data not shown). As IFN-� is character-istic of the Th1 response and IL-4 of the Th2 response, weconcluded that Gal-8 is probably not associated with the activa-tion of either subset. Although other cells such as NKT arealso producers of these cytokines, their numbers (�1% of to-tal T cells) are relatively low, so these results, based on mRNAquantitation, cannot be ascribed to them [21]. These resultsare in agreement with recent findings obtained with the plantlectin Jacalin, which induces T cell activation through CD45with a similar cytokine profile [22], thus suggesting that thislectin might be partially mimicking the endogenous Gal-8 re-activity.

Identification of putative Gal-8 counter-receptorsMany glycosylated molecules at the cell surface may be ableto be recognized by Gal-8 and then transduce an intracellu-lar signal. Among them, CD43, a heavily O-glycosylated gly-coprotein that is highly expressed at the leukocyte surface,is one of the main receptors for Gal-1 implicated in T celldeath regulation by this lectin [23, 24]. Therefore, we won-dered whether CD43 was involved in the proliferation in-duced by Gal-8. No differences were observed in the prolif-eration among splenocytes obtained from B6.CD43null orwild-type mice cultured with Gal-8 (not shown), thus sug-gesting the involvement of other pathways. To screenbroadly for possible splenocyte receptors that interact withGal-8, an affinity-chromatography step followed by massspectrometry analysis was performed. Cell extracts were pre-pared from C57BL/6J mouse splenocytes, passed through aGal-8 affinity column, and eluted with lactose. A discretenumber of bands were observed when the lactose-elutedfraction was resolved in 10% SDS-PAGE (Fig. 5). The main

bands were analyzed by MALDI-TOF after in-gel trypsin di-gestion. In agreement with other authors [13–16], we iden-tified several leukocyte surface glycoproteins, including inte-grins and CD45, as counter-receptors (Fig. 5 and Table 1).Actin was also present, probably as a result of its binding tointegrins. CD45 are abundant and highly glycosylated pro-teins located at the surface of different cells from the hema-topoietic lineage, including lymphocytes. As a result of al-ternative splicing, multiple CD45 isoforms are identified ondifferent cell types depending on their stage of differentia-tion and/or activation [25–27]. The CD45R (also known asB220), found in B cells, together with the CD45RA presentin naive cells (Fig. 5, Band 1, MW�220 kD) and theCD45RO isoform (MW�180 kD) present in activated/mem-ory T cells (Fig. 5, Band 2) were identified in the screening.The reactivity of Gal-8 with the CD45R isoform in B cellswas confirmed by performing MALDI assays in preparationsof splenocytes from athymic nude mice (not shown). In ad-dition, several surface markers from macrophages, neutro-phils (such as sialoadhesin, macrophage mannose receptor,and Mac-1, CD177, and MMP-9), and platelets (Glycopro-tein IIb) were found. Interestingly, this last molecule hasbeen associated recently with the activation of platelets in-duced by Gal-1 [28].

Involvement of CD45 in Gal-8-induced proliferationCD45 is a trans-membrane PTPase, whose main targets arethe Src-kinases Lck and Fyn. Lck, in particular, is a primaryinitiator of signal transduction upon TCR engagement,whose absence leads to severe blocking in T cell differentia-tion and deep impairment of activation in mature T cells[29]. As we showed that Gal-8 is interacting with the extra-cellular domain of CD45, we wondered whether mecha-nisms acting downstream of CD45 activation are indeed in-volved in the proliferation of T cells upon Gal-8 engage-

Figure 4. Gal-8 induces the expression of IL-2, IFN-�, and IL-4.Splenocytes were cultured with Gal-8 alone or in combination withTDG, and a RT-MPCR was performed to determine transcripts for sev-eral ILs and GAPDH as control. The position of control bands is indi-cated on the left.

Figure 5. Identification of putative Gal-8 counter-receptors by MALDI-TOF. Splenocyte extracts were passed through Gal-8-Sepharose affinitycolumns and eluted with 100 mM lactose in PBS. Eluted material wassolved by 10% SDS-PAGE and numbered bands subjected to MALDI-TOF. For description of all identified molecules, see Table 1.



ment. To evaluate this hypothesis, we tested Gal-8-inducedproliferation in the presence of a specific inhibitor of theCD45 PTPase activity [19]. Splenocyte proliferation was pre-vented by the addition of 2 �M of the inhibitor and par-tially reduced when 0.5 �M was assayed (Fig. 6A). In theseexperiments, Gal-8 concentration ranged from 0.25 to 0.8�M. When Gal-8 was tested at higher concentrations (2�M), the effect of the PTPase inhibitor on proliferation wasreduced to only 30% (not shown). This might be a result ofthe fact that a strong cellular agglutination was attainedwith 2 �M Gal-8, which seems to be inducing another kindof cellular signaling in addition to CD45 and thus, not pre-vented by inhibiting its PTPase activity. Activation of theMAPK pathway in Jurkat T cells incubated in the presenceof Gal-8 was readily determined by pERK1/2 [15, 16]. Asshown in Figure 6B, we observed that the activation of thispathway by Gal-8 can be prevented by the addition of theCD45 PTPase inhibitor. We then tested the effect of the in-hibition of pERK1/2 in the proliferation induced by Gal-8on splenocytes. The addition of the U0126 inhibitor to theculture reduced proliferation significantly (Fig. 6C). Thesefindings support the involvement of the CD45 and MAPKpathways in the Gal-8-induced proliferation of T cells.

Costimulatory effect of Gal-8 on T cellsAs described above, Gal-8 was able to stimulate the prolifera-tion of T cells, an outcome prevented by CD45 phosphataseand pERK1/2 inhibition. As CD45 is a modulator of T cell ac-

tivation, these findings suggest that Gal-8 might be able to es-calate the TCR signaling, thus lowering the threshold of TCRactivation. To test this hypothesis, splenocytes from TCROVA

transgenic DO11.10 mice were stimulated for 48 h with subop-timal doses of Gal-8 and the cognate OVA323–339 peptide.When both stimuli were present simultaneously, a synergisticresponse was observed (Fig. 7A), thus supporting that Gal-8 isindeed providing a costimulatory signaling jointly with theTCR. Noticeably, even when higher doses of Gal-8, which aremitogenic per se, were tested, this costimulatory effect was alsoevidenced when cells were collected after 24 h of culture. Atthis time-point, naive splenocyte proliferation was not yet trig-gered fully (Fig. 7B). These results, together with those fromFigure 2, clearly indicate that Gal-8 is able to induce two dif-ferent activation pathways: one in the presence of the cognateantigen and an antigen-independent one. When assayed at 2�M, the Gal-8 costimulatory signal was absent at 24 h (Fig. 7B)and at 48 h (data not shown), perhaps as a result of a saturat-ing effect on the TCR-independent pathway. The tyrosine ki-nase ZAP70 is phosphorylated rapidly in response to TCR en-gagement and induces cell activation. As expected from thesefindings, pZAP70 was increased in cells receiving either stimu-lus (Fig. 7C). Moreover, the synergistic proliferative activity(Fig. 7B) and pZAP70 (Fig. 7C) were prevented by the inhibi-tion of the CD45 PTPase, thus supporting its involvement. Thepossible participation of the MAPK pathway in the costimula-tory property of Gal-8 was tested by addition of the U0126 in-hibitor to the culture. Like in the antigen-independent prolif-

TABLE 1. Gal-8 Protein Binders Identified by MALDI-TOF from Splenocytes

Banda Common name(s) IDMW

(kD) Function

1 PTPase, receptor type C (CD45 RA andB220 isoforms)

NP_035340 220 Lymphocyte activation

1 Sialoadhesin, (CD169, Siglec-1) AAB95641 193 �(2,3) Sialylconjugates binding. Presentin macrophages

2 PTPase, receptor type C (CD45 RO isoform) NP_035340 180 Lymphocyte activation2 Macrophage mannose receptor CAA78028 166 Binding of mannosylated molecules.

Receptor for several infectious agents3 Integrin �-L (CD11a) P24063 133 CD11a/CD18 (LFA-1) leukocyte

adhesion to endothelium; T cell-APC adhesion; T cell costimulation

3 Integrin �-M (CD11b) P05555 131 CD11b/CD18 (MAC-1) leukocyteadhesion and phagocytosis; cell-matrix adhesion; fibrinogen and iC3breceptor

4 Glycoprotein IIb AAF06996 114 GPIIb/IIIa (CD41/CD61) Plateletadhesion and aggregation

4 Neutrophil-specific antigen (CD177) AAH27283 90 PECAM-1 binder5 Integrin �-2 precursor (CD18) CAA33077 88 LFA-1 and MAC-1 complex formation5 Transferrin receptor AAH54522 85.7 Fe metabolism; growth factor5 MMP-9 NP_038627 81.5 Matrix proteolysis; migration6 Hydroacyl-Coenzyme A dehydrogeanase/

3.ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase � subunit

AAH58569 87 Trifunctional enzyme

6 Lactotransferrin AAH06904 80 Growth factor7 Actin CAA31455 41 Cytoskeletal anchorage

a Bands numbered as denoted in Figure 5. APC, antigen presenting cell


eration induced by Gal-8 (Fig. 6), we found that the inhibitionof pERK1/2 was able to prevent the proliferation inducedwhen the OVA peptide and the Gal-8 stimuli were included(Fig. 7D).

DISCUSSION

The ability of Gal to manipulate the immune system is receiv-ing strong interest. There is supportive evidence regardingGal-1 and its capacity of promoting apoptosis of T cells duringtheir development in the thymus as well as in the peripheryafter activation [2, 30–33]. Gal-2, structurally related to Gal-1,also promotes apoptosis of activated T cells [34]. Gal-3, theonly chimera-type Gal, promotes T cell apoptosis when exog-enously added [35, 36], and Gal-9, which belongs to the samegroup as Gal-8, has been shown to induce apoptosis of imma-ture thymocytes and of activated peripheral Th1 cells [4, 37].In another work, Gal-9 has been shown to induce apoptoticdeath in Jurkat T cells in contrast with Gal-8, which showed noapoptosis [38]. This is in agreement with the proliferative re-sponse and the absence of apoptosis induced by Gal-8 in pe-ripheral lymphocytes reported here. These data highlight fur-ther the differences observed in the induced cell behavioramong the different Gal, which in spite of their similar sugarrecognition [39], may induce different and even contrastingeffects on the immune cells.

The use of different lectin concentrations and culture peri-ods allowed us to discriminate between two different biologicalactivities for Gal-8 on peripheral T cells. The addition of Gal-8to cultures induced strong antigen-independent proliferationon T cells (Figs. 2, 3, and 6) and a costimulatory effect on agiven T cell response (Fig. 7). Although all populations in thespleen were targeted by Gal-8 (Fig. 2A), the proliferative effectcould be associated only with CD4� T cells by cell depletionand assays with athymic mice. No proliferation was observed inB cells, although the CD45R isoform was found as a counter-receptor of Gal-8 by MALDI assays performed with splenocytesfrom euthymic (Fig. 5 and Table 1) and athymic mice (notshown). CD4� T cells were stimulated by the Gal, and the in-creased expression of IL-2, IFN-�, and IL-4 suggests that Th1and Th2 populations were induced to proliferate (Fig. 4). Sev-eral integrins are targets of Gal-8 (see refs. [13–16] and Table1) and induce biological responses such as adhesion andspreading in Jurkat and other cells. Therefore, they may alsobe potentially responsible for the proliferative outcome re-ported here. In fact, stimulation of LFA-1, identified here as aGal-8 counter-receptor, induces IL-2 production in Jurkat cells[40]. CD45 was also identified as a Gal-8 counter-receptor byMALDI-TOF analysis, and inhibition of its PTPase activity abro-gated Gal-8-induced proliferation almost completely. Theseresults support that Gal-8 acts as a T cell-activating molecule byagonistic binding to CD45. Perhaps it is associated with theeffect exerted by CD45 reported recently, which lowers the Tcell activation threshold [41, 42]. In contrast, anti-CD45 anti-bodies did not prevent the apoptosis induced by Gal-9 [38].These data denote a biological function for Gal-8 on mature Tcells that distinguishes it from other Gal studied until now.Jacalin, a plant lectin that binds to CD45, induces the secre-

Figure 6. Involvement of CD45 in the induction of cell proliferationby Gal-8. (A) Inhibition of Gal-8-induced splenocyte proliferation.Splenocytes were incubated with the indicated amounts of Gal-8. TDG(30 mM) and CD45 PTPase inhibitor (Inh; 0.5 and 2 �M) were tested.*, P � 0.05; **, P � 0.01. Control cpm values corresponding to Gal-8plus TDG were discounted in all groups. No significant differenceswere found with basal cpm in cultures where only TDG or PTPase in-hibitor was added. (B) Gal-8-induced pERK1/2 in Jurkat T cells. (C)Inhibition of pERK prevents splenocyte proliferation. The U0126 in-hibitor was added at 10 �M 1 h before Gal-8. LPS and Con A wereused at 2.5 �g/ml and included as controls. LPS- or Con A-inducedproliferation alone was not inhibited by the CD45 PTPase inhibitoraddition (not shown). cpm values corresponding to U0126 alone weresimilar to basal cpm. *, P � 0.0002 ; **, P � 0.0004. Assays were per-formed at least three times.



tion of the same pattern of ILs and pERK1/2 as Gal-8 [22],suggesting that this lectin might be mimicking the endoge-nous Gal-8 partially.

Gal-8 was able to synergize the TCR-specific signaling, thusenhancing a specific T cell response induced in the presenceof a low dose of the cognate peptide (Fig. 7A). This ability wasdisclosed when the lectin was assayed at such low doses that itwas unable to induce the polyclonal proliferation. As the lectindose increases, the differential effects between costimulationand polyclonal proliferation become overlapped as a result ofthe strong proliferation induced by Gal-8 in the absence ofantigen (Fig. 2). However, the costimulatory effect was ob-served readily when cultures were collected earlier at 24 h ofstimulation instead of at 48 h (Fig. 7, A vs. B). In these assays,the antigen-independent stimulation showed a delay in theonset, which allowed us to discriminate both activities, indicat-ing that other activation pathways different from the fast TCRsignaling are involved in that process. When high Gal-8 dosessuch as 2 �M were tested, no costimulatory activity was ob-served (Fig. 7B); this might be ascribed to the observed agglu-tination of the cells that precluded the proper antigen presen-tation. This absence of costimulation is in fact supporting theactivation of independent but synergizing signaling pathways,

as at this high dose, the proliferative capacity of Gal-8 was ob-served readily (Fig. 2). In agreement, CD45 PTPase inhibitionprevented the proliferation only partially (not shown), andpERK1/2 was fully abrogated by TDG but reduced only par-tially by CD45 PTPase inhibition in Jurkat cells treated withGal-8 (Fig. 6). The costimulatory activity was observed at lowerGal-8 concentrations (Fig. 7A), thus suggesting that might bethe primary in vivo activity of this lectin. This costimulatoryeffect was prevented by the addition of the CD45 PTPase in-hibitor, probably as Gal-8 triggered the TCR pathway by work-ing as an endogenous agonist for the extracellular domain ofCD45 [41, 42]. In agreement, pZAP70 was demonstratedreadily with Gal-8 at levels similar to those observed with thecognate peptide stimulus (Fig. 7C). These findings allow us topostulate that in vivo, this lectin might help trigger some oth-erwise borderline signals. Activities reported here help to un-derstand the opposite outcome observed on thymocytes, whereeven when binding to all populations, Gal-8 induces apoptoticdeath only in the CD4highCD8high cells [7]. The binding of theTCR with high affinity is an event that in the thymus, mostprobably leads to apoptosis, whereas in the periphery, leads tocell activation. This might be mimicked by the activation ofthe CD45 PTPase, which is known to lower the TCR signaling

Figure 7. Costimulatory activity ofGal-8. (A) Splenocytes (1.5�105 nucle-ated cells) from DO11.10 mice werecultured for 48 h in the presence of thecognate OVA323–339 peptide (OVA;0.4–1 �g/ml) together with a lowamount of Gal-8 (0.1 �M). Assays wereperformed at least three times. *, P �0.01. (B) Same as before, but higherconcentrations of Gal-8 were tested andcultures collected at 24 h. When Gal-8was assayed at 0.5 or 1 �M, the OVA(black bars) versus OVA � inhibitor(dark gray bars) groups: P � 0.05. Nosignificant differences were found be-tween Gal-8 2 �M groups. (C) pZAP70.The experiment was performed as pre-viously, but 5 � 106 cells were incu-bated for 1 h. Cells were collected andprocessed for Western blotting. (D)Splenocytes from DO11.10 mice werepreincubated with 10 �M of the ERKinhibitor U0126 for 1 h before the addi-tion of Gal-8 and/or OVA. Experimen-tal conditions are the same as in A. *, P� 0.0002.


threshold and to exacerbate the response [41, 42]. Therefore,the external stimulation of this molecule by Gal-8 will urge Tcells to proliferate and to induce the double-positive thymo-cyte apoptosis reported previously [7]. In support, Gal-1 hasbeen involved recently in agonistically signaling in thymic se-lection and in peripheral activation of CD8� T cells [43].

Gal-8 is expressed abundantly in many tissues [13], includ-ing the injured endothelium [44], and has been shown re-cently to reach high concentrations (estimated at 25–65 nM)in the inflamed sinovia [45], which are close to those usedhere in vitro. Although Gal-8 was postulated to exert an apo-ptotic effect on inflammatory sinovial cells, this is based on theincrease in Annexin-V binding [45], an event described morerecently as not necessarily associated with cell death induction[38, 46, 47]. Interestingly, the exposure of phosphatidylserineand Ca2� uptake in Jurkat T cells is observed at high concen-trations of Gal-8 but in the absence of DNA fragmentation[38]. Instead, exposure of phosphatidylserine is related to cellactivation by this lectin [47], thus supporting the proliferativeactivity reported here as probably involved in inflammatoryoutcomes. In agreement with this, Gal-4 is able to stimulateCD4� T cells to activate and exacerbate inflammatory boweldisease [48]. In addition, several markers from inflammatorycells such as neutrophils (Mac-1 and MMP-9 reported previ-ously as counter-receptors for Gal-8 [17] and the CD177 anti-gen) as well as macrophages and platelets were also identifiedas Gal-8 counter-receptors (Table 1). Therefore, Gal-8 seemsto be involved in the migration or activation of these inflam-matory cells, which together with the T cell-proliferative activ-ity reported here, supports a fueling role in the pathogenesisof inflammatory diseases. Importantly, Gal-8 was also able tocostimulate T cells, even at low concentrations, an activity thatmight be functional, not only under normal conditions butalso in altered responses such as in the induction of autoim-munity where the lowering of the threshold of T cell activationseems crucial.

ACKNOWLEDGMENTS

This work was supported by grants from the Agencia Nacionalde Promocion de la Ciencia y la Tecnologıa (ANPCyT), Uni-versidad Nacional de San Martın, and Consejo Nacional deInvestigaciones Cientıficas y Tecnicas (CONICET; Argentina).M. V. T. is a Fellow, and J. M. and O. C. are researchers fromCONICET. V. C. is a Fellow from ANPCyT. We thank Dr.Yehiel Zick (The Weizmann Institute of Science) for providingus with an aliquot of the mouse anti-rat Gal-8 mAb and Dr.Anne I. Sperling (University of Chicago) for providing us withthe B6.CD43null mice. We are indebted to Dr. Carlos Buscagliafor his critical reading of the manuscript. The technical assis-tance of Mr. Fabio Fraga in animal care is appreciated.

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KEY WORDS:cell surface molecules � cell activation � CD45 � integrins


Galectin-8 provides costimulatory and proliferative signals to T lymphocytes

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