of June 10, 2018.This information is current as

and Associates with Receptor PhosphatasesSynapse, Disrupts Stable Synapse Formation,Protein 3 Is Recruited to the Immune

Containing−T Cell Ig and Mucin Domain

Ndhlovu and Mario A. OstrowskiDouglas-Vail, Shariq Mujib, Glen M. Chew, Lishomwa C. Kiera L. Clayton, Matthew S. Haaland, Matthew B.

http://www.jimmunol.org/content/192/2/782doi: 10.4049/jimmunol.1302663December 2013;

2014; 192:782-791; Prepublished online 13J Immunol 

MaterialSupplementary

3.DC1http://www.jimmunol.org/content/suppl/2013/12/13/jimmunol.130266

Referenceshttp://www.jimmunol.org/content/192/2/782.full#ref-list-1

, 17 of which you can access for free at: cites 48 articlesThis article

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The Journal of Immunology

T Cell Ig and Mucin Domain–Containing Protein 3 IsRecruited to the Immune Synapse, Disrupts Stable SynapseFormation, and Associates with Receptor Phosphatases

Kiera L. Clayton,* Matthew S. Haaland,* Matthew B. Douglas-Vail,* Shariq Mujib,†

Glen M. Chew,‡ Lishomwa C. Ndhlovu,‡ and Mario A. Ostrowski*,x,{,‖

CD8+ CTLs are adept at killing virally infected cells and cancer cells and releasing cytokines (e.g., IFN-g) to aid this response.

However, during cancer and chronic viral infections, such as with HIV, this CTL response is progressively impaired due to a process

called T cell exhaustion. Previous work has shown that the glycoprotein T cell Ig and mucin domain–containing protein 3 (Tim-3)

plays a functional role in establishing T cell exhaustion. Tim-3 is highly upregulated on virus and tumor Ag-specific CD8+ T cells, and

antagonizing Tim-3 helps restore function of CD8+ T cells. However, very little is known of how Tim-3 signals in CTLs. In this study,

we assessed the role of Tim-3 at the immunological synapse as well as its interaction with proximal TCR signaling molecules in

primary human CD8+ T cells. Tim-3 was found within CD8+ T cell lipid rafts at the immunological synapse. Blocking Tim-3 resulted

in a significantly greater number of stable synapses being formed between Tim-3hiCD8+ T cells and target cells, suggesting that Tim-3

plays a functional role in synapse formation. Further, we confirmed that Tim-3 interacts with Lck, but not the phospho-active form of

Lck. Finally, Tim-3 colocalizes with receptor phosphatases CD45 and CD148, an interaction that is enhanced in the presence of the

Tim-3 ligand, galectin-9. Thus, Tim-3 interacts with multiple signaling molecules at the immunological synapse, and characterizing

these interactions could aid in the development of therapeutics to restore Tim-3–mediated immune dysfunction. The Journal of

Immunology, 2014, 192: 782–791.

The immune system has developed multiple mechanismsto limit T cell responses to self-proteins to prevent auto-immunity. However, these mechanisms also limit T cell

responses to cancer Ags and chronic infection (1, 2). Duringchronic infections, such as HIV type 1 and hepatitis C virus, CD8+

T cells become exhausted, causing CD8+ T cells to lose theireffector function. Loss of production of IL-2 and TNF-a charac-terizes early exhaustion, whereas the production of IFN-g isusually maintained until late-stage exhaustion (3–5). Increasedexpression of multiple negative, coinhibitory checkpoints, in-cluding programmed death receptor 1 (PD-1), has been associatedwith the exhausted phenotype. In addition, antagonizing thesereceptors with Abs or soluble fusion proteins results in partialrescue of effector function (6). We have previously shown that

T cell Ig and mucin domain–containing protein 3 (Tim-3) is highlyexpressed on exhausted HIV-specific CD8+ T cells (4). Similar to

the other coinhibitory receptors, Tim-3 blockade partially rescues

the proliferation, cytokine production, and cytotoxicity of virus-

specific CD8+ T cells, suggesting that Tim-3 plays a functional

role in T cell exhaustion (4, 7). However, unlike PD-1, Tim-3 is

relatively uncharacterized in terms of how it manipulates the cell

to dampen T cell responses.Human Tim-3 is a type I transmembrane protein with extra-

cellular Ig V-like and mucin domains with two N- and oneO-linked

glycosylation sites (8). Tim-3 is expressed at low levels on naive

CD8+ T cells, Th1 and Th17 cells, and regulatory CD4 T cells,

increased on activated CD8+ T cells, and constitutively expressed

on NK cells, dendritic cells, monocytes, and macrophages (4, 9–

15). Known ligands for murine Tim-3 include phosphatidylserine

(16) and galectin-9 (17). The interaction between galectin-9 and

Tim-3 is carbohydrate dependent (17), and as such, galectin-9’s

carbohydrate-binding, lectin properties suggest that it may also

interact or cointeract with other surface glycoproteins including

CD44 (18) and integrins (19), allowing association with Tim-3.Currently, the galectin-9–induced Tim-3 signaling cascade is

unknown. We and others have shown that exhausted Tim-3hi CD8+

T cells respond more efficiently to TCR stimulation when the

Tim-3 pathway is blocked, suggesting that Tim-3 engagement

antagonizes TCR signaling pathways (4, 5, 7, 20–23). Previous

studies have investigated Tim-3 signaling in artificial systems such

as cell lines and transfection systems. Tyrosine phosphorylation

of the Tim-3 cytoplasmic tail and enhancement of this phosphor-

ylation with addition of galectin-9 have been reported in epithelial

cell lines (24, 25). In addition, Tim-3 was shown to bind to Fyn,

p85 (the PI3K adaptor), and Lck (26, 27), further suggesting a role

for Tim-3 in TCR proximal signaling. Finally, Tim-3 expression

was shown to suppress NFAT dephosphorylation and AP-1 tran-

*Department of Immunology, University of Toronto, Toronto, Ontario M5S 1A8,Canada; †Institute of Medical Sciences, University of Toronto, Toronto, OntarioM5S 1A8, Canada; ‡Hawaii Center for AIDS, Department of Tropical Medicine,John A. Burns School of Medicine, University of Hawai‘i, Honolulu, HI 96813;xClinical Sciences Division, University of Toronto, Toronto, Ontario M5S 1A8,Canada; {Maple Leaf Clinic, Toronto, Ontario M5B 1L6, Canada; and ‖Li Ka ShingKnowledge Institute, St. Michael’s Hospital, Toronto, Ontario M5B 1W8, Canada

Received for publication October 1, 2013. Accepted for publication November 13,2013.

This work was supported by a grant from the Canadian Institutes for Health Re-search. K.L.C. and M.A.O. received salary support from the Ontario HIV TreatmentNetwork.

Address correspondence and reprint requests to Dr. Mario A. Ostrowski, Room 6352,Medical Sciences Building, University of Toronto, 1 King’s College Circle, Toronto,ON M5S 1A8, Canada. E-mail address: mario.ostrowski@gmail.com

The online version of this article contains supplemental material.

Abbreviations used in this article: PD-1, programmed death receptor 1; SEB, Staphy-lococcal enterotoxin B; SH2, Src homology 2; sTim-3, soluble T cell Ig and mucindomain–containing protein 3; Tim-3, T cell Ig and mucin domain–containing protein 3.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302663

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scription (28). However, these reports did not study Tim-3 in thecontext of intact TCR signaling with concurrent galectin-9 en-gagement. In addition, the pathway still remains to be studied inprimary human CD8+ T cells. In this study, we characterized theinteraction between Tim-3 on primary human CD8+ T cells andearly signaling events, which may lead to the exhausted phenotypeof CD8+ T cells found in association with chronic viral infectionor cancer.

Materials and MethodsStudy participants

Healthy HIV-seronegative human volunteers were recruited for bloodspecimens obtained via leukophoresis. PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare Biosciences, Uppsala, Sweden). Informedconsent was obtained in accordance with the guidelines for conduction ofclinical research at the University of Toronto and St. Michael’s Hospitalinstitutional ethics boards.

T cell isolation, culture, and activation

CD8+ T cells were isolated using EasySep Human CD8+ T Cell NegativeEnrichment Kit (StemCell Technologies, Vancouver, BC, Canada). Cellsachieved a purity of at least 95%, assessed via flow cytometry. IsolatedCD8+ T cells were cultured in R-10 medium (RPMI 1640, 10% FBS, 1 U/mlpenicillin, 100 mg/ml streptomycin, 2 mM glutamine; Wisent, Saint-Bruno,QC, Canada) and stimulated with 1 mg/ml LEAF-purified anti-CD3 mAb(clone OKT3; BioLegend, San Diego, CA) and 1 mg/ml LEAF-purifiedanti-CD28 mAb (clone 28.8; BioLegend) with 50 U/ml rIL-2 (NationalInstitutes of Health, Bethesda, MD) for 5–7 d to upregulate Tim-3 (29).Cells were then washed three times in R-10 and rested overnight in R-10with no additional stimulation. For Tim-3 blocking experiments, cells werewashed in R-10 medium and treated with 10 mg/ml LEAF-purified anti–Tim-3 antagonistic mAb (clone 2E2; BioLegend) or 10 mg/ml LEAF-purified isotype control Ab (BioLegend) overnight without additionalstimulation. EBV-transformed patient B cells were cultured in R-10 for upto 20 passages.

Lipid raft isolation

A total of 5 3 106 of rested Tim-3hi CD8+ T cells were harvested andresuspended in 20 ml TBS. The cells were lysed in 250 ml 0.5% Brij-58TKM Lysis Buffer (50 mM Tris-HCl [pH 7.4], 25 mM KCl, 5 mM MgCl2,1 mM EDTA, 1 mM NaVO4, and 5 mM NaF with 13 Roche CompleteMini EDTA-free Protease Inhibitor) for 30 min at 4˚C and mixed withequal parts of 80% sucrose in TKM Buffer. The 36% sucrose in TKMBuffer (4.3 ml) was layered on top of the 40% sucrose cell lysate solutionand subsequently layered with 5% sucrose in TKM Buffer (0.2 ml). Thesamples were ultracentrifuged at 50,000 rpm for 16 h at 4˚C in a SW Ti-55rotor (Beckman Coulter, Brea, CA) to isolate lipid rafts, which float to thetop of the gradient (fraction 1), whereas nonlipid raft membrane shouldremain at the bottom of the gradient (fraction 10). The 0.5-ml fractionswere collected from each 5-ml gradient. To assess the presence of lipidrafts, 100 ml each fraction was blotted onto nitrocellulose using a dot blotapparatus and probed with a 1:3000 dilution of cholera toxin–HRP (Sigma-Aldrich, Germany) to detect GM-1, a lipid raft–resident ganglioside.

Soluble Tim-3 staining and flow cytometry

Soluble Tim-3 (sTim-3) was produced and purified as previously described(4) and biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation Kit(Thermo Scientific, Rockford, IL). B cells were washed twice in 2% FBS/PBS and incubated with varying amounts of biotinylated sTim-3 per 3 3105 cells for 30 min at 4˚C with or without a-lactose followed by stainingwith streptavidin-APC (Invitrogen, Carlsbad, CA). B cells were alsostained with Galectin-9–PE (BioLegend). Expression of Tim-3 on acti-vated CD8+ T cells was assessed via staining with anti-CD8 and anti–Tim-3–PE (R&D Systems, Minneapolis, MN; catalog number FAB2365P).Data were acquired on a BD FACSCalibur (BD Biosciences) and analyzedon FlowJo software (Tree Star).

Conjugate formation and immunofluorescent staining

B cell–T cell conjugates were prepared as described in (30) with somemodifications. Briefly, B cells were labeled with Cell Tracker Blue CMAC(Invitrogen) and incubated with 2 mg/ml Staphylococcal enterotoxin B(SEB; Sigma-Aldrich) for 30 min at 37˚C. Rested or Tim-3–blocked Tim-3hi CD8+ T cells were mixed 2:1 with the B cells and spun at 200 3 g for

5 min, gently resuspended, and further incubated for 10 min at 37˚C. Thecells were placed onto poly-L-lysine–coated coverslips and allowed toadhere for 15 min. The coverslips were gently rinsed with PBS and fixedwith 4% methanol-free formaldehyde for 15 min at 37˚C. Cells werewashed with PBS and then permeabilized using 0.2% Triton X-100 for5 min at room temperature then washed with PBS. Blocking was performedusing 10% BSA for 1 h at room temperature followed by primary Abincubation overnight at 4˚C using combinations of anti–Tim-3 (R&DSystems; catalog number AF2365), anti-CD3 (Abcam, Cambridge, U.K.;catalog number ab5690), anti–phospho-ERK1/2 (R&D Systems; catalognumber MAB1018), phospho-Lck Y394 (R&D Systems; catalog numberMAB7500), anti-CD45 (Abcam; catalog number ab33533), and anti-CD148 (Abcam; catalog number ab140236) Abs. Following subsequentwashes in PBS, the coverslips were then incubated with appropriate sec-ondary Ab mixes for 45 min at 37˚C. Coverslips were mounted on slidesusing N-propyl gallate mounting buffer and sealed.

Microscopy and synapse counting

The slides were analyzed using a Zeiss LSM700 Confocal Microscope (CarlZeiss) with a 633 oil immersion objective. Images of T cells, whichformed an immunological synapse, were analyzed using LSM Zen LE2011 Acquisition Software. For semiquantification of stable synapses,slides were analyzed using an epifluorescent microscope with 603 oilimmersion objective. Representative pictures were taken and analysis wasperformed using ImageJ software (National Institutes of Health). Pictureswere taken of multiple fields of view, and 25 pairs of T cell–B cell con-jugates were counted per slide. Stable synapses were defined as a T cell–B cell conjugate with concentrated CD3 at the interface between the twocells, and the T cell was phospho-ERK+. Unstable synapses were definedas a T cell–B cell conjugate in which CD3 was not concentrated at theinterface between the two cells or the T cell was not phospho-ERK+. Onlyconjugates with a Tim-3+ T cell were included in the counts. The numberof stable synapses was blindly counted per 25 pairs of T cell–B cellconjugates.

Immunoprecipitations and Western blot analysis

A total of 1.5 3 107 rested Tim-3hi CD8+ T cells were washed in PBS,resuspended in 1 ml serum-free media, and then incubated at 37˚C for10 min. Cells were treated with or without 1 mM pervanadate for 5 min atroom temperature and then lysed in 0.5 ml Nonidet P-40 Lysis Buffer(Invitrogen) at 4˚C for 30 min. Cleared lysate was incubated with isotypeor Tim-3 Ab (R&D Systems; catalog number AF2365) coupled Protein-GDynabeads (Invitrogen) for 3 h at 4˚C. Beads were washed five times with1 ml ice-cold lysis buffer, and bound protein was eluted in LDS samplebuffer with 50 mM DTT (Invitrogen). For anti-CD3 immunoprecipitations,2 3 106 rested Tim-3hi CD8+ T cells were pretreated with anti-CD3 Ab-coupled Dynabeads (Invitrogen) for 30 min followed by addition of bufferor in-house produced recombinant galectin-9 for 1 h. The cells were lysedin 0.5% Brij-58 TKM lysis buffer, and anti-CD3 beads were collected.CD3-immunoprecipitated protein was eluted in LDS sample buffer with50 mM DTT (Invitrogen). Samples were prepared in LDS sample bufferand 50 mM DTT as per the manufacturer’s instructions and analyzed via4–12% Bis-Tris SDS-PAGE with subsequent Western blotting. Membraneswere probed using anti-phosphotyrosine (EMD Millipore, Billerica, MA;catalog number 05-321), anti–Tim-3 (R&D Systems; catalog numberAF2365), anti-Lck (Abcam; catalog number ab3885), anti-CD45 (Abcam;catalog number ab33533), anti-CD148 (R&D Systems; catalog numberAF1934), and anti-CD3 (Abcam; catalog number ab5690) Abs followedby the appropriate L-chain–specific secondary Abs (Jackson Immuno-Research Laboratories, West Grove, PA). Detection was performed usingchemiluminescence (Thermo Scientific).

Production and purification of human galectin-9

The medium isoform of human galectin-9 (UNIPROT ID: O00182-2) witha C-terminal 3XFLAG tag was codon optimized and subcloned into apET15b vector. Following expression in ArcticExpress (DE3) competentcells (Agilent Technologies, Palo Alto, CA), the cells were induced with0.5 mM isopropyl b-D-thiogalactoside and incubated at 12˚C for 24 h. Cellswere harvested and lysed using B-PER protein extraction reagent (ThermoScientific) as per the manufacturer’s instructions. The lysate was clearedvia centrifugation at 25,000 3 g for 30 min at 4˚C. Functional galectin-9was isolated using a-lactose agarose (Sigma-Aldrich) and eluted using200 mM a-lactose. Polishing purification was performed using a Superdex75 (GE Healthcare, Buckinghamshire, U.K.) via AKTA HPLC. A purity.90% was obtained (assessed via SDS-PAGE and Coomassie and silverstaining analysis), and endotoxin was ,1 EU/mg protein. Identity of the

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protein was confirmed via galectin-9 Western blot (R&D Systems; catalognumber AF2045).

Surface plasmon resonance

The binding of recombinant human galectin-9 to recombinant humansTim-3 was tested using a Biacore X instrument (GE Healthcare). A CM5 chipwas coupled with 1.82 ng sTim-3 at pH 4.5 (equivalent to 1820 responseunits). A 2-fold serial dilution of recombinant human galectin-9, from 563to 18 nM, was injected over the sTim-3 surface, and the change in responseunits was measured.

Galectin-9 affinity precipitation and mass spectrometryanalysis

A total of 10 mg in-house–produced recombinant galectin-9 was immo-bilized onto magnetic anti-FLAG beads (Sigma-Aldrich) and incubatedwith RIPA (Cell Signaling Technology, Beverly, MA) lysate prepared from53 106 rested Tim-3hi CD8+ T cells for 3 h at 4˚C. Following collection ofthe flow-through and subsequent washing with lysis buffer, bound proteinswere first eluted with 200 mM a-lactose in lysis buffer followed bya subsequent denaturing elution in LDS sample buffer with 50 mM DTT.Lysate, flow-throughs, and elutions were analyzed via SDS-PAGE andWestern blotting or in-gel silver staining (Sigma-Aldrich). Silver-stainedbands were excised from the gel and sent for in-gel tryptic digestionand identification via liquid chromatography-tandem mass spectrometry.Analysis of peptide hits was performed using Scaffold 4 software.

ResultsTim-3 is a lipid raft–resident protein and found at theimmunological synapse

TCR signaling predominantly occurs within distinct membranemicrodomains called lipid rafts (31, 32). Productive TCR signalingrequires shuttling of essential proximal signaling molecules inand out of the rafts during initial interaction with the target cell(33). We tested whether Tim-3 could be found within lipid raftsof CD8+ T cells. To do this, primary human CD8+ T cells wereobtained from peripheral blood of normal human volunteers, andstimulated in culture for 5–7 d with anti-CD3, anti-CD28, and IL-2to upregulate Tim-3 as previously described in Mujib et al. (29).Following in vitro stimulation, the cells were washed three timesto remove residual Abs and IL-2, and the cells were rested over-night in plain medium to dampen any residual proximal signaling.The representative flow cytometry plots in Fig. 1A confirm Tim-3expression on the CD8+ T cells used in all assays. Assessment ofTim-39s membrane location in these primary rested Tim-3hi CD8+

T cells using sucrose density gradient purification revealed thepresence of a large amount of Tim-3 in lipid rafts (Fig. 1B).Approximately 40% of total cellular Tim-3 was found in the firstfraction collected from the sucrose gradient, which contained∼87% of GM-1, a lipid raft ganglioside. Lipid raft clustering isrequired for immunological synapse assembly (34), which pro-vided us with a rationale to further investigate the role of Tim-3 atthe immunological synapse.Formation of an immunological synapse requires a T cell in

conjugate with a target cell to provide a TCR stimulus and sur-face ligands/receptors on both cells to coordinate multiplecostimulatory/inhibitory and immune accessory molecules (35,36). In order to assess for immunological synapse formation, weused and modified a previous system (30) in which effector CD8+

T cells are coincubated with B cell lines as APCs that are pulsedwith SEB as the Ag. To confirm the presence of endogenousTim-3 ligand expressed on APCs, we employed a biotinylatedrecombinant sTim-3 with labeled streptavidin secondary to stainour target cells (human B cell line). The B cells bound labeledsTim-3, indicating the presence of the Tim-3 ligand on their sur-face. In addition, the cells stained for an Ab to galectin-9, a Tim-3ligand (Supplemental Fig. 1A, 1B), indicating that surface asso-ciated galectin-9 was present on these cells. To confirm that

galectin-9 was acting as the ligand, we treated the cells withsTim-3 in the presence of a-lactose, a galectin-9 substrate. This sig-nificantly inhibited the binding of the sTim-3 (Supplemental Fig.1B), suggesting that galectin-9 was a major ligand for Tim-3 onthese cells. However, a-lactose treatment of B cells did notcompletely abrogate sTim-3 binding, indicating that either therewere noncarbohydrate-dependent ligands for Tim-3 on theseB cells, as previously described (37), or the affinity of galectin-9for a-lactose was less than that for Tim-3. Thus, we have con-firmed that in the CTL/B cell line system that we will be using tomodel TCR stimulation, the requisite receptors and ligands arepresent to allow for simultaneous Tim-3 engagement.Following conjugate formation with rested Tim-3hi CD8+ T cells

and the target (i.e., SEB-loaded B cells), the cells were stainedfor CD3 (as a marker for the central supramolecular activationcluster) and Tim-3 and then analyzed via confocal microscopy.As shown in Fig. 1C, Tim-3 was recruited to the immunologicalsynapse, suggesting a role for Tim-3 in proximal TCR signaling.Similarly, PD-1 has also been found at the immunological synapseand negatively effects synapse stability, as defined by concentratedand centered CD3 at the T cell synapse (38). Using our conjugate-forming assay, we assessed whether blocking Tim-3 had an effecton synapse stability. Using epifluorescence microscopy, we de-fined stable synapses as conjugates with concentrated CD3 at theinterface between the T cell and APC. In addition, to increase thestringency of our approach, we included phospho-ERK signaling,which results from an integration of signals from both the TCRand activated LFA-1 (39), as an additional parameter to definea stable synapse, which is capable of eliciting downstream sig-naling. Representative pictures of a stable and unstable synapseare shown in Fig. 2A. Blocking Tim-3 overnight on resting Tim-3hi CD8+ T cells using the antagonistic Tim-3 mAb (10) signifi-cantly increased the number of stable synapses formed betweenT cells and their SEB-pulsed targets to 54(6 3.8)% compared with32(6 2.8)% for the isotype control (Fig. 2B). This suggests thatTim-3 signaling plays a role in stable synapse formation.

Tim-3 can be phosphorylated and associates with Lck

Given that Tim-3 is localized in lipid rafts, which are crucial toimmunological synapse formation, and that Tim-3 is located at theimmunological synapse, we postulated that Tim-3 plays a role inproximal signaling events. Proximal signaling is regulated, in part,by tyrosine phosphorylation. Although previous studies haveshown that Tim-3 can be phosphorylated in epithelial cell lines andis enhanced by addition of galectin-9 (24, 25), it is still undeter-mined if this is the case in primary CD8+ T cells. Althoughattempts were made to determine Tim-3’s phosphorylation statuswith ligand treatment, we found that the addition of galectin-9rendered Tim-3 insoluble even in harsh RIPA buffer, which hin-dered subsequent Tim-3 immunoprecipitation (data not shown). Inaddition, we could not detect Tim-3 phosphorylation during TCRstimulation, with or without galectin-9 (data not shown). However,it is possible that only a very small fraction of Tim-3 is beingphosphorylated during TCR stimulation. To assess the potentialfor Tim-3 phosphorylation, we immunoprecipitated Tim-3 fromrested Tim-3hi CD8+ T cells treated with or without pervanadate,a potent phosphatase inhibitor, and analyzed the elutions viaWestern blot. Under these conditions, Tim-3 was found to containphosphorylated tyrosine motifs (Fig. 3A), further suggesting Tim-39s involvement in proximal signaling. Probing for additionalproximal signaling molecules revealed that Tim-3 coimmunopre-cipitated with Lck, confirming previous findings (27), but thisbinding, however, was not dependent upon Tim-3 phosphorylation(lane 1, Fig. 3A). In addition, assessment of conjugates between

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Tim-3hi CD8+ T cells and SEB-loaded B cells revealed that Tim-3colocalized minimally, if not at all, with the active form of Lck(p-Y394) at the immunological synapse (Fig. 3B, top panel). Inter-estingly, large, concentrated areas of active phospho-Lck appearedto exclude Tim-3 (Fig. 3B, bottom panel). This suggests the pos-sibility that Tim-3 may be recruiting a phosphatase to dampen Lckphosphorylation and subsequent activity.

Galectin-9 serves as the canonical human ligand for Tim-3 andreceptor phosphatases in T cells

A previous study has demonstrated that mouse Tim-3 binds tomouse galectin-9 (17); however, the data are unresolved on thisinteraction in humans. To assess the binding of human Tim-3 tohuman galectin-9, we first produced and purified a FLAG-taggedrecombinant human galectin-9 (Supplemental Fig. 2A). Next, weperformed two independent surface plasmon resonance experi-

ments to show that immobilized recombinant human Tim-3 wasable to bind to recombinant human galectin-9 (Supplemental Fig.2B). Results were comparable using our in-house producedrecombinant human galectin-9 and human soluble Tim-3 andrecombinant proteins obtained from R&D Systems (data notshown). However, because the interaction between galectin-9 andTim-3 is carbohydrate dependent, endogenous, surface-expressedTim-3 might have a different glycosylation state, which mayprevent it from binding to galectin-9. We performed a galectin-9affinity precipitation to determine if our recombinant humangalectin-9 was able to precipitate endogenous Tim-3 from Tim-3hi

CD8+ T cells. Galectin-9 bound to endogenous Tim-3 in a carbo-hydrate-dependent manner, as shown by the presence of Tim-3 inthe a-lactose elution (Fig. 4A, lane 4). Following the a-lactoseelution, any remaining proteins were removed via boiling/denaturing elution (Fig. 4A, lane 5). A large portion of Tim-3

FIGURE 1. Tim-3 is found in lipid

rafts and is recruited to the immuno-

logical synapse. (A) Tim-3 expression

on in vitro–activated CD8+ T cells. Iso-

lated CD8+ T cells from whole PBMCs

were activated in vitro for 5–7 d then

washed, rested overnight in plain me-

dium, and Tim-3 expression was assessed

via flow cytometry. (B) Rested Tim-3hi

CD8+ T cells were lysed and subjected to

sucrose gradient centrifugation followed

by fraction collection to isolate lipid

rafts. Fractions were subjected to dot

blot analysis using cholera toxin–HRP

to detect lipid raft–resident protein

GM-1 or Tim-3 using Western blot

(WB). Fraction 1 contains the lipid rafts.

Densitometry analysis using ImageJ

(National Institutes of Health) was used

to determine the fraction of total Tim-3

and GM-1 in fractions 1–3 and 8–10. (C)

Rested Tim-3hi CD8+ T cells were used

to form conjugates with Cell-Tracker Blue–

stained, SEB-loaded B cells followed by

indirect immunofluorescent staining

for CD3 (magenta, marker of the central

supramolecular activation cluster) with

Tim-3 (green) and analyzed via confocal

microscopy.White indicates colocalization.

Original magnification 3630. Figure is

representative of five experiments. FSC,

Forward light scatter; SSC, side scatter.

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was found in the denaturing elution, indicating that either the 200mM a-lactose elution could not completely disrupt the interactionbetween Tim-3 and galectin-9 or that Tim-3 is also able to bind togalectin-9 in a noncarbohydrate-dependent manner. In order todetermine other binding partners of galectin-9, besides Tim-3,the a-lactose elutions were analyzed via SDS-PAGE and silverstaining. As shown in Fig. 4B, multiple proteins bound to galectin-9in a carbohydrate-specific manner. Mass spectrometry analysisidentified most of these proteins as immune accessory molecules,including the phosphatases CD45 and CD148 (Fig. 4C). Thissuggests that although galectin-9 is the ligand for Tim-3, it is ableto coordinate multiple surface receptors, including receptorphosphatases.

Galectin-9 enhances the interaction between Tim-3 and CD45

Because galectin-9 has two carbohydrate recognition domainsjoined by a flexible linker, it is possible that galectin-9 maycolocalize Tim-3 with CD45 or CD148, allowing Tim-3 to asso-ciate with a phosphatase in a ligand-dependent manner. Assessmentof conjugates between Tim-3hi CD8+ T cells and SEB-loadedB cells showed that Tim-3 colocalized with CD45 and CD148at the immunological synapse formed between Tim-3hi CD8+

T cells and Tim-3 ligand–expressing APCs (Fig. 5A). Interest-ingly, addition of recombinant galectin-9 to Tim-3hi CD8+ T cellsduring CD3 engagement resulted in accumulation of Tim-3,

CD45, and CD148 within CD3 signaling complexes, as shown byCD3 immunoprecipitation and subsequent Western blotting (Fig.5B). These data suggest that Tim-3 may mediate its inhibitoryfunction through the recruitment of the receptor phosphatases tothe synapse via its ligand, galectin-9.

DiscussionOur study provides novel insight into the Tim-3 receptor coordi-nation during immunological synapse formation and involvementin proximal TCR signaling. In this study, we show that human Tim-3is found in lipid rafts and recruited to the immunological synapse.Further, Tim-3 may be involved in the process of stable synapseformation, suggesting a role for Tim-3 in proximal signaling. Tim-3was found to have the potential for tyrosine phosphorylationin primary CD8+ T cells and bind to Lck, further confirming thefinding by Rangachari et al. (27) that Tim-3 associates with thisessential proximal signaling molecule. However, Tim-3 was notfound to associate with the active form of Lck, phospho-Y394, atthe synapse, suggesting that Tim-3 may be recruiting a phospha-tase. Interestingly, in addition to confirming galectin-9 as the Tim-3ligand, we described novel interactions between galectin-9 andreceptor phosphatases CD45 and CD148. Finally, galectin-9 wasable to enhance the interaction between these phosphatases andTim-3 within CD3 signaling complexes. These data suggest the

FIGURE 2. Blocking Tim-3 enhances

synapse formation. (A) Tim-3hi CD8+

T cells were rested overnight with either

an isotype control or the antagonistic

Tim-3 mAb 2E2 and subjected to con-

jugate formation and subsequent immuno-

fluorescence staining with Cell-Tracker

Blue–stained, SEB-loaded B cells. Depicted

are a T cell–B cell conjugate with a stable

synapse (top panel) and a T cell–B cell

conjugate with an unstable synapse (bot-

tom panel). Stable synapses were defined

as a T cell–B cell conjugate with concen-

trated CD3 at the interface between the

two cells, and the T cell was phospho-

ERK+. Unstable synapses were defined as

a T cell–B cell conjugate in which CD3

was not concentrated at the interface be-

tween the two cells or the T cell was not

phospho-ERK+. Only conjugates with

a Tim-3+ T cell were included in the

counts. Shown are representative synapses

taken from the anti–Tim-3 Ab–treated

conditions. Original magnification 3600.

(B) Bar graph showing the difference in

frequency of stable synapse formation

between cells treated with the isotype

control or antagonistic Ab 2E2. For semi-

quantification of stable synapses, pictures

were taken of multiple fields of view, and

25 pairs of T cell–B cell conjugates were

blindly counted per slide. Statistical analy-

sis was performed using a two-tailed paired

Student t test from four independent

experiments.

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possibility that Tim-3 may regulate Lck function via interactionwith CD45 or CD148 at the synapse, an interaction mediated byits ligand galectin-9.Proximal TCR signaling is a complex process regulated not only

by posttranslational modifications, including tyrosine phosphory-lation, but also by lipid raft dynamics and compartmentalization ofproteins. For T cells to initiate signaling, first the peptide-loadedMHC interacts with the TCR. This results in recruitment to thelipid rafts and exclusion of CD45 (33). Fyn and/or non-CD8–bound Lck become associated with the TCR–CD3 signalingcomplex, resulting in phosphorylation of CD3 ITAMs and re-cruitment of ZAP70. Following ZAP70 phosphorylation, the Srchomology 2 (SH2) domain of CD8-bound Lck binds to phospho-ZAP70, recruiting CD8 to the MHC–TCR complex. CD8 binds to

the MHC, increasing the stability of the MHC–TCR complex (40),sustaining proximal signaling and initiation of downstream sig-naling.The finding that .40% of Tim-3 resides in lipid rafts and is

recruited to the immunological synapse provides evidence thatTim-3 is involved in proximal signaling. Although this studyassessed membrane location of Tim-3 on resting cells, furtherwork is warranted to determine if Tim-3 accumulates within or isexcluded from lipid rafts during TCR signaling, similar to Lck andCD45, respectively. However, because TCR triggering results inthe accumulation of lipid rafts at the synapse (34), it is likely thatTim-3 would accumulate. In addition, we found that blockingTim-3 using the antagonistic Ab 2E2 resulted in significantly moreconjugates that had stable synapses. Initial proximal TCR sig-

FIGURE 3. Tim-3 interacts with Lck. (A) Rested Tim-3hi CD8+ T cells were treated with or without pervanadate, and Tim-3 was immunoprecipitated

following lysis of the cells. Elutions and lysate were analyzed via SDS-PAGE and Western blotting (WB) for phospho-tyrosine (pY), stripped, and reprobed

for Tim-3, followed by Lck. (B) Rested Tim-3hi CD8+ T cells were used to form conjugates with Cell-Tracker Blue–stained, SEB-loaded B cells followed

by indirect immunofluorescent staining for CD3 (magenta), phospho-Y394 Lck (p-Y394 Lck; red), and Tim-3 (green) and analyzed via confocal mi-

croscopy. Yellow indicates colocalization of p-Y394 Lck and Tim-3, whereas white indicates colocalization of CD3 and Tim-3. Figure is representative of

three experiments. Original magnification 3630.

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naling, such as Lck phosphorylation and subsequent downstreamsignaling to linker for activation of T cells, is required for stablesynapse formation (41), further providing evidence that Tim-3 isinvolved in TCR proximal signaling. Although the exact mecha-nism of how Tim-3 effects proximal signaling is still under in-vestigation, our work provides preliminary evidence that Tim-3may be involved in Lck regulation.Our finding that Tim-3 binds to Lck is not novel (27); however,

we did show that this interaction occurred in primary human CD8+

T cells. In addition, we showed that Tim-3 did not localize withthe active form of Lck, phospho-Y394. Lck activity is regulatedvia phosphorylation of the positive regulatory tyrosine Y394 in thekinase domain and the negative regulatory tyrosine Y505 in theC-terminal end (33). When Y505 is phosphorylated, it forms a bondwith its own SH2 domain, preventing substrate access to the ki-nase domain. However, when Y394 is phosphorylated, the proteinachieves full kinase activity. It would be prudent to assess Tim-39slocation in relation to the inactive form of Lck (pY505) versus theactive form of Lck (pY394); however, the inactive form pY505 isbound to its own SH2 domain, preventing access to the phos-phorylated motif by any Ab used for immunofluorescence. Thus,we are unable to assess, given currently available reagents,whether Tim-3 preferentially binds pY505 Lck. However, directlyassessing Lck activity in Tim-3 immunoprecipitates using immune

complex kinase assays would be more accurate rather than in-ferring activity based on the phosphorylation state of Tim-3–precipitated Lck. Thus, further studies to characterize the activityof Tim-3–associated Lck are warranted. Phosphorylation of Y505and Y394 are achieved by the kinase Csk and via trans/autophosphorylation, respectively (33). Dephosphorylation of ei-ther tyrosine is mediated by the receptor phosphatase CD45. In-terestingly, CD45 plays a dual role in positively and negativelyregulating Lck, depending on the expression level of CD45 (42).Low to intermediate concentrations of CD45 result in dephos-phorylation of Lck at Y505, resulting in an increase in Lck ac-tivity. However, high levels of CD45 result in dephosphorylationof Y394, effectively decreasing Lck kinase activity.Despite a recent study that claimed human Tim-3 was not the

receptor for human galectin-9 (43), we confirmed that galectin-9was the physiological ligand for Tim-3 using two different bio-chemical assays. In addition, we discovered that galectin-9 boundto receptor phosphatases CD45 and CD148, suggesting a possiblemechanism of how Tim-3 could regulate Lck activity. Inducinghigh local concentrations of CD45 in proximity to Tim-3 andassociated Lck could result in dephosphorylation of Lck Y394 andY505. In addition, we showed that galectin-9 was able to increaselevels of Tim-3, CD45, and CD148 within CD3 signaling rafts.Thus, although CD45 and CD148 are normally excluded from the

FIGURE 4. Galectin-9 binds to

multiple immune accessory mole-

cules including receptor phospha-

tases CD45 and CD148. (A) Bead-

immobilized recombinant galectin-9

or beads alone (negative control) were

incubated with lysate from rested

Tim-3hi CD8+ T cells. Bound pro-

teins were eluted with 200 mM

a-lactose followed by a subsequent

boiling/denaturing elution in reducing

sample buffer. Lysate, flow-throughs,

and elutions were analyzed via SDS-

PAGE and Western blotting (WB)

for Tim-3. (B) In addition, a-lactose

elutions were analyzed via SDS-

PAGE and subsequent silver stain-

ing. Differential bands were excised

from the galectin-9 affinity precipi-

tation a-lactose elution and sent for

liquid chromatography-tandem mass

spectrometry identification. (C) Anal-

ysis of the mass spectrometry results

using Scaffold 4 software yielded

identification of multiple immune

accessory molecules and receptor

phosphatases.

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synapse (33, 44), concurrent ligation of these receptor phospha-tases with Tim-3 via galectin-9 could enhance the presence ofthese phosphatases at the synapse, resulting in negative regulationof Lck as well as dampening of TCR signaling.Importantly, all of this work was performed using primary CD8+

T cells with minimal manipulation of the T cells or target cells.Although Tim-3 expression was induced via in vitro stimulation,no proteins were ever overexpressed, which can lead to artificialresults and false positives, such as chaperones binding to over-expressed proteins (45). In addition, this work studied Tim-3 un-der near physiological conditions. Use of primary CD8+ T cellsprovides an intact TCR signaling platform, including presence ofproximal signaling molecules, regulation via lipid raft dynamics,and presence of receptor phosphatases that are essential for theinitiation and final dampening of TCR signaling (44, 46, 47). Thisis absent in many of the cell lines used to study Tim-3 thus far (24,25, 27). In addition, our system uses SEB loaded on target cells toprovide a TCR stimulus, which does not bypass proximal sig-naling, unlike the PMA/ionomycin stimulus used to study Tim-3(28). Finally, we provide a target cell, which expresses the phys-iological Tim-3 ligand as shown by sTim-3 binding to the B cell ina carbohydrate-specific manner. This is essential because previousstudies have engaged Tim-3 using putative agonistic Abs (13, 27).

Galectin-9 is a soluble protein, not a transmembrane protein, likeprogrammed death ligand-1 and programmed death ligand-2. Itcan associate with the membrane via interaction with other gly-cans; however, its behavior during T cell engagement and synapseformation is currently unknown, stressing the importance to un-derstand how galectin-9 effects Tim-3 before using Abs to engageTim-3. Indeed, using a galectin-9 affinity precipitation, we didshow that galectin-9 was able to bind to various immune accessorymolecules. Thus, the Tim-3–galectin-9 interaction is not a closedsystem but a complex interplay with various other surface gly-cosylated molecules. Although tempted to use agonistic Abs tostudy the effects of Tim-3, the reality is that when Tim-3 is en-gaged under physiological conditions, so are other receptors, in-cluding receptor phosphatases like CD45.Using the data we have collected, we wish to propose a working

model. In the case of Tim-3 expressed on the T cell and galectin-9expressed on the surface of the APCs, both CD45 and Tim-3 getrecruited to the TCR signaling clusters at the immunologicalsynapse. This results in CD45-mediated dephosphorylation of theactive tyrosine (Y394) of Lck, and accumulation of inactive Lckwithin TCR signaling clusters. This increases the ratio of inactiveLck to active Lck and eventual dampening of TCR signaling. Theseresults, thus far, are observational, and future functional inves-

FIGURE 5. Tim-3 colocalizes with

receptor phosphatases, an interaction

that is enhanced in the presence of

galectin-9. (A) Rested Tim-3hi CD8+

T cells were used to form conjugates

with Cell Tracker Blue–stained, SEB-

loaded B cells followed by indirect

immunofluorescent staining for CD3

(magenta), Tim-3 (green), and either

CD45 (red, top panel) or CD148

(red, bottom panel) and analyzed via

confocal microscopy. Yellow indi-

cates colocalization. The following

are two representative pictures of four

experiments. Original magnifica-

tion 3630. (B) Rested Tim-3hi

CD8+ T cells were pretreated with

anti-CD3 beads followed by addition

of buffer or recombinant galectin-9.

The cells were lysed, and anti-CD3

beads were collected. CD3-immuno-

precipitated protein was eluted using

reducing sample buffer and analyzed

via SDS-PAGE and Western blot

(WB) for Tim-3. The blot was stripped

and reprobed for CD45, CD148, and

CD3. Figure is representative of three

experiments.

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tigations focusing on the Tim-3 intracellular pathway and galectin-9–mediated effects using our physiological T cell/APC system arewarranted.Efforts need to be made to understand the signaling pathways

associated with coinhibitory receptors to allow for better devel-opment of therapeutic interventions. The signaling pathways forcoinhibitory receptors CTLA-4 and PD-1 are currently known. Inaddition, these proteins have antagonistic Abs currently used inclinic or in phase III trials, respectively, for cancer treatment (48).Coinhibitory molecules are not redundant, as shown by the syn-ergistic effects of dual blockade. This stresses the importance ofunderstanding what specific effects each inhibitory receptor has onT cell function to better formulate blockades that are most ef-fective against different diseases such as cancer and chronic viralinfections.

AcknowledgmentsWe thank Dr. Michael Julius for help and expertise with the lipid raft iso-

lation, Dr. Andrew Wilde for the use of microscope equipment, Dr. Walid

Houry for help and expertise with the protein purifications and use of

equipment, and Dr. R. Brad Jones for helpful comments.

DisclosuresThe authors have no financial conflicts of interest.

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