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the Buildup of the Early TCR SignalosomeSequential Cooperation of CD2 and CD48 in

Wolfgang Paster and Hannes StockingerGerhard J. Zlabinger, Maria Sibilia, Alois Sonnleitner,Paul Eckerstorfer, Rene Geyeregger, Vladimir Leksa, Arshad Muhammad, Herbert B. Schiller, Florian Forster,

http://www.jimmunol.org/content/182/12/7672doi: 10.4049/jimmunol.0800691

2009; 182:7672-7680; ;J Immunol 

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Sequential Cooperation of CD2 and CD48 in the Buildup ofthe Early TCR Signalosome1

Arshad Muhammad,* Herbert B. Schiller,* Florian Forster,* Paul Eckerstorfer,*Rene Geyeregger,† Vladimir Leksa,*‡ Gerhard J. Zlabinger,§ Maria Sibilia,† Alois Sonnleitner,¶

Wolfgang Paster,2* and Hannes Stockinger2*

The buildup of TCR signaling microclusters containing adaptor proteins and kinases is prerequisite for T cell activation. Onehallmark in this process is association of the TCR with lipid raft microdomains enriched in GPI-proteins that have potential toact as accessory molecules for TCR signaling. In this study, we show that GPI-anchored CD48 but not CD59 was recruited to theimmobilized TCR/CD3 complex upon activation of T cells. CD48 reorganization was vital for T cell IL-2 production by mediatinglateral association of the early signaling component linker for activated T cells (LAT) to the TCR/CD3 complex. Furthermore, weidentified CD2 as an adaptor linking the Src protein tyrosine kinase Lck and the CD48/LAT complex to TCR/CD3: CD2 associatedwith TCR/CD3 upon T cell activation irrespective of CD48 expression, while association of CD48 and LAT with the TCR/CD3complex depended on CD2. Consequently, our data indicate that CD2 and CD48 cooperate hierarchically in the buildup of theearly TCR signalosome; CD2 functions as the master switch recruiting CD48 and Lck. CD48 in turn shuttles the transmembraneadapter molecule LAT. The Journal of Immunology, 2009, 182: 7672–7680.

E fficient activation of T cells entails numerous interactionsof a plethora of receptors and signaling molecules andresults in production of cytokines, such as IL-2, and

clonal expansion and differentiation to effector T lymphocytes.This process is induced by a primary Ag-specific signal via TCRengagement by peptide-MHC as well as a second signal providedby costimulatory receptors engaged by their respective ligands onan APC (1). T cell activation commences with an early wave ofprotein tyrosine kinase activity, which is mediated by the Src-family kinase Lck, the Syk family kinase ZAP70 and members ofthe Tec kinase family. A central target for ZAP70 phosphorylationis the transmembrane adapter molecule linker for activated T cells(LAT)3 (2). Phosphorylated LAT serves as crucial link betweenTCR-proximal events and distal signaling pathways and is indis-pensable for T cell development and function (3, 4).

T cells reorganize their surface molecules to form a well-struc-tured contact zone with APCs known as immunological synapse

(IS) (5, 6). It is an emerging concept in T cell biology that TCR-microclusters in the periphery of the IS are the main source ofsustained TCR signaling, whereas the center of the synapse mightplay a role in signal termination (7). Lipid rafts may play a role inthe formation of these signaling microclusters by providing a plat-form for molecular association and segregation processes (8, 9).

Central components of lipid rafts are GPI-anchored receptorsthat are enriched in the rafts via their glycolipid anchors. Othersand we have shown that cross-linking of GPI-anchored proteins onT cells, such as CD90, Ly-6, CD48, and CD59 results in phos-phorylation of protein tyrosine kinases like Lck (10, 11). Thus,GPI-anchored proteins have potential to provide costimulatory sig-nals during T cell activation. CD48 in particular was firmly dem-onstrated to amplify TCR signaling (12, 13).

Recently, we analyzed the dynamics of the TCR, the lipid raft-associated GPI-proteins CD48 and CD59, and the major leukocytephosphatase CD45 in living naive human peripheral blood T lym-phocytes by a noninvasive single-molecule imaging approach. Wefound that T cell stimulation on planar CD3 mAb coated surfacesinduced the immobilization of CD48 in the center of the T cellinterface whereas the second GPI protein, CD59, remained highlymobile (14). Heterogeneity in the behavior of lipid raft moleculesand different lipid composition of microdomains has been alreadydemonstrated by others (15–17), however, it is still not clear whatmolecular interactions determine this diversity. In the murine andhuman system, CD48 has been described as a ligand for CD2 inboth cis-and trans-configuration (18–21), and CD2 has been as-cribed to signaling microdomains built up of Lck and LAT inJurkat cells stimulated on TCR mAb coated surfaces (22). CD2may additionally serve as a ligand for the GPI-molecule CD59 (23,24); however, this binding is highly questionable as it could not beconfirmed by others (21, 25). Therefore, we hypothesized that CD2could be responsible for the specific immobilization of CD48 uponT cell activation. Indeed, according to our new data, CD2 is crucialfor localization of CD48 to the TCR/CD3 complex, whereas CD48in turn is not required for TCR recruitment of CD2. Both CD48

*Department of Molecular Immunology, Centre for Physiology, Pathophysiology andImmunology, Medical University of Vienna, Vienna, Austria; †Institute of CancerResearch, Medical University Vienna, Vienna, Austria; ‡Institute of Molecular Biol-ogy, Slovak Academy of Sciences, Bratislava, Slovak Republic; §Institute of Immu-nology, Medical University of Vienna, Vienna, Austria; and ¶Center for BiomedicalNanotechnology, Upper Austrian Research, Linz, Austria

Received for publication February 29, 2008. Accepted for publication April 6, 2009.

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 funded by the GEN-AU program of the Austrian Federal Ministry ofScience and Research, the PhD program Cell Communication in Health and Disease,the Competence Centre for Biomolecular Therapeutics and the Higher EducationCommission of Pakistan.2 Address correspondence and reprint requests to Dr. Wolfgang Paster or Dr. HannesStockinger, Centre for Physiology, Pathophysiology and Immunology, Medical Uni-versity of Vienna, Lazarettgasse 19, Vienna, Austria. E-mail address: wolfgang.paster@meduniwien.ac.at or hannes.stockinger@meduniwien.ac.at3 Abbreviations used in this paper: LAT, linker for activated T cell; IS, immunolog-ical synapse; SPSP, solid phase signalosome precipitation; AF, Alexa Fluor; shRNA,short hairpin RNA; SEE, staphylococcus enterotoxin E.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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and CD2 knockdown cells display similar defects in IL-2 expres-sion due to defective recruitment of essential signaling moleculesto the TCR/CD3 complex. Thus, the lateral association of CD2with CD48 is required for productive T cell activation.

Materials and MethodsCells

The human Jurkat IL-2-Luc cells, stably transfected with an IL-2 promoterluciferase reporter construct, were a gift of Dr. Thomas Baumruker (No-vartis Institutes for BioMedical Research, Vienna, Austria). The Jurkat Tcell line E6.1 and the B cell line Raji were purchased from the AmericanType Culture Collection. Human PBMCs were isolated from blood ofhealthy donors by standard density-gradient centrifugation using Lym-phoprep (Nycomed). Cells were maintained in RPMI 1640 medium(Sigma-Aldrich) supplemented with 100 �g/ml penicillin, 100 �g/mlstreptomycin, 2 mM L-glutamine (Invitrogen), and 10% heat inactivatedFCS (PAA). For Jurkat IL-2-Luc cells, 1 mg/ml G418 was added addi-tionally. HEK 293T cells were maintained in DMEM (Sigma-Aldrich) sup-plemented with 100 �g/ml penicillin, 100 �g/ml streptomycin, 2 mM L-glutamine (Invitrogen), and 10% heat inactivated FCS (PAA). All cellswere grown at 37°C and 5% CO2 in a humidified atmosphere.

Antibodies

The mAbs to CD3 (MEM-57; IgG2a and MEM-92; IgM), CD48 (MEM-102;IgG1), CD59 (MEM-43/5; IgG2b), CD2 (MEM-65; IgG1), and MHC class II(MEM-136; IgG1) were a kind gift of Dr. Vaclav Horejsí (Institute of Mo-lecular Genetics, Academy of Sciences of the Czech Republic, Prague, CzechRepublic); the anti-TCR �-chain mAb C305 (IgM) was provided by Dr.Arthur Weiss (University of California, San Francisco, CA). The mAb OKT3to CD3 was obtained from Ortho Pharmaceuticals. The mAb Leu28 toCD28 was purchased from Becton Dickinson (BD Biosciences). TheCD3 mAb UCH-T1 to the CD3 �-chain was from Santa Cruz Biotech-nology, rabbit polyclonal Abs to Lck and LAT and the anti-phosphotyrosine mAb 4G10 (HRP conjugated) were from Upstate Bio-technology. The mAb to phosphorylated Tyr 505 on Lck (pY505) waspurchased from Cell Signaling Technology and Ab to pLAT was pur-chased from Biosource. Goat anti-rabbit IgG and rabbit anti-mouse IgG,both HRP conjugated, were from Bio-Rad and Sigma-Aldrich,respectively.

Immunoprecipitation and Western blotting

Jurkat T cells (5 � 106 to 1 � 107/ml) were lysed for 25 min at 4°C inice-cold lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mMEDTA and protease inhibitors (5 �M aprotinin, 5 �M leupeptin, 5 �Mpepstatin, 1 mM PMSF; all from Sigma-Aldrich) containing 1% NonidetP-40 (Pierce) or 0.5% N-dodecyl-D-maltoside]. The insoluble material wasremoved by centrifugation at 13,000 rpm for 2 min at 4°C and the super-natant was distributed into the wells of a 96-well plate which were pre-coated with the respective mAbs (20 �g/ml in PBS) as described (14).After incubating the wells with the cell lysate (50 �l/well) over night at4°C, the wells were washed with PBS, after which 1� SDS-PAGE loadingbuffer was added (7–10 �l/well). The immunoprecipitates were collectedand analyzed by SDS-PAGE and Western blotting.

Solid phase signalosome precipitation (SPSP)

Jurkat T cells (7 � 106/ml) were incubated in ice-cold RPMI 1640 mediumwith 5% FCS and 20 mM HEPES for 1 h at 4°C. Then, the cells were addedinto six-well plates (1 ml/well) coated with the CD3 mAb OKT3 (10 �g/ml) and shortly spun down. The six-well plates were incubated at 37°C forthe indicated periods of time. After stimulation, the medium was carefullyremoved and the attached cells were lysed for 12 min at 4°C using ice-coldlysis buffer (see above) supplemented with 1 mM sodium orthovanadateand 50 mM NaF. Subsequently, the lysate was collected and the insolublematerial removed by centrifugation at 13,000 rpm for 2 min at 4°C. Af-terward, the six-well plates with the precipitated protein complexes werewashed three times with ice-cold washing buffer [20 mM Tris-HCl (pH7.5), 150 mM NaCl, 5 mM EDTA, 0.1 mM sodium orthovanadate] con-taining 0.1% Tween 20 (Sigma-Aldrich). The immunoprecipitated proteincomplexes were harvested in 150 �l of 1� SDS-PAGE loading buffer.Finally, both the lysates and the immunoprecipitates were analyzed bySDS-PAGE and immunoblotting.

Analysis of protein tyrosine phosphorylation

Before stimulation, Jurkat T cells were incubated in RPMI 1640 mediumsupplemented with 1% FCS for 4 h at 37°C. Cells were stimulated for

different durations at 37°C using CD3 mAb MEM-92 (10 �g/ml). Thereactions were stopped by addition of ice-cold washing buffer (see above).After centrifugation (2 min, 850 � g, 4°C), cells were immediately lysedfor 30 min in ice-cold lysis buffer (see above) supplemented with 1 mMsodium orthovanadate, 50 mM NaF. After centrifugation, lysates were an-alyzed by reducing SDS-PAGE conditions (10% gel) and immunoblottingusing HRP-labeled anti-pTyr mAb 4G10 (Upstate Biotechnology).

Lipid-raft separation by sucrose-gradient centrifugation

Cell lysates were adjusted to 40% (w/v) sucrose by adding an equal volumeof an 80% sucrose solution (TBS containing 80% w/v sucrose, 2 mMEDTA, and protease inhibitors). These preparations were placed on top ofa 60% sucrose layer in a centrifuge tube (Sorvall Instruments-DuPont). Ontop of this, layers of 20%, 10% and 5% sucrose were placed. After ultra-centrifugation (180,000 � g, 16 h, 4°C), 375 �l fractions were collectedfrom the top. Aliquots of each fraction were diluted in a 4� gel loadingSDS-buffer. Proteins were separated by SDS-PAGE and immunoblottedfollowed by probing the membranes with the respective mAbs.

Flow cytometry

Cells were washed with staining buffer (PBS/1% BSA/0.02% NaN3) andincubated for 30 min with 4 �g/ml human Ig on ice to prevent nonspecificbinding of the mAb to Fc receptors. Primary Ab (10 �g/ml) was added andthe cells were incubated for 30 min on ice. Cells were washed with stainingbuffer and incubated with secondary Ab for 30 min on ice. After a finalwash, cells were analyzed on an LSRII or FACSCalibur flow cytometer(BD Biosciences).

Coverslip preparation and confocal microscopy

For confocal microscopy, epoxide-bearing glass slides were prepared asdescribed (26). The cells were washed twice in PBS containing 2% FCS(PBS-FCS) and then resuspended (2 � 106 cells/ml) in HBSS containing1 mM Ca2� and 1 mM Mg2�. The cell suspension was gently placed on theCD3 mAb (MEM-57)-coated cover slips. Cells were stimulated at 37°C forvarious time intervals. The incubation was stopped by fixing the cells with3.75% paraformaldehyde in PBS. After washing twice with PBS-FCS, thecells were blocked with 4 �g/ml human Ig in PBS for 20 min. Then, thecells were incubated for 30 min with directly labeled Alexa Fluor (AF)mAbs against CD3 (MEM-57/AF647), CD48 (MEM-102/AF555), orCD59 (MEM-43/5/AF555) at a final concentration of 10 �g/ml. Followingstaining, the cells were washed twice with PBS-FCS and analyzed on aconfocal LSM 510 Zeiss microscope (Carl Zeiss AG) by making serial zstacks from bottom to top.

Knockdown of CD2 and CD48 expression by RNA interference

The following oligonucleotides were annealed and cloned via EcoRI/AgeI to the lentiviral short hairpin RNA (shRNA) expression vectorpLKOpuro1 (provided by Dr. Sheila Stewart, Washington UniversitySchool of Medicine, St. Louis, MO): shCD2 sense at position 341 (5�-CCGGTGACCGATGATCAGGATATCTTCAAGAGAGATATCCTGATCATCGGTCTTTTTG-3�) and shCD2 anti-sense at position 341 (5�-AATTCAAAAAGACCGATGATCAGGATATCTCTCTTGAAGATATCCTGATCATCGGTCA-3); shCD2 sense at position 864 (5�-CCGGTCCTCAGAATCCAGCAACTTTTCAAGAGAAAGTTGCTGGATTCTGAGGTTTTTG-3�) and shCD2 anti-sense at position 864 (5�-AATTCAAAAACCTCAGAATCCAGCAACTTTCTCTTGAAAAGTTGCTGGATTCTGAGGA-3�); shCD48 sense at position 884 (5�-CCGGTGCATGCTGCTGAATTATCATTCAAGAGATGATAATTCAGCAGCATGCTTTTTG-3�) and shCD48 anti-sense at position 884 (5�-AATTCAAAAAGCATGCTGCTGAATTATCATCTCTTGAATGATAATTCAGCAGCATGCA-3�); shCD48 sense at position 193 (5�-CCGGTGCCTGCCTGAGAACTACAATTCAAGAGATTGTAGTTCTCAGGCAGGCTTTTTG-3�)and shCD48 anti-sense at position 193 (5�-AATTCAAAAAGCCTGCCTGAGAACTACAATCTCTTGAATTGTAGTTCTCAGGCAGGCA-3�). Gene-specific shRNA-target sequences are indicated on the sense strand initalic. As control, we used the MISSION Non-Target pLKOpuro1 ControlVector (Sigma-Aldrich). Production of lentiviral supernatants was per-formed in 293T cells by cotransfection of the appropriate pLKOpuro1vector with the packaging plasmids psPAX2 and pMD2.G (Addgeneplasmids 10703 and 12259; constructed by Dr. Didier Trono, EcolePolytechnique Federal de Lausanne; obtained via Addgene). Forty-eighthours after transfection, viral supernatants were harvested, filtered, andused freshly for the transduction of cells. The transduction was doneovernight in the presence of 5 �g/ml polybrene. Transduced cells wereselected with puromycin (1 �g/ml).

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For shRNA-mediated gene silencing in human peripheral blood T cells,freshly isolated PBMCs were stimulated with plate-bound CD3 mAbOKT-3 (5 �g/ml) and soluble CD28 mAb Leu28 (2 �g/ml) for 24 h andinfected with freshly prepared virus, which was concentrated by ultracen-trifugation from the supernatant of the producer cells. Twenty-four hourslater, a second round of infection was performed. Transduced T cells wereselected by stimulation for further 3 days under puromycin (1 �g/ml).Then, the T cells were washed and rested for 5 days. For the cytokineassay, the T cells were restimulated with plate-bound CD3 mAb OKT-3and soluble CD28 mAb Leu28 as indicated.

Luciferase assay

Jurkat IL-2-Luc cells were stimulated as indicated at 37°C. Luciferase as-says were performed using the luciferase reporter gene assay kit (RocheDiagnostics) according to the manufacturer’s instructions. Luciferase ac-tivity was measured in the Berthold Lumat LB 9501 device (BertholdTechnologies GmbH) and normalized for protein content with the Bio-Rad

protein assay (Bio-Rad). Each luciferase assay experiment was performedin triplicates and repeated at least three times. The bars represent triplicateswith indicated SD. Statistical significance was evaluated using a two-tailed,paired Student’s t test.

Measurement of secreted IL-2

Samples were analyzed using the Luminex xMAP suspension array tech-nology. Thirty microliters of culture supernatants were used undiluted.Standard curves were generated using rIL-2 (R&D Systems). All incuba-tion steps were performed at room temperature and in the dark to protectthe beads from light. To avoid inconsistencies, all samples from an indi-vidual experiment were measured in the same run. Control samples wereincluded in all runs. Detection limits were 30 pg/ml for IL-2. The exper-iments were performed at least two times in at least triplicates, and the datawere expressed as mean values with SD. Statistical significance was eval-uated using a two-tailed, paired Student’s t test.

FIGURE 1. Recruitment of CD48 tothe TCR/CD3 complex requires expres-sion of CD2. A, Lentiviral shRNA-medi-ated gene knockdown of CD2 expressionin Jurkat IL-2-Luc cells. Staining of con-trol shRNA transduced cells (shCtr) isshown in filled gray histograms, CD2knockdown cells (shCD2) are in black.The dashed line represents staining of theisotype matched control mAbs. B,Confocal imaging of a semiartificialIS formed by control and CD2 si-lenced Jurkat IL-2-Luc cells on aCD3 mAb (MEM-57) coated glasssurface. Jurkat cells were incubatedon the stimulatory surface for the in-dicated times at 37°C. Afterward,cells were fixed and stained withAF-labeled mAbs to CD3 (MEM-57; AF647), CD48 (MEM-102;AF555), and CD59 (MEM-43/5;AF555). The analysis was per-formed using an LSM 510 Meta la-ser scanning microscope systemfrom Zeiss. Individual cells werescored for central or peripheral dis-tribution of CD48 and CD59 in thesemiartificial IS. The table summa-rizes 34 cells from two independentexperiments. C, Analysis of TCR/CD3-complex associated moleculesby standard immunoprecipitation inresting Jurkat IL-2-Luc cells (left)and by SPSP (right) in control andCD2-silenced Jurkat IL-2-Luc cells.The SPSP method makes use ofCD3 mAb (OKT3) coated surfacesfor both T cell stimulation and pre-cipitation of CD3-associated signal-ing complexes. Cells were allowedto settle on the stimulatory surfaceof a six-well plate for the indicatedtimes and then were lysed. The su-pernatant containing nonprecipitatedmaterial was removed and the CD3-as-sociated complexes were harvested bythe addition of SDS-PAGE samplebuffer to the wells. Both the precipitatedand the nonprecipitated materials wereanalyzed under reducing conditions us-ing a 10% SDS-PAGE gel followed byimmunoblotting.

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Calcium flux measurement

Cells were stained with 5.5 �M Fura Red AM-ester and 2.6 �M Fluo-4AM-ester (Molecular Probes) in RPMI 1640 medium for 45 min at 37°C.After washing twice with medium, cells were again incubated for 30 minat 37°C. Fluo-4/Fura-red loaded cells were then analyzed by flow cytom-etry using a LSRII flow cytometer (BD Biosciences). Calcium mobilizationwas monitored within the first three minutes after TCR cross-linking withthe anti-TCR mAb C305 by analyzing the fluorescence emission ratio ofFura Red and Fluo-4 as previously described (27).

Immune synapse assay

Raji B cells transduced with either control shRNA or CD48 specificshRNA were labeled with CellTracker Orange (chloromethyl-tetramethyl-rhodamine methyl ester; Molecular Probes) and pulsed with 5 �g/ml staph-ylococcal enterotoxin E (SEE; Toxin Technology) in HBSS at 37°C for 20min. After washing, the Raji cells were incubated with shRNA vector-transduced Jurkat E6.1 T cells (shCtr, shCD2, or shCD48) at a ratio of 1:1at 37°C for 20 min. The reaction was stopped by adding ice-cold HBSS.Cells were plated on poly-L-lysine-coated slides (Marienfeld) and allowedto settle for 15 min on ice. For staining of CD3� and LAT, cells were fixedwith 4% formaldehyde in PBS for 15 min at room temperature, perme-abilized with 0.1% Triton X-100 in PBS for 15 min at room temperature(except for staining with CD3�), and treated with the mouse anti-humanCD3 mAb UCH-T1 and the rabbit anti-human polyclonal LAT Ab fol-

lowed by the appropriate AF488-labeled goat-anti-mouse or goat-anti-rabbit Abs (Molecular Probes) for 30 min at room temperature. Using a�40 magnification on an Aristoplan fluorescence microscope (Leica Mi-crosystems), two individuals determined independently the percentage ofcells showing CD3� and LAT relocalization to the IS by counting at least100 conjugates per blinded sample.

ResultsCD2 is necessary for recruitment of CD48 to the TCR/CD3complex

We have previously demonstrated that CD48 but not CD59 getsimmobilized in the center of a CD3 mAb-induced artificial immu-nological synapse together with the TCR/CD3 complex (14). In the

FIGURE 2. CD2 and CD48 are key mediators of LAT recruitment to theTCR/CD3 complex. A, Lentiviral shRNA-mediated gene knockdown of CD48expression in Jurkat IL-2-Luc cells. Staining of control shRNA transducedcells (shCtr) is shown in filled gray histograms, CD48 knockdown cells(shCD48) are in black. The dashed line represents staining of the isotypematched control mAbs. B, SPSP of control and CD48 silenced Jurkat IL-2-Luccells, stimulated and precipitated on CD3 mAb (OKT3) surfaces for the indi-cated times. The precipitated as well as the nonprecipitated material was sub-jected to SDS-PAGE and immunoblotted with the indicated Abs. C, Samplematerial obtained from SPSP with CD2 silenced cells was subjected to specificimmunoblotting using CD3�, Lck, and LAT Abs.

FIGURE 3. Lipid raft partitioning of the TCR, LAT phosphorylation,and calcium mobilization upon T cell activation depend on CD2 and CD48.A, Jurkat control and CD2 silenced cells were stimulated with the CD3mAb MEM-92 in solution (10 �g/ml). At the indicated time points, cellswere lysed and the lipid raft partitioning of CD3 was analyzed by sucrosegradient ultracentrifugation and immunoblotting as described in Materialsand Methods. B, LAT phosphorylation is dependent on CD2 expression.Immunoblot analysis of control and CD2 silenced Jurkat IL-2-Luc cellsstimulated for the indicated times with the CD3 mAb MEM-92 in solution(10 �g/ml). Cells were lysed, subjected to SDS-PAGE and immunoblottedwith the indicated Abs. C, Reduced calcium flux upon TCR cross-linkingin CD2 and CD48 silenced Jurkat cells. Jurkat cells were stained withFluo-4 and Fura-red. Calcium transients in response to the anti-TCR mAbC305 and the calcium ionophore ionomycin were measured by flowcytometry.

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present study, we asked whether the differential behavior of thesetwo lipid raft markers was dependent on interaction with CD2,which is a central molecule in TCR signaling microclusters (22).To study this, we performed lentiviral shRNA-mediated geneknockdown of CD2 in the human T leukemia cell Jurkat IL-2-Luc,stably transfected with an IL-2 promoter luciferase reporter con-struct. We obtained a quantitative knockdown of almost 100% ofCD2 expression by the shRNA targeting position 341; the stainingof CD3, CD48, and CD59 remained unaffected (Fig. 1A). A similareffect but with weaker down-regulation of CD2 was obtained withan shRNA targeting position 864 (data not shown).

First, we subjected these cells to confocal microscopy analysis.We allowed Jurkat T cells to settle and spread on CD3 mAb-coatedglass slides. The cells were fixed after different time points fol-lowed by staining with specific mAbs. In accord with our previousstudy (14), in Jurkat cells transduced with the control shRNA, after20 min of stimulation we found CD48 in the centre of the spread-ing zone colocalized with CD3. In the CD2-silenced cells reorga-nization and colocalization of CD48 with CD3 did not occur.CD59 was mostly restricted to peripheral regions of the artificialimmunological synapse in the control as well as the CD2 silencedcells (Fig. 1B).

As the next step, we analyzed the role of CD2 for CD48 re-cruitment to the TCR/CD3 complex at the biochemical level. Wedid not observe an interaction of CD3 with CD2, CD48, or CD59in resting Jurkat cells when using standard immunoprecipitation(Fig. 1C, left). We next analyzed molecules associating with theTCR/CD3 complex upon stimulation. To preserve signaling asso-ciates we used SPSP described earlier by us (14). This methodallows T cell stimulation with a surface-immobilized Ab and iso-lation of molecules associated with the ensuing semiartificial im-munological synapse. In brief, to synchronize cell spreading, thecells were shortly centrifuged (at 4°C) onto tissue culture platescoated with a CD3 specific mAb (OKT3). After different timeperiods of stimulation at 37°C, the cells were lysed with 1%

FIGURE 4. Both CD2 and CD48 are essential for a stable associate oftyrosine phosphorylated signaling proteins in the TCR signalosome. A,SPSP of control and CD2 silenced Jurkat IL-2-Luc cells, stimulated, andprecipitated on CD3 mAb (OKT3) surfaces for the indicated times. Theprecipitated as well as the nonprecipitated material was subjected to SDS-PAGE and immunoblotted with the phosphotyrosine specific mAb 4G10.B, CD48 silenced cells were analyzed as in A.

FIGURE 5. Impaired recruitment of LAT to the immune synapse ofCD2 or CD48 silenced Jurkat cells. A, Expression analysis of CD2, CD48,and MHC class II on control shRNA (shCtr, filled gray histograms) andshCD48 (black lines) transduced Raji B cells. The dashed line representsstaining of the isotype matched control mAbs. B, CellTracker Orange la-beled Raji B cells were pulsed with or without SEE and mixed with JurkatT cells. Then, the B cell/T cell conjugates were fixed and stained witheither a CD3 mAb or an Ab against LAT. C, shCtr- or CD48-silenced RajiB cells were pulsed with SEE and incubated with either shCtr-, shCD2-, orshCD48-silenced Jurkat T cells. The B cell/T cell conjugates were analyzedfor redistribution of LAT to the B cell/T cell interface. Two individualsanalyzing at least 100 conjugates counted the number of conjugates withredistributed LAT from two independent blinded samples.

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Nonidet P-40 as a detergent, and both the precipitated and non-precipitated material was analyzed by SDS-PAGE and Westernblotting. Using mAbs against other T cell surface markers such asMHC class I we confirmed the specificity of the assay (14) (datanot shown). A similar approach using magnetic beads coated withTCR mAb and mechanical cell disruption has been also success-fully used for isolation of TCR-associated membrane patches (28).

When we subjected shRNA expressing control cells to SPSP,CD48 readily coprecipitated with CD3 after 10 min of incubation(Fig. 1C, right). The association of CD48 was transient because wecould not detect CD48 molecules in the CD3 precipitates of thesecells after 60 min of stimulation. At this time point, only CD2stayed in complex with CD3. CD59 did not associate at any timewith CD3 confirming our data from single molecule microscopy(14). In contrast, in the CD2 knockdown cells the CD3 precipitateswere completely devoid of CD48 (Fig. 1C). CD2 is thus crucial forrecruitment of CD48 to the TCR/CD3 complex upon T cellactivation.

CD48 is dispensable for recruitment of CD2 but necessary forassociation of LAT to the TCR complex

Next, we were interested whether the CD2 ligand CD48 mightinfluence the recruitment of CD2 and TCR signalosome compo-nents to the TCR complex. To answer this question, we subjectedthe Jurkat IL-2-Luc cells to shRNA-mediated gene knockdown ofCD48. With an shRNA targeting position 884, again we obtaineda sufficient knockdown efficiency of �90% (Fig. 2A). An shRNAtargeting position 193 reduced also significantly CD48 expressionbut the effect was weaker as found with position 884 (data notshown).

Using the CD48 knockdown cells, we analyzed the influence ofCD48 on association and reorganization of CD2, CD59, LAT, andLck with the TCR/CD3. After 10 min of stimulation on the CD3mAb-coated surface, we found quantitative association of CD3,CD2, CD48, LAT, and Lck in control cells (Fig. 2B). In the CD48silenced cells, CD2 coprecipitated with CD3, while Lck recruit-ment was reduced and that of LAT completely lost. When we usedinstead of 1% Nonidet P-40, 0.5% N-dodecyl-D-maltoside as adetergent, we detected no interaction of CD3 with either CD48 orCD2 both in control and silenced cells (data not shown). As sol-

ubilization of lipids from membrane compartments by N-dodecyl-D-maltoside is significantly enhanced compared with NonidetP-40 (29), the differential results indicate a role for protein-lipidinteractions, i.e., lipid rafts, in the recruitment of these molecules.We also tested the recruitment of Lck and LAT to the TCR/CD3complex of CD2-silenced cells and found a complete loss ofLAT recruitment. In contrast to CD48 silenced cells, CD2 si-lencing also led to a defect in Lck association to the TCR/CD3complex (Fig. 2C).

Because CD2 was needed for association of CD48 with theTCR/CD3 complex, we assumed that the CD2-CD48 interactionmight be important for recruiting the TCR to lipid rafts. If thiswere true, we would expect reduced distribution of the CD3/TCRto the lipid raft containing low-density fractions in a sucrose gra-dient. Therefore, we stimulated control and CD2-silenced Jurkatcells in solution with a soluble CD3 IgM mAb (MEM-92). Then,we subjected the cell lysates to ultracentrifugation on a sucrosegradient. CD3 stimulation led to redistribution of CD3 to lipid raftfractions of the sucrose gradient, and indeed, CD2 silencing led toa significant decrease of CD3 in lipid raft fractions (Fig. 3A). Asimilar result was obtained with CD48 silenced cells (data notshown).

The observed defects of LAT and Lck recruitment and the ab-errant raft distribution of CD3 in CD2 or CD48 silenced cellsshould affect tyrosine phosphorylation and calcium mobilizationupon TCR engagement. To assess the influence of CD2 on prox-imal TCR signaling, we stimulated the CD2-silenced Jurkat cellswith CD3 mAb MEM-92 and analyzed the total cell lysates byWestern blotting. We found a strongly diminished phosphorylationof LAT at all time points in CD2-silenced cells compared with thecontrol cells. We did not detect any difference in phosphorylationof the negative regulatory Tyr 505 of Lck when comparing CD2silenced with control Jurkat cells (Fig. 3B). Next, we analyzed thecalcium mobilization of control, CD2- or CD48-silenced Jurkatcells upon TCR cross-linking with the anti-TCR mAb C305 andfound a significant reduction of the calcium flux in both silencedcells (Fig. 3C).

To get hold of the TCR-proximal signalosome dependent onCD2 and CD48, we again applied the SPSP method and analyzedthe pool of phosphorylated proteins coprecipitated with CD3

FIGURE 6. cis-interaction of CD2 and CD48 is critical for T cell IL-2-expression. A, Control, CD2 or CD48 silenced Jurkat IL-2-Luc cells werestimulated in different ways or left unstimulated. For stimulation, the cells were either cocultured for 8 h at a 1:1 ratio with SEE pulsed Raji B cells as APCs,or stimulated for 16 h with plate-bound CD3 mAb OKT3 (5 �g/ml), with or without soluble CD28 mAb Leu28 (3 �g/ml). The CD2-negative Raji B cellswere either transduced with control shRNA or CD48 specific shRNA to silence CD48 on the APC. After stimulation, the cells were lysed and luciferaseactivity was measured. One representative experiment of three is shown. In this experiment, for silencing of CD2 and CD48 the shRNA constructs targetingpositions 341 and 864, respectively, were used. Similar results were obtained when using the alternative constructs targeting positions 884 and 193,respectively. B, ELISA measurement of IL-2 secretion of control, CD2-, or CD48-silenced Jurkat IL-2-Luc cells upon stimulation with SEE-pulsed RajiB cells.

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(Fig. 4). Anti-phosphotyrosine blots showed an overall reductionin phosphoprotein bands associated with TCR/CD3 in both CD2and CD48 silenced Jurkat cells, especially at early time points ofstimulation (Fig. 4, A and B). Interestingly, we did not detect anydifference in the phosphoprotein patterns of control and silencedcells in the nonprecipitated material. This indicates that signaltransduction still occurs in the absence of CD2 and CD48 but thelack of formation of a stable signalosome at the TCR impairs chan-neling the signals into the required downstream signaling path-ways. These data are in accord with depletion studies of choles-terol that destroyed lipid rafts resulting in uncontrolled T cellactivation by transient tyrosine phosphorylation of multiple pro-teins (30).

Next, we tested whether CD2 or CD48 silencing resulted in adefect of LAT reorganization to the IS. To test this, we induced aT/B cell synapse by stimulating the Jurkat T cells with Raji B cellspulsed with SEE. As verified by flow cytometry, Raji B-cells were

negative for CD2 but expressed CD48 (Fig. 5A). Therefore, toexclude a potential costimulatory effect of CD48 by trans interac-tion, we silenced the CD48 expression on Raji B cells using theCD48 shRNA constructs. Similar to Jurkat cells, the shRNA con-struct targeting position 884 had the strongest down-regulatoryeffect. Other markers such as MHC class II were not affected (Fig.5A). Fixed B/T cell conjugates were stained for CD3 and LAT(Fig. 5B). Indeed, we found decreased recruitment of LAT to thecontact plane between Jurkat T cells silenced for either CD2 orCD48 and SEE pulsed Raji B cells. This microscopic observationis in accord with the biochemical signalosome precipitation datashown in Fig. 2, B and C.

Then, we analyzed the influence of CD2 and CD48 silencing onthe IL-2 expression of T cells in a functional assay. In agreementwith earlier data (12, 31), CD2- and CD48-silenced Jurkat T cellsshowed strongly decreased IL-2 promoter activity (Fig. 6A) andIL-2 levels in the culture supernatant when stimulated with RajiB-cells SEE (Fig. 6B). We found the same or an even strongerinhibition of IL-2 promoter activity when CD48 was silenced inthe Raji cells (Fig. 6A). Furthermore, we found decreased IL-2promoter activity in CD2 and CD48 silenced cells when stimulatedvia plate-bound OKT3 against CD3, with or without soluble CD28Abs (Fig. 6A).

Finally, to exclude the possibility that the functions of CD2 andCD48 in organizing the TCR signalosome in Jurkat T cells arepeculiar for and restricted to this cell line, we silenced CD2 orCD48 in primary human peripheral blood T cells. To down-regu-late the target molecules in these cells, we performed a primarystimulation via CD3 and CD28 in the presence of control, CD2, orCD48 shRNA containing lentiviruses over a period of 6 days. Af-ter a resting period of 5 days, we found a significant reduction ofsurface expression of both CD2 (35% down-regulation) and CD48(50% down-regulation) (Fig. 7A). Then, we restimulated thesecells via CD3 plus CD28 and measured the IL-2 secretion. Inagreement with the results obtained with Jurkat T cells, CD2 si-lencing resulted in �90% and CD48 silencing in �65% inhibitionof IL-2 secretion (Fig. 7B). Taken together, our data are pointingto the engagement of CD2 in lateral interactions of the TCR/CD3complex with other T cell membrane molecules, in particularCD48, for signal transduction.

DiscussionGPI-anchored proteins are well-characterized components ofmembrane microdomains, so-called lipid rafts that are rich in sig-naling molecules (9). Engagement of GPI-anchored surface pro-teins can deliver TCR-like signals leading to a rise in intracellularcalcium and a wave of protein tyrosine phosphorylation. GPI-an-chored proteins have thus potential to play a role as co-stimulatorymolecules in T cell activation (11). Indeed, for GPI-anchoredCD48, a role in amplification of TCR signaling has been firmlydemonstrated (12). By using specific mAbs, a similar role has beenascribed to CD59, the second abundant GPI-protein expressed byT cells (32, 33). However, recent data using cells and mice defi-cient in CD59 rather indicate an inhibitory role of this molecule inT cell biology (34).

Heterogeneity in protein content and lipid composition of lipidrafts has been reported (15–17). Whether the lipid environment orprotein-protein interactions are the major determinants of this het-erogeneity is a matter of debate. We recently showed by singlemolecule microscopy and immunobiochemistry that the GPI-an-chored molecules CD48 and CD59 reorganized and arranged dif-ferently in semiartificial synapses of human peripheral blood Tlymphocytes induced on CD3 mAb coated glass slides. Although

FIGURE 7. Silencing of CD2 or CD48 in human peripheral blood Tcells impairs TCR signaling for IL-2 expression. Human peripheral bloodT lymphocytes silenced for CD2 or CD48 were generated as described inMaterials and Methods. A, Surface expression analysis of CD2 and CD48on control shRNA, shCD2-, and shCD48-treated T cells blasts. The blacklines represent the staining with the specific CD2 and CD48 mAbs; thedashed lines represent staining of the isotype matched control mAbs. B, Forthe analysis of IL-2 secretion, the cells were stimulated for 24 h with theindicated concentrations of plate-bound CD3 mAb OKT3 plus solubleCD28 mAb Leu28 (2 �g/ml). After 24 h, the culture supernatants wereharvested and secreted IL-2 quantified by the Luminex assay.

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CD48 was rapidly immobilized and pulled to the TCR/CD3 com-plex, CD59 stayed highly mobile and away from CD3 (14). It isnow generally believed that the TCR complex, which resides out-side lipid rafts in resting T cells, gets associated with rafts uponTCR engagement (35). As commonly used biochemical extractionmethods assign CD48 and CD59 to the same “detergent resistant”membrane fraction, in a simplified view, raft-recruitment of theTCR should lead to equal colocalization with different raft mark-ers. However, this is not the case and hence lipid-mediated proteinassociations alone cannot fully explain the differential behavior ofCD48 and CD59.

Protein-protein interactions may be an additional determinantfor CD48 recruitment to the TCR. Work of Douglass and Valedescribed that CD2, Lck, and LAT coclustered in microdomainson Jurkat cells stimulated on TCR Ab coated surfaces (22). Clus-tering was dependent on phosphorylation of LAT and functionalLck, thus protein-protein interactions are a likely explanation forthe microcluster formation.

In the murine system, CD48 is a well-described ligand for CD2in both trans- and cis-configurations (18–20). Human CD2-CD48interactions were reported to be of lower affinity (21). We thusreasoned that CD48 recruitment to the TCR/CD3 complex couldbe associated with the above-described CD2/LAT/Lck clusters viaa cis-interaction of CD2 with CD48 on the surface of T cells.Indeed, our CD2 knockdown completely abrogated TCR/CD3 as-sociation with CD48 (Fig. 1), Lck, and LAT (Fig. 2C). This meansthat CD2 is required for efficient association of the TCR with Lck,CD48, and LAT. CD2 was found to physically interact with the Srcfamily kinases Lck and Fyn and treatment of T cells with CD2 mAbsled to an increase in Lck kinase activity (12, 36). This interactionmight be mediated by binding of the kinases’ SH3-domains to aseries of proline-rich stretches in the cytoplasmic domain of CD2.A similar mechanism of Lck activation via proline-containing se-quences has been proposed for CD28 (37). However, in contrast tohumans, this proline stretch might not be sufficient in the murinesystem where CD2 contains further a CIC motif at the membrane-proximal region for interaction with Lck (38).

The impact of CD48 on T cell activation has been recognizedwith the description of severe T cell signaling defects in the CD48knockout mouse (13). Coengagement of the TCR with CD48 viamAb enhances localization of tyrosine-phosphorylated TCR-� tolipid rafts and boosts Ag-induced T cell activation, most probablyvia its association to Lck (12). In accordance with published data,our CD48 silenced T cells showed reduced IL2 synthesis uponTCR-stimulation. We thus conclude that TCR-CD48 clustering isan important early event for an efficient initiation of T cell signal-ing. In fact, CD48 knockdown in Jurkat cells led to a completeabsence of LAT-recruitment to the TCR/CD3 complex (Figs. 2 and5), providing a mechanistic insight into CD48-mediated modula-tion of TCR-signaling.

Taken together, our results reveal a hierarchy in the recruitmentof early signaling molecules to the TCR/CD3 complex mediatedby a cis-interaction of CD2 and CD48. We could demonstrate thatCD2 acts as master regulator of Lck and CD48 association to theTCR/CD3 complex. CD48, in turn, assists as a molecular carrierof LAT.

AcknowledgmentsWe thank Vaclav Horejsi for providing Abs, Thomas Baumruker for pro-viding Jurkat IL-2-Luc cells, and Sheila Stewart for providing the plasmidpLKO-puro1. We are grateful to Eva Steinhuber and Margarethe Merio forgeneral technical assistance and LUMINEX quantification of cytokines,respectively.

DisclosuresThe authors have no financial conflict of interest.

References1. Weiss, A., and D. R. Littman. 1994. Signal transduction by lymphocyte antigen

receptors. Cell 76: 263–274.2. Lindquist, J. A., L. Simeoni, and B. Schraven. 2003. Transmembrane adapters:

attractants for cytoplasmic effectors. Immunol. Rev. 191: 165–182.3. Zhang, W., C. L. Sommers, D. N. Burshtyn, C. C. Stebbins, J. B. DeJarnette,

R. P. Trible, A. Grinberg, H. C. Tsay, H. M. Jacobs, C. M. Kessler, et al. 1999.Essential role of LAT in T cell development. Immunity 10: 323–332.

4. Zhang, W., B. J. Irvin, R. P. Trible, R. T. Abraham, and L. E. Samelson. 1999.Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line. Int. Immunol. 11: 943–950.

5. Monks, C. R., B. A. Freiberg, H. Kupfer, N. Sciaky, and A. Kupfer. 1998. Three-dimensional segregation of supramolecular activation clusters in T cells. Nature395: 82–86.

6. Bunnell, S. C., D. I. Hong, J. R. Kardon, T. Yamazaki, C. J. McGlade, V. A. Barr,and L. E. Samelson. 2002. T cell receptor ligation induces the formation ofdynamically regulated signaling assemblies. J. Cell Biol. 158: 1263–1275.

7. Saito, T., and T. Yokosuka. 2006. Immunological synapse and microclusters: thesite for recognition and activation of T cells. Curr. Opin. Immunol. 18: 305–313.

8. Montixi, C., C. Langlet, A. M. Bernard, J. Thimonier, C. Dubois, M. A. Wurbel,J. P. Chauvin, M. Pierres, and H. T. He. 1998. Engagement of T cell receptortriggers its recruitment to low-density detergent-insoluble membrane domains.EMBO J. 17: 5334–5348.

9. Horejsi, V. 2003. The roles of membrane microdomains (rafts) in T cell activa-tion. Immunol. Rev. 191: 148–164.

10. Stefanova, I., V. Horejsi, I. J. Ansotegui, W. Knapp, and H. Stockinger. 1991.GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Sci-ence 254: 1016–1019.

11. Loertscher, R., and P. Lavery. 2002. The role of glycosyl phosphatidyl inositol(GPI)-anchored cell surface proteins in T-cell activation. Transplant. Immunol. 9:93–96.

12. Moran, M., and M. C. Miceli. 1998. Engagement of GPI-linked CD48 contributesto TCR signals and cytoskeletal reorganization: a role for lipid rafts in T cellactivation. Immunity 9: 787–796.

13. Gonzalez-Cabrero, J., C. J. Wise, Y. Latchman, G. J. Freeman, A. H. Sharpe, andH. Reiser. 1999. CD48-deficient mice have a pronounced defect in CD4� T cellactivation. P. Natl. Acad. Sci. USA 96: 1019–1023.

14. Drbal, K., M. Moertelmaier, C. Holzhauser, A. Muhammad, E. Fuertbauer,S. Howorka, M. Hinterberger, H. Stockinger, and G. J. Schutz. 2007. Single-molecule microscopy reveals heterogeneous dynamics of lipid raft componentsupon TCR engagement. Int. Immunol. 19: 675–684.

15. Schade, A. E., and A. D. Levine. 2002. Lipid raft heterogeneity in human pe-ripheral blood T lymphoblasts: a mechanism for regulating the initiation of TCRsignal transduction. J. Immunol. 168: 2233–2239.

16. Brugger, B., C. Graham, I. Leibrecht, E. Mombelli, A. Jen, F. Wieland, andR. Morris. 2004. The membrane domains occupied by glycosylphosphatidyli-nositol-anchored prion protein and Thy-1 differ in lipid composition. J. Biol.Chem. 279: 7530–7536.

17. Kiyokawa, E., T. Baba, N. Otsuka, A. Makino, S. Ohno, and T. Kobayashi. 2005.Spatial and functional heterogeneity of sphingolipid-rich membrane domains.J. Biol. Chem. 280: 24072–24084.

18. Kato, K., M. Koyanagi, H. Okada, T. Takanashi, Y. W. Wong, A. F. Williams,K. Okumura, and H. Yagita. 1992. CD48 is a counter-receptor for mouse CD2and is involved in T cell activation. J. Exp. Med. 176: 1241–1249.

19. Kaplan, A. J., K. D. Chavin, H. Yagita, M. S. Sandrin, L. H. Qin, J. Lin,G. Lindenmayer, and J. S. Bromberg. 1993. Production and characterization ofsoluble and transmembrane murine CD2: demonstration that CD48 is a ligand forCD2 and that CD48 adhesion is regulated by CD2. J. Immunol. 151: 4022–4032.

20. Zhu, B., E. A. Davies, P. A. van der Merwe, T. Calvert, and D. E. Leckband.2002. Direct measurements of heterotypic adhesion between the cell surface pro-teins CD2 and CD48. Biochemistry 41: 12163–12170.

21. Arulanandam, A. R., P. Moingeon, M. F. Concino, M. A. Recny, K. Kato,H. Yagita, S. Koyasu, and E. L. Reinherz. 1993. A soluble multimeric recombi-nant CD2 protein identifies CD48 as a low affinity ligand for human CD2: di-vergence of CD2 ligands during the evolution of humans and mice. J. Exp. Med.177: 1439–1450.

22. Douglass, A. D., and R. D. Vale. 2005. Single-molecule microscopy revealsplasma membrane microdomains created by protein-protein networks that ex-clude or trap signaling molecules in T cells. Cell 121: 937–950.

23. Hahn, W. C., E. Menu, A. L. Bothwell, P. J. Sims, and B. E. Bierer. 1992.Overlapping but nonidentical binding sites on CD2 for CD58 and a second ligandCD59. Science 256: 1805–1807.

24. Deckert, M., J. Kubar, D. Zoccola, G. Bernard-Pomier, P. Angelisova, V. Horejsi,and A. Bernard. 1992. CD59 molecule: a second ligand for CD2 in T cell adhe-sion. Eur. J. Immunol. 22: 2943–2947.

25. van der Merwe, P. A., A. N. Barclay, D. W. Mason, E. A. Davies, B. P. Morgan,M. Tone, A. K. Krishnam, C. Ianelli, and S. J. Davis. 1994. Human cell-adhesionmolecule CD2 binds CD58 (LFA-3) with a very low affinity and an extremely fastdissociation rate but does not bind CD48 or CD59. Biochemistry 33:10149–10160.

26. Pammer, P., R. Schlapak, M. Sonnleitner, A. Ebner, R. Zhu, P. Hinterdorfer,O. Hoglinger, H. Schindler, and S. Howorka. 2005. Nanopatterning of biomol-ecules with microscale beads. Chemphyschem. 6: 900–903.

7679The Journal of Immunology

by guest on June 4, 2017http://w

ww

.jimm

unol.org/D

ownloaded from

27. Bailey, S., and P. J. Macardle. 2006. A flow cytometric comparison of Indo-1 tofluo-3 and Fura Red excited with low power lasers for detecting Ca2� flux.J. Immunol. Methods 311: 220–225.

28. Harder, T., and M. Kuhn. 2001. Immunoisolation of TCR signaling complexesfrom Jurkat T leukemic cells. Sci. STKE 2001: PL1.

29. Davidson, D., M. Bakinowski, M. L. Thomas, V. Horejsi, and A. Veillette. 2003.Phosphorylation-dependent regulation of T-cell activation by PAG/Cbp, a lipidraft-associated transmembrane adaptor. Mol. Cell Biol. 23: 2017–2028.

30. Kabouridis, P. S., J. Janzen, A. L. Magee, and S. C. Ley. 2000. Cholesteroldepletion disrupts lipid rafts and modulates the activity of multiple signalingpathways in T lymphocytes. Eur. J. Immunol. 30: 954–963.

31. Teh, S. J., N. Killeen, A. Tarakhovsky, D. R. Littman, and H. S. Teh. 1997. CD2regulates the positive selection and function of antigen-specific CD4� CD8� Tcells. Blood 89: 1308–1318.

32. Deckert, M., M. Ticchioni, B. Mari, D. Mary, and A. Bernard. 1995. The gly-cosylphosphatidylinositol-anchored CD59 protein stimulates both T cell receptor�/ZAP-70-dependent and -independent signaling pathways in T cells. Eur. J. Im-munol. 25: 1815–1822.

33. Korty, P. E., C. Brando, and E. M. Shevach. 1991. CD59 functions as a signal-transducing molecule for human T cell activation. J. Immunol. 146: 4092–4098.

34. Longhi, M. P., B. Sivasankar, N. Omidvar, B. P. Morgan, and A. Gallimore.2005. Cutting edge: murine CD59a modulates antiviral CD4� T cell activity ina complement-independent manner. J. Immunol. 175: 7098–7102.

35. Miceli, M. C., M. Moran, C. D. Chung, V. P. Patel, T. Low, and W. Zinnanti.2001. Co-stimulation and counter-stimulation: lipid raft clustering controls TCRsignaling and functional outcomes. Semin. Immunol. 13: 115–128.

36. Marie-Cardine, A., I. Maridonneau-Parini, M. Ferrer, S. Danielian, B. Rothhut,R. Fagard, A. Dautry-Varsat, and S. Fischer. 1992. The lymphocyte-specific ty-rosine protein kinase p56lck is endocytosed in Jurkat cells stimulated via CD2.J. Immunol. 148: 3879–3884.

37. Holdorf, A. D., J. M. Green, S. D. Levin, M. F. Denny, D. B. Straus, V. Link,P. S. Changelian, P. M. Allen, and A. S. Shaw. 1999. Proline residues in CD28and the Src homology (SH)3 domain of Lck are required for T cell costimulation.J. Exp. Med. 190: 375–384.

38. Nunes, R. J., M. A. Castro, C. M. Goncalves, M. Bamberger, C. F. Pereira,G. Bismuth, and A. M. Carmo. 2008. Protein interactions between CD2 and Lckare required for the lipid raft distribution of CD2. J. Immunol. 180: 988–997.

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