the late endosomal transporter cd222 directs the spatial ...hannes stockinger,* and vladimir...

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of LckDirects the Spatial Distribution and Activity The Late Endosomal Transporter CD222

Hannes Stockinger and Vladimir LeksaHerbert B. Schiller, Gabriela Ondrovicová, Oreste Acuto, Alexander Zwirzitz, Clemens Donner, Cyril Boulegue,Supper, Anna Ohradanova-Repic, Paul Eckerstorfer, Karin Pfisterer, Florian Forster, Wolfgang Paster, Verena

http://www.jimmunol.org/content/193/6/2718doi: 10.4049/jimmunol.1303349August 2014;

2014; 193:2718-2732; Prepublished online 15J Immunol

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The Journal of Immunology

The Late Endosomal Transporter CD222 Directs the SpatialDistribution and Activity of Lck

Karin Pfisterer,* Florian Forster,* Wolfgang Paster,*,† Verena Supper,*

Anna Ohradanova-Repic,* Paul Eckerstorfer,* Alexander Zwirzitz,* Clemens Donner,*

Cyril Boulegue,‡ Herbert B. Schiller,*,‡ Gabriela Ondrovi�cova,x Oreste Acuto,†

Hannes Stockinger,* and Vladimir Leksa*,x

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell

activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central

component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling

cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions,

which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-

deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphory-

lation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting

T cells, which guarantees operative T cell effector functions. The Journal of Immunology, 2014, 193: 2718–2732.

Tcell signaling molecules have to be tightly controlledspatially and temporally to ensure proper T cell activationwithout hyperresponsiveness (1). A fundamental step

during the onset of T cell signaling is the recruitment of thelymphocyte-specific protein tyrosine kinase (Lck) to the im-munological synapse (IS) and the subsequent phosphorylation ofthe ITAMs of the CD3 z-chain (CD3z). ZAP70 can thereafter bindto the phosphorylated ITAMs of the TCR complex where it isphosphorylated by Lck to initiate further downstream signaling(2). For fine-tuning of T cell activation active and inactive Lck poolsmust be kept at equilibrium. It has been shown that Lck activity isnot directly linked with TCR engagement (3, 4). Lck rather existsin several conformational and activity states: 1) an unphosphory-lated “primed” form; 2) an active form phosphorylated at the

tyrosine 394 (pY394); 3) an inactive “closed“ form phosphory-lated at the COOH-terminal tyrosine 505 (pY505); and 4) adouble phosphorylated form (pY394 + pY505) that is thought topossess an equal kinase activity as single phosphorylated Lck atY394 (3). Hence, active Lck is constitutively present in restingT cells, and it is its coordinated intracellular distribution that iscrucial to balance T cell activity and nonreactivity (5–7). Theantagonistic activities of the phosphatase CD45 and the kinaseCsk control Lck’s mode of action (2, 8, 9). CD45 plays a pivotalrole in the activation of Lck via dephosphorylation of Y505 (10,11), but it is not clear where the interaction takes place and how itis regulated in space and time.The late endosomal transmembrane molecule CD222, also known

as the cation-independent mannose 6-phosphate/insulin-like growthfactor 2 receptor, is a multifunctional broadly expressed regulator ofprotein trafficking (12). It binds mannose 6-phoshate–bearing proteinsincluding acid hydrolases and TGF-b (13), the insulin-like growthfactor 2 (14) and plasminogen (15). On delivering its cargo, CD222traffics between the trans-Golgi network (TGN), endosomes, and theplasma membrane (16). CD222 is barely expressed on the surface ofresting T cell but has been reported to be upregulated at the plasmamembrane uponT cell activation in rodents (17). In this study,we showthat CD222 is also upregulated on the surface of human T cellsupon stimulation. Remarkably, we found that CD222 transportsLck intracellularly and directs its distribution to CD45 at the plasmamembrane. The loss of CD222 causes a decrease in signal transduc-tion and subsequentlya profoundblockadeofTcell effector functions.

Materials and MethodsAbs

The mAb to CD222, unlabeled and conjugated with AF647 (MEM-238),CD45, both unlabeled (MEM-28) and conjugated with Pacific Orange(HI30), and CD4, unlabeled (MEM-241), were purchased from EXBIO(Prague, Czech Republic); the mAb to CD3 (MEM-57), CD59 (MEM-43/5),pTag (H902), and a-fetoprotein (AFP-12) were provided by Dr. V. Ho�rej�sı(Institute of Molecular Genetics, Academy of Sciences of the CzechRepublic, Prague, Czech Republic). The anti–TCRb-chain mAb C305was a gift from Dr. A. Weiss (University of California, San Francisco,San Francisco, CA). The stimulatory CD3 mAb OKT3 was from Ortho

*Molecular Immunology Unit, Institute for Hygiene and Applied Immunology,Center for Pathophysiology, Infectiology and Immunology, Medical University ofVienna, Vienna A-1090, Austria; †Sir William Dunn School of Pathology, Universityof Oxford, Oxford OX1 3RE, United Kingdom; ‡Department of Molecular Medicine,Max-Planck Institute of Biochemistry, Martinsried 82152, Germany; and xLaboratoryof Molecular Immunology, Institute of Molecular Biology, Slovak Academy of Sci-ences, 84551 Bratislava, Slovak Republic

Received for publication December 17, 2013. Accepted for publication July 3, 2014.

This work was supported by the Austrian Science Fund (FWF Fonds zur Forderungder Wissenschaftlichen Forschung) P22908 (to V.L.), European Union FrameworkProgram 7 “NANOFOL” Grant NMP4-LA-2009-228827 (to H.S.), Wellcome TrustGrant GR076558MA, and European Union Framework Program 7 Grant EC-FP7-SYBILLA-201106 (to O.A.). W.P. was supported by the Erwin Schroedinger Fellow-ship from the Austrian Science Fund.

Address correspondence and reprint requests to Dr. Vladimir Leksa and Dr. HannesStockinger, Institute for Hygiene and Applied Immunology, Center for Pathophysi-ology, Infectiology, and Immunology, Medical University of Vienna, Lazarettgasse19, 1090 Vienna, Austria. E-mail addresses: [email protected] (V.L.)and [email protected] (H.S.)

The online version of this article contains supplemental material.

Abbreviations used in this article: AF, Alexa Fluor; AFP, a-fetoprotein; CLSM,confocal laser scanning microscopy; CTR, control; EEA1, early endosome Ag 1;IS, immunological synapse; LAT, linker of activated T cells; LC-MS, liquidchromatography–mass spectrometry; Luc, luciferase; MAL, myelin and lymphocyteprotein; mut, mutant; OST, One-Strep-tag; pY, phospho-tyrosine; SEE, staphylococ-cal enterotoxin E; sh, short hairpin; TGN, trans-Golgi network; wt, wild-type.

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

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Pharmaceuticals (Raritan, NJ). The CD28 mAb Leu-28, anti–phospho-CD3z/CD247 (Y142) mAb K25-407.69, and anti–phospho-linker of acti-vated T cells (LAT; Y171) were from BD Biosciences (San Jose, CA). TheHRP-conjugated anti-phosphotyrosine mAb 4G10 was from MerckMillipore (Billerica, MA). The anti-GAPDH mAb 14C10, anti–phospho-ZAP70 Ab (Y319)/Syk (Y352), anti-ZAP70 mAb 99F2 and L1E5, anti–phospho-p44/42 MAPK Ab ERK1/2 (Y202/Y204), anti-p44/42 MAPK AbERK1/2, anti–phospho-Src mAb (Y416), anti–non-phospho-Src mAb(Y416), anti–phospho-Lck Ab (Y505), anti-Lck mAb 73A5, and the rabbitmAb to early endosome Ag 1 (EEA1) C45B10 were from Cell SignalingTechnology (Danvers, MA). The anti-Lck mAb H-95 was from Santa CruzBiotechnology (Santa Cruz, CA). Biotinylation of unconjugated mAb wasperformed in our laboratory. As secondary reagents we used HRP-con-jugated goat anti-mouse and anti-rabbit IgG (Sigma-Aldrich, St. Louis,MO), streptavidin-HRP conjugate (GE Healthcare, Buckinghamshire,U.K.), and goat anti-mouse IgG+IgM (H+L)-FITC conjugate (An der Grub,Kaumberg, Austria).

Cell culture

The T cell line Jurkat E6.1 and the B cell line Raji were from the AmericanType Culture Collection. The Lck-deficient Jurkat T cell line JCaM 1.6(18) was from the European Collection of Cell Culture (Salisbury, U.K.).The immortalized CD222 negative mouse fibroblasts were provided byE. Wagner (Spanish National Cancer Research Centre, Madrid, Spain).The Jurkat IL-2 Luc T cells were stably transfected with a luciferaseexpressing IL-2 promoter reporter construct as described previously (19).The Jurkat NFAT Luc cells containing an artificial promoter region withfour NFAT binding sites were created in our laboratory. Human PBMCwere isolated from purchased buffy coats of healthy adult volunteers (RotesKreuz, Vienna, Austria) by density gradient centrifugation on Lymphoprep(Nycomed). All primary human T cells as well as mouse and humanT cell lines were maintained in RPMI 1640 medium, the HEK-293 cellsin DMEM, all supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 2 mmol/L L-glutamine (Life Technologies, Carlsbad, CA),100 mg/ml penicillin (Life Technologies), and 100 mg/ml streptomycin(Life Technologies). Cells were grown in a humidified atmosphere at 37˚Cand 5% CO2 and passaged every 2–3 d.

T cell activation assay

Primary human T cells isolated by CD14-depletion from PBMC werecultured for 2 d to separate non- from adherent cells. T cell lines and primaryhuman T cells (5 3 104) were seeded in 96-well plates and kept unstimu-lated or stimulated by: 1) plate-bound CD3 mAb OKT3 (1 mg/ml) alone ortogether with soluble CD28 mAb Leu-28 (0.5 mg/ml); 2) CD3 Ab-coupledbeads; 3) staphylococcal enterotoxin E (SEE)–loaded Raji B cells; or 4)PMA (5 ng/ml) plus ionomycin (1 mmol/l). After indicated time intervals,the cells were harvested and analyzed via flow cytometry, immunoblotting,microscopy, or functional cellular assays. To measure proliferation, beforestimulation, the cells were resuspended in PBS, incubated with CFSE(0.5 mmol/l; Molecular Probes, Eugene, OR) at 37˚C for 10 min, andthereafter washed twice with medium.

Immunoprecipitation, immunoblotting, and mass spectrometricanalysis

For immunoprecipitation of CD222, 2–20 3 107 cells were lysed in lysisbuffer (20 mmol/l Tris and 140 mmol/l NaCl [pH 7.5]), containing 1%lauryl maltoside or 1% Nonidet P-40 as detergent, complete protease in-hibitor mixture (Roche, Basel, Switzerland), and benzonase (Novagen,Merck Millipore) for 30 min on ice. Lysates were incubated for 2 h at 4˚Cwith cyanogen bromide–activated beads coupled with CD222 mAb MEM-238 or isotype control mAb AFP-12. The beads were washed three timeswith ice-cold lysis buffer, and the bound proteins were eluted with 1.23nonreducing Laemmli sample buffer at 95˚C for 5 min.

For immunoprecipitation of Lck, the Lck-deficient Jurkat T cell cloneJCaM 1.6 was transduced with a lentiviral expression construct for Lckfused to the One-Strep-tag epitope (Lck-OST). Lysates of resting or sodiumpervanadate–stimulated cells were subjected to precipitation with Strep-tactin–Sepharose beads. Biotin was used to elute bait proteins from thecapture beads. Biotin-blocked beads served as a negative control.

For the analysis of theCD45–Lck interaction at theplasmamembrane, JCaM1.6 T cells expressing Lck-GFP were silenced for CD222. Then, the silencedandcontrol cellswere surface labeledwithCD45mAbon ice,washed, and lysedwith lysis buffer containing detergent 1% Nonidet P-40. The mAb complexeswere then precipitated by using proteinA/G beads (Santa Cruz Biotechnology).

For specific immunoprecipitation of cell surface CD222, unstimulatedPBMC were labeled with mAb against CD222, CD45 (positive control), or

AFP-12 (negative control). Proteins were cross-linked with 1 mM DSP(Pierce, Thermo Scientific Fisher, Rockford, IL), and cells were lysed withdetergent 1% lauryl maltoside on ice. The Ab–protein complexes werefished with protein A/G beads overnight. Beads were washed three times,and proteins were eluted with Laemmli sample buffer.

The immunoprecipitates were analyzed by Western blotting. Briefly,proteins were separated by SDS-PAGE, followed by transfer of the proteins toImmobilon polyvinylidene difluoride membranes (Merck Millipore). Themembranes were blocked with 5% nonfat milk (Maresi, Vienna, Austria) inTBS-Tween and incubated with specific Ab dilutions for detection of indicatedproteins. For visualization, blots were developed either with HRP secondaryconjugates and a HRP substrate (Biozym, Hessisch Oldendorf, Germany) orwith secondary fluorescenceAbs, followed by detection at either the Fuji LAS-400 imager (Fuji, Tokyo, Japan) or the Odyssey infrared imaging system(LiCor Biosciences, Lincoln, NE). Band intensities were quantified via MultiGauge Software Version 3.1 (Fuji), Image Studio Version 2.0, or ImageJ.

For mass spectrometric analysis, CD222 coimmunoprecipitates and cor-responding controls were digested in-solution as described previously (20),and samples were prepared, according to the filter aided sample preparationprocedure following analysis via liquid chromatography–mass spectrometry(LC-MS). Lck coimmunoprecipitates were separated on 4–12% gradientBis-Tris NuPAGE gels (Invitrogen Life Technologies), the gels were washedin distilled water, lightly stained with Colloidal Blue (Invitrogen LifeTechnologies), and subjected to Gel-LC-MS as described previously (21).

Gene knockdown via RNA interference and retroviraltransduction

Stable knockdown cell lines were produced via lentiviral introduction ofshort hairpin (sh)RNA specific for the CD222 mRNA. The following CD222silencing sequences were used for cloning into the vector pLKOpuro1 (a giftfrom S. Steward, Washington University School of Medicine, St. Louis, MO):shCD222/P1 at position 6588 with the sequence 59-GCCCAACGATCAG-CACTTCttcaagagaGAAGTGCTGATCGTTGGGC-39 and shCD222/P2 atposition 4525 with the sequence 59-GAGCAACGAGCATGATGACttcaaga-gaGTCATCATGCTCGTTGCTC-39. TheMISSIONnontarget shRNAcontrolvector (pLKOpuro1; Sigma-Aldrich) was used as a negative control (shCTR).Virus particles were generated via transfection of HEK-293 cells with the si-lencing constructs and the lentiviral envelope coding plasmid pMD2.G and thepackaging vector psPAX2 (bothhelping vectorswere constructedbyD.Trono,Ecole Polytechnique Federal de Lausanne, and obtained from Addgene,Cambridge, MA) as described previously (22). After 48 h, the virus particleswere harvested, filtered, and used for the target cell line infections in thepresence of 5 mg/ml polybrene (Sigma-Aldrich). The next day, the cells werewashed with and maintained in medium containing 1 mg/ml puromycin(Sigma-Aldrich) to select transduced cells. CD222 knockdown cells wereused from 10 d to 2 mo postinfection for all experiments.

Lck constructs were prepared in the retroviral expression vector pBMN-Zfor transfection of the Phoenix amphotropic virus producer cell line (bothprovided by G. Nolan, Stanford University School of Medicine, Stanford,CA) (15). The target cells were transduced one to three times with the viralsupernatants depending on the efficacy of transduction and maintainedafterward in normal culture medium.

Gene knockout via zinc finger nucleases

Cys2His2-based zinc fingers were created via the Context DependentAssembly method provided by the free software tool ZiFiT to identify andtarget the genomic sequence of CD222 specifically (23). The zinc fingerconstructs targeting exons 18 and 22 were cloned into pMLM290/pMLM292 and pMSM800/pMLM802 (both from Addgene) to generatezinc finger nuclease expression constructs. These constructs were used forthe transfection of Jurkat T cells via electroporation. For this, 23 106 cellswere resuspended in 100 ml Cytomix (120 mM KCl, 0.15 mM CaCl2,5 mM MgCl2, 10 mM K2HPO4, 25 mM HEPES [pH 7.6], 2 mM EGTA,5 mM Glutathion, 1.25% DMSO [v/v], and 100 mM sucrose), mixed withthe plasmids (each 0.5 mg), transferred in electroporation cuvettes, andtransfected with the Amaxa System program S-18 (Amaxa, Lonza, Basel,Switzerland). Cells were immediately removed from the cuvettes, shortlyspun down, and cultured in 12-well plates containing prewarmed medium.After cell recovery and expansion, CD222-negative cells were repeatedlysorted with a FACSAria (BD Biosciences), expanded in Jurkat T cell–conditioned medium, and cultured in RPMI 1640 medium until analysis.

Immunofluorescence staining and confocal laser scanningmicroscopy

Primary T cells or T cell lines were let to adhere on adhesion slides(Superior-Marienfeld Laboratory Glassware, Lauda-Koningshofen, Germany),

The Journal of Immunology 2719


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fixed, and permeabilized with 4% formaldehyde and 0.1% saponin, re-spectively, and stained with the following Ab: the rabbit anti-Lck AbH-95 and the rabbit anti-human mAb EEA1 (C45B10), followed by agoat anti-rabbit AF488 Ab, the anti-Lck mouse Ab (73A5), followed byanti-mouse AF488 Ab, the AF647-conjugated CD222 mAb MEM-238,and the Pacific Orange-conjugated CD45 mAb HI30 (all diluted 1:50)in PBS containing 2% BSA. For stimulation of T cells before staining,cells were incubated with SEE-loaded Raji B cells at 37˚C for the indi-cated time points. Mouse fibroblasts were either cultured in 8-well chamberslides (Nunc, Thermo Scientific Fisher) until confluency or trypsinizedand cytospun onto glass slides prior to fixation and staining as describedabove. For each cell, one vertical and one horizontal cut was made throughthe cell, and the Lck distribution was analyzed using the ZEN 2011 blueedition software (Zeiss, Jena, Germany). Nuclei were stained with DAPI.The slides were washed with 13 PBS and mounted with mounting me-dium for fluorescence analysis (Vectashield; Vector Laboratories, Burlingame,CA). Isotype-matched controls were included. Pictures were captured witha confocal laser scanning microscopy (CLSM) 700 (Zeiss).

Flow cytometry

For the analysis of cell surface Ags, cells were washed and resuspended instaining buffer (13 PBS containing 1% BSA and 0.02% NaN3). Subse-quently, the cells were incubated for 20 min at 4˚C with a fluorochrome-conjugated mAb. For indirect immunofluorescence staining, the cells wereadditionally incubated with secondary conjugates for 20 min at 4˚C. Be-fore analysis, the cells were washed with staining buffer. Flow cytometrywas performed using a LSR II (BD Biosciences) and analyzed with FlowJoversion 8.8.6 for Macintosh or FlowJo version 7.2.5 for Windows.

Luciferase assays

To assess the IL-2 promoter activity and NFAT binding to DNA, IL-2 lu-ciferase- and NFAT-reporter Jurkat T cells were assayed, respectively. Briefly,the reporter cells (23 105) were either kept unstimulated or were stimulatedfor 6 h in triplicates in CD3 mAb OKT-3–coated (1 mg/ml) 96-well plateswith or without soluble CD28 mAb Leu-28. The cells were washed with PBSand subsequently lysed in 100 ml luciferase lysis reagent (Promega, Madi-son, WI) for 30 min on ice. Seventy-five microliters of lysate were transferredinto white 96-well microplates, and luminescence was measured after ad-dition of the reaction reagent at a Mithras LB940 Elisa Reader (BertholdTechnologies, Bad Wildbach, Germany). The protein concentration wasdetermined via Bradford assay (Bio-Rad, Hercules, CA), and the lumines-cence intensity was normalized to the protein content of each sample.

Cytokine measurement

Culture supernatants (30 ml) of primary T cells were harvested after 24 hof stimulation and analyzed via the Luminex xMAP suspension arraytechnology. Briefly, the cells were stimulated with plate-bound OKT3(1 or 5 mg/ml) and soluble Leu-28 (2 mg/ml) or kept unstimulated. Stan-dard curves were generated using recombinant cytokines (R&D Systems,Minneapolis, MN). Experiments were performed at least twice with eachsample in triplicates. Data are expressed as mean values of triplicates.

Calcium mobilization assay

Cells (1 3 106) were washed, resuspended in 100 ml RPMI 1640 mediumcontaining 1 mmol/l Indo-1 AM (Invitrogen Life Technologies) and incubatedfor 30 min at 37˚C. The cells were washed with medium, incubated in 1 mlmedium for next 30 min at 37˚C, and rested thereafter on ice until data ac-quisition at a LSR II flow cytometer. For the analysis of intracellular calciumflux, 300 ml Indo-1–loaded cells were prewarmed for 5 min at 37˚C, the base-line response was recorded for 30 s, before the cells were stimulated with a1:300 dilution of the hybridoma supernatant of the anti-TCR mAb C305for 3 min to analyze the increase in calcium mobilization. The indo-violet/indo-blue ratio was calculated with the FlowJo software and plotted against thetime. Ionomycin was used to check the overall responsiveness and cell viability.

Analysis of T cell signaling molecules

Jurkat T cells or primary T cells (1.5 3 106) were starved for 1 h in RPMI1640 medium containing 1% FCS at 37˚C, 5% CO2 and 80% humidity.Cells were rested on ice for 10 min, stimulatory mAb were added, allowedto bind for 5 min, and then, the cells were incubated for several time pointsat 37˚C. The cells were quickly washed with ice-cold lysis buffer withoutdetergent and lysed in complete lysis buffer containing 1% detergentNonidet P-40 (Promega), a protease inhibitor mixture (Roche), sodiumorthovanadate (Sigma-Aldrich), sodium fluoride, and benzonase for 30 minon ice. Lysates were sampled with 43 reducing or nonreducing samplebuffer and analyzed by Western blot analysis.

PCR analysis

For real-time PCR analysis, primary human T cells, both resting andstimulated for 1 d with plate-bound CD3 mAb OKT3 (1 mg/ml) togetherwith soluble CD28 mAb Leu-28 (0.5 mg/ml), were lysed in TRIzol(Invitrogen Life Technologies), and RNA was extracted according to themanufacturer’s instructions. cDNA was synthesized from 500 ng totalRNA with SuperScript VILO cDNA Synthesis Kit (Invitrogen Life Tech-nologies). Gene expression was measured by the 22DDCT method (24),based on quantitative real-time PCR using the CFX96 Real-Time PCR sys-tem (Bio-Rad) with TaqMan primer sets for human CD222, and YWHAZ asan endogenous control. For conventional PCR, 20 ng genomic DNA purifiedby standard isopropanol precipitation was used as a template of PCR am-plification with Taq polymerase (40 cycles at 95˚C for 30 s, 61˚C for 30 s, and72˚C for 30 s) and with primer sets for human CD222 bridging an exon–exonjunction, and intronless gene endosialin, as an endogenous control (TaqManGene Expression Assay; ABI, Life Technologies).

Lipid raft preparation

Cells (2–3 3 107) were lysed for 30 min on ice in lysis buffer containing1% detergent Brij-58 and protease inhibitors. Lysates were adjusted with80% sucrose solution to 40% sucrose in 1 ml, placed on a 1 ml 60% su-crose layer and overlaid by 20, 10, and 5% sucrose fractions (all 1 mlcontaining 0.5% detergent Brij-58 and protease inhibitors). Gradients wereultracentrifuged for 16–18 h at 150,000 3 g at 4˚C. Fractions (500 ml)were taken from the top to the bottom and denatured at 95˚C with samplebuffer and analyzed by SDS-PAGE, followed by Western blot analysis.

Gel filtration analysis

The GE Healthcare AKTA FPLC system was used for the gel filtrationexperiments. Cell lysates were prepared by solubilization with 1% detergentTriton X-100 (Promega). Samples were loaded at 4˚C in a volume of 500 mlonto a Superose 6 HR 10/300 GL column (GE Healthcare) equilibratedwith Triton X-100 (0.5%) in PBS. The absorbance at 280 nm was moni-tored, and 500-ml fractions were collected and analyzed by immunoblot-ting. The following standards of molecular mass (all from GE Healthcareor Sigma-Aldrich) were used: Blue dextran (2000 kDa), thyroglobulin(669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), BSA(66 kDa), and carbonic anhydrase (29 kDa).

Membrane–cytosol separation

Cells (6 3 107) were incubated in 2 ml hypotonic buffer (42 mmol/l po-tassium chloride, 5 mmol/l magnesium chloride, 10 mmol/l HEPES, andprotease inhibitor mixture [pH 7.5]) for 30 min on ice and thereafterpassaged nine times through a 30-g needle. Cell fragments were centri-fuged twice at 300 3 g, and the resulting supernatant was centrifuged for40 min at 35,000 3 g at 4˚C. The cytosolic supernatant was collected andthe pellet containing membranous cellular parts was solubilized in ex-traction buffer (13 lysis buffer containing 1% Triton X-100, 0.1%NaDodSO4, 0.5% sodium deoxycholate, and the protease inhibitor mix-ture) for 30 min on ice and centrifuged at 300 3 g to remove debris. Thefractions were analyzed via Western blotting.

Statistical analysis

Data analysis was performed in GraphPad Prism5 (GraphPad Software, LaJolla, CA). Different experiments were analyzed by one-sample or standardStudent t test or two-way ANOVA, followed by the Bonferroni posttest. Ap value , 0.05 was considered as significant (*). Statistical analyses wereperformed in GraphPad Prism. In general, data are expressed as mean 6SEM (or mean 6 SD, when indicated).

ResultsCD222 surface expression on human T cells increases uponTCR stimulation

CD222 is upregulated on the cell surface of rodent T cells upon T cellactivation (17). Therefore, we hypothesized that this molecule mightplay a role in T cell activation. We first investigated the expressionof CD222 on human lymphocytes. Isolated human PBMC werelabeled with CFSE and analyzed for surface expression of CD222via flow cytometry using a specific CD222 mAb. Upon cross-linking of the TCR complex with an activatory CD3 mAb,CD222 was upregulated on the cell surface on days 2, 3, and 4(Fig. 1A). However, analysis of CD222 mRNA levels in resting

2720 CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck


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and CD3/CD28 mAb–stimulated T cells revealed no significantincrease in CD222 expression upon activation (Fig. 1C). This sug-gests the regulation of CD222 via subcellular distribution and notvia gene expression. Regardless of the cell type, ∼90% of CD222have been reported to be localized in the TGN and endosomalcompartments at steady state (25). Therefore, the majority ofCD222 is kept intracellular and only a small proportion is dis-played, dependent on the cellular condition, at the cell surface.Investigation of the T cell proliferation kinetics showed that

CD222 upregulation on the plasma membrane preceded T celldivision cycles. Both stimulated, yet not blasting T cells and

blasting T cells displayed more CD222 on the surface comparedwith unstimulated T cells (Fig. 1B). Notably, the majority ofproliferating cells was positive for CD222, which pointed towarda possible involvement of CD222 in T cell activation and/or dif-ferentiation.

CD222 knockdown dampens T cell effector functions

To investigate the putative role of CD222 in T cell activation, pri-mary human PBMC were silenced for the expression of CD222via the lentiviral-based introduction of shRNA. Total CD222protein content was analyzed for shCTR and CD222-silenced

FIGURE 1. Cell surface upregulation of CD222 upon T cell stimulation and abrogation of T cell effector functions upon CD222 knockdown. (A) Human

primary T cells were isolated from PBMC, stained with CFSE, stimulated with OKT3-coated beads, and analyzed via flow cytometry on days 2–4.

Stimulated and unstimulated cells were harvested and stained with Alexa Fluor (AF)647-conjugated CD222 mAb MEM-238. Dot plots show the CFSE

dilution profile versus CD222 staining. (B) The histogram overlay depicts the surface expression of CD222 in unstimulated cells (gray) and stimulated yet

not blasting cells incubated with OKT3-coated beads for 2 d (dark gray) and blasting cells after 4 d (black histogram) versus the isotype control (light gray,

filled). Shown is one representative experiment out of three. (C) Primary human T cells, both resting and stimulated, were lysed, and RNA was extracted.

cDNAwas synthesized from total RNA, and gene expression was measured by real-time PCR as described in Materials and Methods with TaqMan primer

sets for human CD222 and YWHAZ as endogenous control. CD222 mRNA expression was assessed 1 d poststimulation and normalized to the CD222

levels at the beginning of the experiment. The means 6 SD of duplicates are shown and are representative of two independent experiments. (D–F) Human

PBMC were silenced for CD222 expression and analyzed for CD222 protein content via Western blotting (D) and surface expression via flow cytometry

(E, histogram overlay). Isotype, light gray filled histogram; shCTR cells, black solid line; and shCD222/P1 cells, dark gray solid line. (F) Cells were stimulated

for 24 h with CD3 mAb OKT3 with or without costimulatory CD28 mAb Leu-28. Supernatants of CD222-silenced and control-silenced cells were analyzed

via the Luminex technology for the presence of TNF-a, IL-2, and IFN-g. Data are expressed as means of triplicates 6 SEM and are representative for two

different donors and individual experiments. Significance was evaluated via a two-way ANOVA, followed by the Bonferroni posttest; *p , 0.05.



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(shCD222/P1) primary T cells via Western blotting and revealeda reduction by ∼75% (Fig. 1D). Subsequent flow cytometricanalysis revealed that CD222 surface expression was downregu-lated by ∼50% on shCD222/P1 T cells compared with shCTRT cells (Fig. 1E). To assess their effector functions, both shCTRand shCD222/P1 T cells were stimulated with low or high con-centrations of plate-bound CD3 mAb (1 and 5 mg/ml) in combi-nation with costimulatory soluble CD28 mAb (2 mg/ml). Secretedcytokine levels were measured via the Luminex system. Secretionof TNF-a, IL-2, and IFN-g was reduced by .50% in shCD222/P1;higher concentrations of CD3 mAb could not rescue the response inshCD222 cells (Fig. 1F). For further experiments, the wild-type (wt)Jurkat T cell line as well as the IL-2 and NFAT luciferase reporterJurkat T cell lines were used because the overall CD222 silenc-ing efficiency was mostly moderate in primary T cells in our hands,whereas in Jurkat T cells, the efficiency of silencing was muchhigher. Furthermore, CD222 is a very stable molecule. It takes ∼7–10 d upon silencing for the complete degradation of CD222 so thatthe functional effects become observable. As primary T cells arevery sensitive to prolonged in vitro culture, we used the T cell linesfor further experiments.To determine whether the cytokines were less synthesized or the

transport/secretion was hampered upon CD222 downregulation, weused human luciferase reporter T cell lines: first, CD222- andcontrol-silenced Jurkat T cells that expressed luciferase under thecontrol of the IL-2 promoter were stimulated via CD3 or CD3/CD28, and the lysates were analyzed for luciferase activity. Si-lencing of CD222 at two distinct positions (shCD222/P1 andshCD222/P2) led to a reduction of the luciferase activity uponT cell stimulation via CD3 (Fig. 2A). Even activation of the co-stimulatory pathway via CD28 ligation, which quantitatively en-hances TCR-induced signals (26), could not rescue the IL-2 pro-moter activity in the reporter cells silenced at the first position(P1) and resulted in a significant luciferase reduction. Althoughsilencing at the second position (P2) did not reduce CD222 tran-scripts as effective as P1 (Fig. 2A, inset), the corresponding cellsstill displayed a reduced IL-2 promoter activity, although notsignificant. Second, we used human reporter Jurkat T cells thatexpressed the luciferase gene driven by an artificially designedpromoter region containing multiple binding sites for the tran-scription factor NFAT (27). The NFAT-driven luciferase expres-sion was significantly reduced in shCD222/P1 cells comparedwith shCTR cells upon stimulation with CD3/CD28 mAb (Fig.2B). These data revealed that CD222 knockdown affected cyto-kine production at the transcriptional level.To analyze whether decreased Ca2+ mobilization was respon-

sible for reduced NFAT promoter activity, we measured intracel-lular Ca2+ fluxes by flow cytometry with the Ca2+ sensitive dyeIndo-1. To enable an equal Lck expression in control- and CD222-silenced T cells, we used the Lck-deficient Jurkat T cells JCaM1.6 that we reconstituted before silencing with a GFP-tagged Lckfollowed by expressing gating. We found that silencing of CD222at P1 and P2 led to a significant reduction in the cytosolic Ca2+

mobilization capacity compared with control-silenced Lck-GFP+

JCaM 1.6 T cells (Fig. 2C). Detailed analysis of Ca2+ fluxexperiments showed that the peak values of the 390/495 ratio andthe overall mean values of shCD222/P1-silenced cells were sig-nificantly reduced, whereas the baseline values remained un-changed (Fig. 2D). Silencing of CD222 at the less efficient P2 siteinfluenced the Ca2+ flux to a lesser extent, which might beexplained via a dose-dependent mode of action of CD222. Indeed,this assumption was supported by other experiments: titration ofvirus, carrying the silencing construct, correlated with lower ex-pression of CD222 and reduced Ca2+ mobilization in Jurkat

T cells (Fig. 2E) suggesting a direct correlation between the totalamount of the CD222 protein and the T cell response to TCRstimuli. Furthermore, the artificial overexpression of CD222 led tosignificantly higher intracellular Ca2+ flux in Jurkat T cells andCD222-overexpressing cells reacted faster than cells expressingthe control vector (Fig. 2F). However, it has to be mentioned thatcells highly overexpressing CD222 are more susceptible for ap-optosis compared with control cells (28). Furthermore, highamounts of CD222 lead to a decreased cellular growth rate (29).Therefore, just cells slightly overexpressing CD222 survived andgrew adequately. Finally, to verify the data obtained via RNAinterference, genomic CD222 was targeted and cut via sequence-specific zinc finger nucleases in Jurkat T cells (Supplemental Fig.1A). Analysis of three independently sorted CD222 knockoutpopulations (C20, C21, and C25; Supplemental Fig. 1B), revealedthat each of them displayed a reduction in intracellular Ca2+ fluxescompared with CTR cells (Supplemental Fig. 1C). Importantly,this phenotype was rescued in knockout cells that were stablytransduced with recombinant human CD222 (C20 cells recon-stituted with CD222; Supplemental Fig. 1D–F). Taken together,these data imply that CD222 is an essential regulator of the TCRsignaling cascade.

CD222 is crucial for early T cell signaling

To untangle the mode of operation of CD222 in TCR signaling, westimulated control- and CD222-silenced Jurkat T cells with the TCR-specific mAbC305 and evaluated the tyrosine phosphorylation in thecell lysates via Western blot analysis: several molecules were lessphosphorylated in CD222-silenced cells, particularly in molecularmass regions corresponding to the TCR proximal signaling mole-cules ZAP70, LAT, and CD3 (Fig. 3A), suggesting a very earlyeffect of CD222 on T cell signal transduction. To verify this, weused specific mAb in a multicolor fluorescence multiplexing ap-proach. Indeed, the earliest T cell signaling proteins CD3z andZAP70 displayed lower tyrosine phosphorylation upon CD222downregulation. Accordingly, all ensuing signaling molecules, suchas LAT and ERK, were less phosphorylated in shCD222 cells(Fig. 3B). Statistical evaluation of multiple experiments analyzingpCD3z and pERK1/2 revealed that the phosphorylation of both(i.e., early and late signaling molecules) was significantly reducedupon CD222 knockdown (Fig. 3C). In addition, Lck’s serine 59,a substrate of active ERK1/2 (30), was less phosphorylated inCD222 knockdown cells, which possibly could underlie an insuf-ficient signal amplification to sustain T cell activation (SupplementalFig. 2A). Supporting evidence for the implication of CD222 inTCR-proximal signaling was provided via CLSM: upon 15-minstimulation of JCaM 1.6 T cells with superantigen SEE-loadedRaji B cells, CD222 redistributed from the cell surface and/orintracellular pericentrosomal compartments (31) to the interfaceof the IS in ∼70% of JCaM 1.6 T cells that were in contact to RajiB cells (n = 24; Fig. 3D).

Lck and CD45 interact with CD222

To unravel possible interaction partners of CD222, we pulled downCD222 with specific mAb-coupled beads after cell lysis of restingJurkat T cells with lauryl maltoside, and analyzed coprecipitatedproteins via LC-MS. We found, along with known CD222 bindingproteins, various T cell–specific proteins. Among these proteinswe found Lck and CD45, molecules implicated in the regulationof T cell signaling (Table I). To verify the protein–protein inter-actions, we precipitated CD222 from lysates of resting JurkatT cells via CD222 mAb-coated beads and analyzed coimmuno-precipitated proteins by Western blotting. We detected both, Lckand CD45, in the CD222 coimmunoprecipitates (Fig. 4A).



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Moreover, we detected the Lck-CD222 interaction also by a re-verse approach performed with resting JCaM 1.6 T cells, ex-pressing Lck fused to the One-Strep-Tag epitope (Lck-OST).Therein, CD222 was reproducibly found in the isolated Lckcomplexes via LC-MS analysis (Mascot protein score = 486.34).The interaction of CD222 and Lck also was confirmed by coim-munoprecipitation studies with JCaM 1.6 T cells, followed byimmunoblotting (Fig. 4B). A truncated nonfunctional mutant of

Lck encompassing only the first 10 aa of Lck (LckN10) did notcoimmunoprecipitate with CD222 (Supplemental Fig. 2B), sug-gesting a specific protein–protein interaction. Furthermore, weverified the interaction of CD222 and Lck in nonstimulated primaryhuman T cells isolated from peripheral blood (Fig. 4C).Immunofluorescence-based in situ localization via CLSM revealedthat Lck colocalized with CD222 in resting Jurkat T cells (Fig. 4D).However, the majority of interactions seemed to take place in in-

FIGURE 2. CD222 knockdown downregulates IL-2 promoter activity, NFAT promoter binding, and Ca2+ fluxes. (A) CD222-silenced (shCD222/P1 and

shCD222/P2) and control-silenced (shCTR) IL-2 luciferase reporter Jurkat T cells were analyzed for CD222 protein content by Western blotting (inset).

GAPDH was used as a loading control. shCD222/P1 (dark gray, n = 3), shCD222/P2 (light gray, n = 2), and shCTR cells (black, n = 3) were stimulated with

OKT3 with or without Leu-28, and lysates were assayed for luciferase activity. Shown is the mean of three individual experiments (mean 6 SEM), and

significance was analyzed via Student t test; **p , 0.01. (B) CD222-silenced and control-silenced NFAT luciferase reporter Jurkat T cells were treated and

analyzed as in (A). Data represent mean of triplicates, and significance was evaluated via Student t test; **p , 0.01. One representative experiment out of

two is shown. (C) CD222-silenced Lck-GFP+ JCaM 1.6 T cells (shCTR black, shCD222/P1 dark gray, shCD222/P2 light gray) were assayed for Ca2+

mobilization upon stimulation with the anti-TCR mAb C305. Shown is one of three representative experiments. For evaluation of significant differences,

adjacent 3 3 5-s-time ranges were selected and the corresponding mean 390/495 ratios were analyzed via Student t test; ***p , 0.001. (D) Equal 5-s-time

ranges were selected from two individual Ca2+ flux experiments and the mean values 6 SEM of shCTR (black), shCD222/P1 (dark gray), and shCD222/P2

cells (light gray) were plotted. Peak, peak 390/490 ratio values; mean y, mean 390/490 ratio values; base, mean of baseline. Differences were calculated via

two-way ANOVA, followed by the Bonferroni posttest; **p , 0.01. (E) Jurkat T cells were transduced with varying titers of virus containing the silencing

construct (low, medium, and high). shCTR (black) and different shCD222/P1 (dark, middle, and light gray) Jurkat T cells were analyzed for their Ca2+

mobilization ability via flow cytometry after stimulation with C305. Plotted is the median value of the Ca2+ response. Shown is one representative ex-

periment out of two. (F) Jurkat T cells were transfected with GFP-tagged CD222 (CD222-GFP). Control vector cells (CTR vector, black) and CD222-GFP

expressing cells (gray) were compared for the C305-induced Ca2+ mobilization. One of two representative experiments is shown, and the mean response is

plotted for each cell type. For evaluation of significant differences, three equal adjacent time ranges were selected, and the mean 390/495 ratios were

analyzed via Student t test; ***p , 0.001. Ionomycin was used for all Ca2+experiments as a control.



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tracellular compartments: Upon conditions allowing the specificimmunoprecipitation of cell surface-bound CD222, no Lck wascoimmunoprecipitated from lysates of primary T cells (Fig. 4E).Furthermore, CD222 and Lck colocalized with the EEA1 in restingJurkat T cells (Supplemental Fig. 3). Moreover, when we stimulatedJCaM 1.6 T cells that ectopically expressed Lck-GFP with SEE-loaded Raji B cells, we found a coordinated intracellular accumu-lation of Lck and CD222 submembrane of the active IS (Fig. 4F),suggesting that the cytoplasmic interaction of CD222 and Lckprevails also upon T cell activation. Analysis of consecutivez-stacks of the depicted cells confirmed that CD222 colocalizedwith Lck almost exclusively within the cell with a Pearson’s co-efficient of ∼0.3, whereas the Pearson’s coefficient was practicallyzero at the plasma membrane edges (Fig. 4F, right panel). Thesefindings suggest that the interaction between CD222 and Lck occurspreferentially in cytoplasmic vesicles and that CD222 is involved inthe initiation of the early signaling cascade through acting on Lck.

CD222 knockdown leads to an inhibitory phosphorylationsignature on Lck

Lck has been reported to exist in resting T cells in four differentpools, each constituting ∼25% of total Lck and constantly main-taining at this equilibrium (3, 4) with CD45 being an importantregulator of the Lck phosphorylation status. Because we foundCD222 to interact with both Lck and CD45, we hypothesized thatCD222 might be a mediator involved in the regulation of Lckphosphorylation. To test this, we investigated the tyrosine phos-phorylation of Lck normalized to total Lck in shCD222/P1,shCD222/P2, and shCTR Jurkat T cells via LiCor infrared fluo-rescence Western blot analysis (Fig. 5A, 5C). CorrespondingCD222 surface expressions were analyzed via flow cytometry(Fig. 5B). Lck was significantly more phosphorylated at Y505 inshCD222/P1 cells compared with shCTR cells (Fig. 5A, 5C). Si-lencing with the second construct (shCD222/P2) as well increasedLck phosphorylation at Y505 (Fig. 5C), although not significantly,which correlated with the less effective silencing at this posi-tion (Fig. 5B). Lck phosphorylation at Y394 was not changed(Fig. 5A, 5D), whereas the amount of Lck not phosphorylated atY394 (non-pY394) significantly increased upon CD222 knockdown(Fig. 5A, 5E). These findings suggest that silencing of CD222causes a shift in Lck pools toward the less active form solelyphosphorylated at the inhibitory Y505.To pinpoint whether the increased phosphorylation at Y505 was

the cause of a blockade in signaling upon CD222 silencing, we useda mutated form of Lck that was made up of a phenylalanine insteadof the tyrosine at the position 505 and consequently could notbe phosphorylated at this site (32). Lckwt and the mutated form ofLck (LckY505mut) had been ectopically expressed in Lck-deficient

FIGURE 3. CD222 knockdown affects the phosphorylation of distal and

proximal TCR signaling molecules. (A) shCTR and shCD222/P1 Jurkat

T cells were stimulated with CD3 mAb (MEM-92) for the indicated time

points, and lysates were analyzed via Western blotting for the presence of

phosphotyrosines. GAPDH was used as a loading control after stripping of

the membrane; kiloDalton ranges are indicated on the left. Arrows depict

kiloDalton areas of 70, 38, and 22 kDa. (B) For the simultaneous detection

of total protein versus the phosphorylated form (green versus red), we

analyzed lysates from shCTR and shCD222/P1 Jurkat T cells with spe-

cifically labeled Abs directed against the indicated proteins via the LiCor

system. (C) The dual fluorescence immunoblots were analyzed by the

LiCor system. Phosphorylated proteins were set relative to total protein

content of the same molecule (except for CD3), and individual experi-

ments were normalized to the maximum value during the stimulation

course, which was set to 100% (% of Max); n = 4 for CD3 and n = 2 for

ERK1/2. Data represent mean percentage of Max 6 SD. Significance was

evaluated via the Bonferroni posttest following two-way ANOVA; *p ,0.05. (D) Upper panels, JCaM 1.6 T cells were stimulated for 15 min with

SEE-loaded Raji B cells, fixed, permeabilized, and stained for CD222

(red). Lower panels, JCaM 1.6 T cells without activated B cells were

treated the same. Pictures were captured at a CLSM. Phase contrast pic-

tures were overlaid with the CD222-staining (right). Nuclei were visual-

ized with DAPI (blue). Shown is one of at least two representative ex-

periments. Scale bar, 5 mm.



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JCaM 1.6 T cells before the cells were silenced for CD222 andintracellular calcium mobilization was measured via flow cytometry.JCaM 1.6 T cells expressing Lckwt showed a reduced calcium fluxupon CD222 compared with control silencing. However, silencingof CD222 did not affect the calcium flux in JCaM 1.6 T cellscarrying the mutated form of Lck (Fig. 5F), suggesting that thealtered phosphorylation of Lck upon CD222-silencing caused theless reactive T cell phenotype.

CD222 regulates the intracellular distribution of Lck

To test whether CD222 was responsible for the efficient transport ofLck, we analyzed the distribution and membrane localization ofLck via immunofluorescent staining and CLSM of resting controland CD222 knockdown Jurkat T cells. The typical homogenoussurface distribution of Lck was disrupted in CD222 knockdowncells (Fig. 6A). Approximately 60–70% of shCD222/P1 andshCD222/P2 Jurkat T cells showed a diminished Lck surfacedistribution when counted manually. Surface plots of Lck stainingshowed a punctuated distribution in CD222-silenced cells withLck-free gaps among Lck-rich membrane areas (Fig. 6B), al-though the total Lck content in crude cell lysates of unstimulatedJurkat T cells was even higher in CD222-silenced cells as detectedby immunoblotting (Supplemental Fig. 2C). Lck is posttransla-tionally modified by palmitoylation and myristoylation at its Nterminus, which directs and anchors the molecule into lipid rafts(33, 34)—organizational platforms of cellular membranes im-portant for T cell signaling (35–38). We investigated total cellularlipid raft fractions in resting shCD222/P1 and shCTR JurkatT cells by means of sucrose-based density gradient flotationanalysis. The comparison of lipid raft- with nonraft fractions didnot reveal any significant changes upon CD222 knockdown (Sup-plemental Fig. 4A). The size exclusion chromatography analysison a Superose 6 column revealed in shCD222 cells a slight shiftof both Lck and CD45 towards higher molecular mass fractions,which also contained EEA1 (Supplemental Fig. 4B) correspondingto endosomal membranes (39).To analyze the intracellular distribution of Lck in more detail, we

evaluated the surface/cytoplasm ratio of Lck. Lck-deficient JCaM

1.6 T cells were retrovirally transduced to ectopically expressGFP-tagged Lck (Lck-GFP) to visualize the molecule in situwithout staining (Fig. 6C, upper panel). CD222 silencing at P1 ledto a significant reduction of surface-localized Lck, as indicated bythe decrease in the surface/cytoplasm ratio (Fig. 6C). shCD222/P2cells also displayed a significantly decreased surface/cytoplasmratio but again less pronounced than shCD222/P1. This phenom-enon was highly specific because the transport of the Lck mutantvariant bearing only the first 10 aa (LckN10-GFP), encompassingmyristoylation and palmitoylation sites indispensable for the cellmembrane targeting (Fig. 6D, upper panel) and lipid raft parti-tioning, was unaffected (Fig. 6D). In addition, the GFP-taggedLck lacking the first 10 aa (LckDN10-GFP), a mutant variantthat in contrast cannot be inserted into the inner leaflet of theplasma membrane (Fig. 6E, upper panel) was distributed evenlywithin the cell of all cell types investigated, as can be seen by theratio around 1 (Fig. 6E). When human Lck-GFP was retrovirallyintroduced in CD222-negative mouse fibroblasts, we observedsimilar patterns: Lck was not sufficiently transported to the sur-face, whereas in cells coexpressing human recombinant CD222,the surface localization of Lck-GFP increased (Fig. 6F). Thesefindings indicate that CD222 does not influence lipid raft parti-tioning of Lck but rather directs its cellular distribution.

CD222 knockdown impairs the interaction of CD45 with Lck

Finally, we investigated the interaction of Lck and CD45. Unsti-mulated JCaM 1.6 T cells ectopically expressing Lck-GFP werestained for CD45 and analyzed for the colocalization of CD45 withLck in shCTR and CD222-silenced cells (shCD222/P1 and /P2;Fig. 7A). The overall Pearson’s coefficient, a measure of coloc-alization, of the CD45-Lck interaction was low, as reported pre-viously (4), but markedly decreased upon silencing of CD222(Fig. 7B). In silenced cells, Lck was apparently retained in in-tracellular membranes and/or accumulated freely in the cyto-plasm. By biochemical separation of total cellular membranesfrom cytosolic cell contents, we found a significant increase incytosolic Lck upon CD222 silencing (Fig. 7C). To test whether theCD45–Lck interaction occurred at the plasma membrane, we la-

Table I. Mass spectrometric identification of T cell–specific interaction partners of CD222

Protein Names D CTR mAb CD222 mAb

CD222 1.22 3 109 6.53 3 106 1.22 3 109

Dipeptidyl-peptidase 1 6.43 3 107 3.53 3 105 6.47 3 107

Voltage-gated potassium channel subunit b-2 2.14 3 107 1.92 3 106 2.33 3 107

Putative uncharacterized protein MAP4K4 8.88 3 106 8.53 3 105 9.73 3 106

14-3-3 protein 3.16 3 106 2.98 3 106 6.14 3 106

Calnexin 2.59 3 106 4.11 3 105 3.00 3 106

Tubulin b-2C chain; Tubulin b-2 chain 1.53 3 106 1.02 3 106 2.55 3 106

Rho guanine nucleotide exchange factor 2 1.47 3 106 3.46 3 105 1.82 3 106

Dedicator of cytokinesis protein 2 1.37 3 106 0.00 3 100 1.37 3 106

Proto-oncogene tyrosine-protein kinase LCK 1.34 3 106 4.92 3 105 1.84 3 106

Leukocyte common Ag (CD45) 1.16 3 106 2.97 3 105 1.46 3 106

Ras-related protein Rab-7a 1.15 3 106 2.24 3 105 1.37 3 106

MHC class I Ag 1.02 3 106 1.46 3 105 1.17 3 106

Serine/threonine-protein phosphatase 2A 9.27 3 105 2.90 3 105 1.22 3 106

Ras-related protein Rab-11B 7.97 3 105 9.81 3 104 8.95 3 105

Stomatin-like protein 2 5.46 3 105 8.42 3 105 1.39 3 106

Sodium/potassium-transporting ATPase subunit a-1 5.28 3 105 0.00 3 100 5.28 3 105

Ras-related protein Rab-1A 4.45 3 105 7.08 3 104 5.15 3 105

Integrin b-chain 3.83 3 105 0.00 3 100 3.83 3 105

Formin-like protein 1 3.63 3 105 0.00 3 100 3.63 3 105

Basigin 3.29 3 105 1.68 3 105 4.97 3 105

CD222 was fished with specific mAb-coated beads (MEM-238) from cell lysates prepared with 1% lauryl maltoside. Theprecipitated proteins were analyzed via LC-MS. The mAb AFP-12 to a-fetoprotein was used as a control (CTR mAb). Proteinswith a MEM-238:CTR mAb ratio .3 were considered as specifically enriched. The table lists proteins reported to be relevantfor T cell regulation sorted via the delta value (D) of MEM-238 and CTR Ab.



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beled the surface of living cells with a CD45 mAb on ice, lysedthe cells, immunoprecipitated cell surface CD45, and analyzed theeluates for coimmunoprecipitated Lck (Fig. 7D). The interactionof cell surface CD45 with Lck was reduced by ∼40 and 20% inshCD222/P1 and shCD222/P2 knockdown cells, respectively (Fig.7D). In contrast, LckN10, the non-functional mutant variant ofLck consisting of the 10 N-terminal amino acids responsible forplasma membrane anchorage, did not coimmunoprecipitate with

surface CD45 (Fig. 7E). These data suggest that CD222 controlsthe transport of the kinase Lck to the plasma membrane andsubsequently its interaction with CD45, the key activatory phos-phatase of Lck.

DiscussionLck is described to exist in four different phosphorylation states,constantly maintained at equilibrium, with ∼40–50% of consti-

FIGURE 4. CD222 interacts with Lck and CD45. (A) CD222 was immunoprecipitated by using MEM-238–coated beads from Jurkat T cell lysates

prepared with 1% detergent lauryl maltoside, and coprecipitated proteins were analyzed via Western blotting. The mAb AFP-12 served as a control (CTR

Ab). The lysates before precipitation (lysate) and the immunoprecipitates (IP) were analyzed. (B) Lck-deficient JCaM 1.6 T cells were transduced with

a lentiviral expression construct for Lck fused to the One-Strep-tag epitope (Lck-OST). Lysates of resting or sodium pervanadate (NaPV)–stimulated cells

were subjected to precipitation with Streptactin–Sepharose beads. Biotin-blocked beads served as negative control. Coprecipitation of CD222 was detected

using mAb MEM-238. (C) Human primary PBMC were subjected to coimmunoprecipitation analysis as in (A). (D) Jurkat T cells were fixed on adhesion

slides, permeabilized, and stained with Abs to detect CD222 (red) and Lck (green). Arrowheads depict areas of colocalization at the cell surface, and arrows

show intracellular areas of colocalization. Scale bar, 5 mm. (E) Immunoprecipitation analysis of cell surface expressed CD222. Resting primary human

PBMC were labeled on ice with mAbs directed against CD222 (MEM-238), CD45 (MEM-28), or AFP-12 as control. Proteins were cross-linked with 1 mM

DSP, and cells were lysed with 1% lauryl maltoside on ice. mAbs were fished with protein A/G beads over night. Beads were washed three times, and

proteins were eluted with Laemmli sample buffer. The lysates (Lys), nonimmunoprecipitates (supernatants - SN), and precipitates (IP) were analyzed for

CD222, CD45, and Lck via Western blotting. (F) JCaM 1.6 T cells transduced with Lck-GFP (green) were stimulated for 15 min with SEE-loaded Raji B

cells, fixed, permeabilized, and stained for CD222 (red). Nuclei are visualized with DAPI (blue) and cell morphologies are shown with phase contrast

(gray). Pictures were captured at a CLSM. Shown is one of at least two representative experiments. Scale bar, 5 mm. To evaluate the colocalization of

CD222 and Lck upon immunological synapse formation, 30 consecutive sections (z-stacks) though the cells (from the bottom to the top) were captured at

a confocal laser scanning microscope. Each section was analyzed via ImageJ for the interaction of CD222 and Lck and plotted as Pearson’s coefficient

(y-axis) versus the cell diameter (x-axis, from the bottom to the top).



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tutive active Lck within resting T cells (3, 40). As active Lckrepresents a high risk for a T cell to get permanently activatedresulting in hyperreactive adaptive immune reactions in an or-ganism, the spatial arrangement of Lck has to be accurately or-ganized in time and space. Limiting the access of possible kinasesubstrates or orchestrating the interaction with regulatory proteinscan prevent overwhelming or self-directed immune responses. Onthe basis of our data, we propose a novel function for the lateendosomal transporter CD222 in the early TCR signaling cascade.CD222 is essential for the efficient recruitment of Lck to theplasma membrane, the place where it interacts with its phospha-tase CD45. The loss of CD222 leads to accumulation of Lck in itsinactive form, which results in diminished T cell activation andeffector functions.Because Lck represents a very powerful kinase that is involved in

various T cell signaling pathways (41–44), its transport and reg-ulation has been investigated extensively (45–49). One importantmolecule implicated in the transport of Lck is the myelin andlymphocyte protein (MAL). Upon knockdown of MAL, Lck was

stuck in the TGN, without significant increase in cytosolic Lck.Furthermore, MAL was essential for lipid raft partitioning of Lck,whereas the transport to nonraft membranes was unhampered (45).In contrast, knockdown of CD222 hampered the efficient transportof Lck to the plasma membrane with an apparent accumulation ofLck within intracellular membranes and freely floating in thecytoplasm but without any significant effect on lipid raft parti-tioning. It has been shown previously via single molecule mi-croscopy and bleaching experiments that single Lck moleculesrecolonize the cell membrane directly from the cytoplasm, ratherthan via two-dimensional transversal diffusion (49), which mayexplain the high amount of non–membrane-bound cytosolic Lck.Lck has been reported previously to be present in endosomes (47,50). Our confocal microscopy and also gel filtration fractionationexperiments revealed an accumulation of Lck and CD222 in theEEA1-positive fractions corresponding to endosomal membranes.Furthermore, via mass spectrometry analysis we identified Rab11,a marker of endosomal membranes critically involved in proteintrafficking, to interact with CD222. This strongly suggests that

FIGURE 5. CD222 knockdown increases Lck phosphorylation at Y505. (A) Crude cell lysates of shCTR and shCD222 Jurkat T cells stimulated for the

indicated time points with CD3 mAb MEM-92 were fluorescently probed for the presence of total Lck and Lck pY505, pY394, and non-pY394, re-

spectively. Shown is one of at least two representative experiments. (B) CD222 surface expression of control and silenced Jurkat T cells (shCD222/P1,

n = 13 and /P2, n = 2) was analyzed by flow cytometry, and mean fluorescence intensities were calculated and set relative to the mean fluorescence intensity

of shCTR cells. One sample t test was used to evaluate significances versus the control; ***p , 0.001. (C) Dual fluorescence immunoblots of shCTR (n =

8), shCD222/P1 (n = 8), and shCD222/P2 Jurkat T cells (n = 2) were analyzed for Lck pY505 (normalized to total Lck), and values were plotted relative to

shCTR cells (assuming 100%). One sample t test was used for statistic calculations; **p , 0.01. (D and E) Crude cell lysates of shCTR and shCD222/P1

Jurkat T cells were analyzed via Western blotting for Lck pY394 (n = 4) and Lck non-pY394 (n = 2). Bound Abs were visualized via HRP-conjugated

secondary Abs and band intensities were normalized to GAPDH signals. Student t test was used to evaluate statistical differences; **p , 0.01. (F) Lck-

deficient JCaM 1.6 T cells ectopically expressing wt (Lckwt-GFP, upper panel) or the constitutive active form of Lck (LckY505mut-GFP, lower panel) were

silenced for CD222 (shCD222). Ca2+ fluxes were compared between GFP+/shCTR and GFP+/shCD222 cells after stimulation with the anti-TCR mAb

C305. Shown is the mean over threshold of one of two representative experiments.



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FIGURE 6. The transport of Lck to the cell surface is blocked in CD222 knockdown cells. (A and B) Jurkat T cells silenced for CD222 (shCD222/P1 and /P2)

and control-silenced cells (shCTR) were placed on adhesion slides, fixed, permeabilized, and stained for Lck (mAb H-95). For detection, AF488-

coupled secondary anti-rabbit Ab was used (green). After blocking, CD222 was stained using AF647-conjugated mAb MEM-238 (blue). Pictures were

captured with a CLSM and are representative of at least two individual experiments. Scale bar, 5 mm. (B) Individual cells in each setting (N in A) were

analyzed for Lck expression via plotting the surface fluorescence intensity in three dimensions with the ImageJ software (surface plot function). (C–E)

Upper panels, Cellular distribution patterns of Lck-GFP, LckDN10-GFP, and LckN10-GFP. JCaM 1.6 T cells expressing GFP-tagged versions of Lck were

let to adhere on adhesion slides, fixed with 4% formaldehyde, and analyzed for the distribution of Lck and Lck variants via CLSM. Nuclei were visualized

with DAPI. Shown are representative pictures out of three independent experiments. Scale bar, 5mm. Lower panels, JCaM 1.6 T cells expressing GFP-tagged wt

full-length Lck (C, Lck-GFP, n = 69), the GFP-tagged 10 N-terminal aa of Lck (D, N10-GFP, n = 20), and the N-terminal deletion (Figure legend continues)



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CD222 coordinates the endosome-dependent transport of Lck thatwas shown to be essential for T cell activation (48). Uncoordi-nated 119 protein has been shown to activate endosomal Rab11 forthe plasma membrane targeting of Lck (48) and activation of Fyn(51). However, unlike MAL and uncoordinated 119 protein,CD222 does not contribute to the IS, but rather builds an earlyvesicular platform submembrane of the IS. Although severalconsecutive devices obviously coordinate Lck transport (52–54),the contribution of CD222 seems to be very specific as human

CD222 can perform its action on human Lck even in mousefibroblasts (i.e., irrespective of other T cell–specific molecules).Moreover, the absence of MAL in fibroblasts (55) suggests thatCD222 operates upstream of MAL.To our knowledge, the role of CD222 in T cell signal trans-

duction has yet not been investigated in detail, but CD222 has beenshown to enhance TCR-induced signaling via CD26 internalization(56). However, we found no evidence for the contribution of CD26to the effect of CD222 on Lck because we have observed the effect

variant of Lck (E, DN10-GFP, n = 20 for shCTR and shCD222/P1, n = 10 for shCD222/P2) were silenced for CD222 at the two positions shCD222/P1

and /P2 or were shCTR. Cells were fixed and pictures were captured with a CLSM. Regions of interest areas of the cell surface and the cytoplasm were

drawn for each cell with ImageJ, mean fluorescence intensities were determined (arbitrary units, AU), and surface/cytoplasm ratios were plotted for each

cell. Statistical differences were evaluated by Student t test; **p , 0.01; ***p , 0.001. Shown data result from two independent experiments. (F) Human

CD222 directs the distribution of human Lck in CD222-negative mouse fibroblasts. Left panel, Human Lck-GFP (green) was retrovirally introduced into

fibroblasts of CD222 knockout mice transfected with (lower panels) or without (upper panels) human CD222. The cells were grown in chamber slides,

fixed, permeabilized, and stained for CD222 (MEM-238-AF647, red). Nuclei were visualized with DAPI (blue). Pictures were captured at a CLSM and are

representative of at least two individual experiments. Scale bar, 10 mm. Right panel, The cells were trypsinized, cytocentrifuged onto glass slides, fixed, and

analyzed for Lck distribution. Vertical and horizontal cuts through the cell were made, creating two values per cell. The intensity profiles of each cell were

exported to Microsoft Excel and adjusted that values started equally. The overlay shows the mean arbitrary units of 15 cells for each position in the cell.

FIGURE 7. Silencing of CD222 results in de-

creased CD45–Lck interaction. (A) JCaM 1.6

T cells expressing wt Lck-GFP (green) were either

shCTR or CD222-silenced (shCD222/P1 and /P2),

fixed, permeabilized, and stained with mAb directed

against CD45 (red). Nuclei were stained with

DAPI (blue), and pictures were captured with a

CLSM. Shown are representative pictures from

one of three independent experiments. Scale bar,

5 mm. (B) Single-cell pictures from JCaM 1.6 T

cells [treated and stained like in (A)] were analyzed

for Lck–CD45 colocalization via the JACoP plug-in

in ImageJ (n = 63 for all cell types). Data points

and mean were calculated from cell images from

two independent experiments. Statistical differences

were determined via Student t test; ***p , 0.001.

(C) After hypotonic cell lysis of shCTR and

shCD222 cells, the membranous parts were sepa-

rated from the cytosol and assayed for the presence

of Lck, CD45, and the cytosolic G protein GBP-1

(69) by Western blotting. The membranes were

developed by HRP-conjugated secondary Abs

and chemiluminescence. Lck band intensities of

shCTR (black) and shCD222/P1 cells (gray) of two

independent experiments were calculated and

normalized to the maximal signal (% of Max) and

the mean 6 SEM plotted in the graph. One sample

t test was used to evaluate statistical differences;

*p , 0.05. (D) Cell surface CD45 was labeled with

mAb, and then, the cells were lysed and CD45

precipitation performed by adding protein A/G

beads. The eluates were analyzed for coimmuno-

precipitated Lck by Western blot analysis. Statis-

tical evaluation of the coimmunoprecipitated Lck

(Lck/CD45 ratios) was determined via ImageJ.

Mean ratios 6 SEM of two independent experi-

ments are shown in the bar graphs. Differences

were evaluated via Student t test, *p , 0.05. (E)

Coimmunoprecipitation analysis of surface-bound

CD45 and LckN10-GFP. Surface-bound CD45

molecules of JCaM 1.6 T cells expressing LckN10-

GFP were labeled and pulled down as described in

(D). Lysates and immunoprecipitated proteins were

analyzed by Western blotting and detected via an

anti-GFP Ab.



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of CD222 knockdown on T cell activation in Jurkat T cells thatlack CD26 expression (57). Interestingly, ligation of the commoncostimulatory molecule CD28, which also quantitatively enhancesT cell responses to TCR stimuli (26), did not rescue, but evenresulted in a more pronounced phenotype induced by silencing ofCD222.The loss of CD222 in resting T cells results in accumulation of

Lck single phosphorylated at residue Y505 that represents theclosed inactive form of Lck (7, 58). Consequently, the homeostasisof the four proposed conformational forms of Lck (3) is shiftedtoward the inhibitory closed conformation of Lck. Recently, it hasbeen proposed that the intracellular distribution of Lck is regu-lated via its phosphorylation state and the corresponding con-formations (4, 6): the open active form (pY394) is thought toinduce clustering, whereas the closed inactive form (pY505)prevents the formation of clusters, which has been shown to belipid independent (4). Putting these data together one might sug-gest that the CD222 knockdown, which results in a 2-fold increaseof inhibitory Lck pY505, does prevent Lck from clustering andT cells from getting activated. The finding that the hyperactivemutated form LckY505mut triggers the activation of the secondarymessenger Ca2+ also in the absence of CD222 supports this model.On the basis of our data, CD222 mediates the shift of Lck towardthe active dephosphorylated form by complexing it with CD45.We pinpoint the CD222-driven CD45–Lck interaction to the cellsurface, which indicates, first, that dephosphorylation of Y505 viaCD45 takes place at the cell membrane, and second, that Csk-induced phosphorylation of Y505 (59) can occur within the cell.Because we found CD222 to interact with both the kinase Lck

and its phosphatase CD45, the question aroused whether thetransport of CD45 might be as well miss-regulated upon silencingof CD222. However, several facts speak against the simultaneoustransport deregulation of Lck and CD45 in the CD222 knockdownphenotype. First, we found just a minor downregulation of CD45at the cell surface. Second, CD45 is not only a positive but alsoa negative regulator of T cell activation and differentiation (59,60). Intermediate levels of CD45 surprisingly produce hyperre-active T cells (59), which we could not detect at all in our ex-perimental setup. Third, CD45 dephosphorylates not only Lck butalso CD3z (4, 59–61). However, the phosphorylation of CD3z wasunaffected in resting, and notably, even significantly reduced instimulated CD222 knockdown cells. Taking these facts into con-sideration, we conclude that the primary function of CD222 inT cell activation is to act as intermediary in the interaction ofCD45 with Lck resulting in the correct dephosphorylation ofLck at the inhibitory Y505.We show in this study that the interaction between CD222 and

Lck occurs preferentially inside the cell and disbands at the cellsurface, where Lck is “handed over” to CD45. This indicates thatCD222 is involved in very early signaling events. However, we donot have at the moment an explanation for CD222´s upregulationon the surface of proliferating and differentiating T cells—in thelater phase of T cell activation, shortly before cell division isinitiated. One can envisage several possible reasons of the in-creased CD222 surface accumulation during later phases of T cellactivation: 1) It might be necessary to fully confer the capacity ofcells to respond. 2) It might result from higher protein transportactivity mediated by CD222. Because upon T cell activation theturnover of Lck is enhanced (50), the fusion of CD222-containingtransport vesicles with the plasma membrane might result in el-evated CD222 on the cell surface. 3) It might be important for thelater signaling events as a negative feedback loop to abrogateT cell activation. The latter could be accomplished via regulatingthe bioavailability of the mitogenic insulin-like growth factor 2

that is both postnatally expressed in thymic epithelial cells (62)and upregulated in diseases (28), namely via internalization andsubsequent degradation in lysosomes (12, 63). 4) Furthermore,CD222’s surface upregulation might play a role in T cell differ-entiation from the double negative to the double positive stage asCD222 Ab was reported to block T cell ontogeny (64). Notably,Lck knockout mice also display a similar reduction in double-positive thymocytes in T lymphocyte development (65) as theone caused by the CD222 Ab treatment. However, because of thelethality of the CD222 knockout in mice (66–68), the function ofCD222 in T cell differentiation has never been shown in vivo. 5)An involvement of CD222 in T cell migration can also not beexcluded as it has been shown that CD222 negatively regulatescell adhesion via uPAR and integrins (22). 6) Alternatively, itcould be the result of a better accessibility of the CD222 mAbcaused by altered interactions that unmask the epitope.Irrespective of the pathways that are amenable in the upregu-

lation of cell surface CD222 upon T cell activation, we show in thisstudy a central function of the endosomal transporter CD222 in theinitiation of T cell signal transduction through controlling Lckdistribution: CD222 targets Lck to CD45 at the plasma membraneand thereby maintains the equilibrium of Lck phosphorylation atthe steady state.

AcknowledgmentsWe thank Peter Steinberger (Institute of Immunology, Medical University

of Vienna) and Thomas Decker (Max F. Perutz Laboratories, Department

of Genetics, Microbiology and Immunobiology, University of Vienna) for

productive discussions and constructive ideas; Erwin Wagner (Spanish

National Cancer Research Centre, Madrid, Spain) for providing mouse

fibroblasts, Vaclav Ho�rej�sı (Institute of Molecular Genetics, Academy of

Sciences of the Czech Republic, Prague, Czech Republic), Nicole Bou-

cheron (Division of Immunobiology, Institute of Immunology, Center for

Pathophysiology, Infectiology and Immunology, Medical University of

Vienna, Austria), and Arthur Weiss (Department of Medicine and De-

partment of Microbiology and Immunology, Rosalind Russell-Ephraim

P. Engleman Medical Research Center for Arthritis, and Howard Hughes

Medical Institute, University of California, San Francisco, San Francisco,

CA) for providing Ab; Reinhard Fassler (Department of Molecular Medicine,

Max Planck Institute of Biochemistry, Martinsried, Germany) for experimen-

tal and intellectual help with mass spectrometric analysis; S. Steward (Wash-

ington University School of Medicine, St. Louis, MO); G. Nolan (Baxter

Laboratory in Stem Cell Biology, Department of Microbiology and Immu-

nology, Stanford University, Stanford, California) for providing cloning

vectors and cell lines; and Cristiana Soldani, Anna Elisa Trovato and

Chiuhui Mary Wang (Humanitas Clinical and Research Center, Rozzano,

Milan, Italy) for practical help with immunofluorescence analysis. We are

grateful to the imaging and flow cytometry core facilities of the Medical

University of Vienna.

DisclosuresThe authors have no financial conflicts of interest.

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the late endosomal transporter cd222 directs the spatial ...hannes stockinger,* and vladimir...

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