The Late Endosomal Transporter CD222 Directs the Spatial Distribution and Activity of Lck

This information is current as Karin Pfisterer, Florian Forster, Wolfgang Paster, Verena of October 6, 2021. Supper, Anna Ohradanova-Repic, Paul Eckerstorfer, Alexander Zwirzitz, Clemens Donner, Cyril Boulegue, Herbert B. Schiller, Gabriela Ondrovicová, Oreste Acuto, Hannes Stockinger and Vladimir Leksa J Immunol 2014; 193:2718-2732; Prepublished online 15 August 2014; Downloaded from doi: 10.4049/jimmunol.1303349 http://www.jimmunol.org/content/193/6/2718 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2014/08/15/jimmunol.130334 Material 9.DCSupplemental References This article cites 69 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/193/6/2718.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Late Endosomal Transporter CD222 Directs the Spatial Distribution 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 Ondrovicova´,x Oreste Acuto,† Hannes Stockinger,* and Vladimir Leksa*,x

The spatial and temporal organization of T signaling molecules is increasingly accepted as a crucial step in controlling 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- Downloaded from 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.

cell signaling molecules have to be tightly controlled 394 (pY394); 3) an inactive “closed“ form phosphory-

spatially and temporally to ensure proper T cell activation lated at the COOH-terminal tyrosine 505 (pY505); and 4) a http://www.jimmunol.org/ T without hyperresponsiveness (1). A fundamental step double phosphorylated form (pY394 + pY505) that is thought to during the onset of T cell signaling is the recruitment of the possess an equal activity as single phosphorylated Lck at -specific (Lck) to the im- Y394 (3). Hence, active Lck is constitutively present in resting munological synapse (IS) and the subsequent of T cells, and it is its coordinated intracellular distribution that is the ITAMs of the CD3 z-chain (CD3z). ZAP70 can thereafter bind crucial to balance T cell activity and nonreactivity (5–7). The to the phosphorylated ITAMs of the TCR complex where it is antagonistic activities of the phosphatase CD45 and the kinase phosphorylated by Lck to initiate further downstream signaling Csk control Lck’s mode of action (2, 8, 9). CD45 plays a pivotal (2). For fine-tuning of T cell activation active and inactive Lck pools role in the activation of Lck via dephosphorylation of Y505 (10, must be kept at equilibrium. It has been shown that Lck activity is 11), but it is not clear where the interaction takes place and how it by guest on October 6, 2021 not directly linked with TCR engagement (3, 4). Lck rather exists is regulated in space and time. in several conformational and activity states: 1) an unphosphory- The late endosomal transmembrane molecule CD222, also known lated “primed” form; 2) an active form phosphorylated at the as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is a multifunctional broadly expressed regulator of protein trafficking (12). It binds mannose 6-phoshate–bearing *Molecular Immunology Unit, Institute for Hygiene and Applied Immunology, including acid and TGF-b (13), the insulin-like growth Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna A-1090, Austria; †Sir William Dunn School of Pathology, University factor 2 (14) and plasminogen (15). On delivering its cargo, CD222 of Oxford, Oxford OX1 3RE, United Kingdom; ‡Department of Molecular Medicine, traffics between the trans-Golgi network (TGN), endosomes, and the x Max-Planck Institute of Biochemistry, Martinsried 82152, Germany; and Laboratory plasma membrane (16). CD222 is barely expressed on the surface of of Molecular Immunology, Institute of Molecular Biology, Slovak Academy of Sci- ences, 84551 Bratislava, Slovak Republic resting T cell but has been reported to be upregulated at the plasma Received for publication December 17, 2013. Accepted for publication July 3, 2014. membrane upon T cell activation in rodents (17). In this study, we show This work was supported by the Austrian Science Fund (FWF Fonds zur Fo¨rderung that CD222 is also upregulated on the surface of human T cells der Wissenschaftlichen Forschung) P22908 (to V.L.), European Union Framework upon stimulation. Remarkably, we found that CD222 transports Program 7 “NANOFOL” Grant NMP4-LA-2009-228827 (to H.S.), Wellcome Trust Lck intracellularly and directs its distribution to CD45 at the plasma Grant GR076558MA, and European Union Framework Program 7 Grant EC-FP7- SYBILLA-201106 (to O.A.). W.P. was supported by the Erwin Schroedinger Fellow- membrane. The loss of CD222 causes a decrease in signal transduc- ship from the Austrian Science Fund. tion and subsequently a profound blockade of T cell effector functions. Address correspondence and reprint requests to Dr. Vladimir Leksa and Dr. Hannes Stockinger, Institute for Hygiene and Applied Immunology, Center for Pathophysi- ology, Infectiology, and Immunology, Medical University of Vienna, Lazarettgasse Materials and Methods 19, 1090 Vienna, Austria. E-mail addresses: [email protected] (V.L.) Abs and [email protected] (H.S.) The online version of this article contains supplemental material. The mAb to CD222, unlabeled and conjugated with AF647 (MEM-238), CD45, both unlabeled (MEM-28) and conjugated with Pacific Orange Abbreviations used in this article: AF, Alexa Fluor; AFP, a-fetoprotein; CLSM, (HI30), and CD4, unlabeled (MEM-241), were purchased from EXBIO confocal laser scanning microscopy; CTR, control; EEA1, early endosome Ag 1; (Prague, Czech Republic); the mAb to CD3 (MEM-57), CD59 (MEM-43/5), IS, ; LAT, linker of activated T cells; LC-MS, liquid chromatography–mass spectrometry; Luc, luciferase; MAL, myelin and lymphocyte pTag (H902), and a-fetoprotein (AFP-12) were provided by Dr. V. Horejsı´ protein; mut, mutant; OST, One-Strep-tag; pY, phospho-tyrosine; SEE, staphylococ- (Institute of Molecular Genetics, Academy of Sciences of the Czech cal enterotoxin E; sh, short hairpin; TGN, trans-Golgi network; wt, wild-type. Republic, Prague, Czech Republic). The anti–TCRb-chain mAb C305 was a gift from Dr. A. Weiss (University of California, San Francisco, Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 San Francisco, CA). The stimulatory CD3 mAb OKT3 was from Ortho www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303349 The Journal of Immunology 2719

Pharmaceuticals (Raritan, NJ). The CD28 mAb Leu-28, anti–phospho- AFP-12 (negative control). Proteins were cross-linked with 1 mM DSP CD3z/CD247 (Y142) mAb K25-407.69, and anti–phospho-linker of acti- (Pierce, Thermo Scientific Fisher, Rockford, IL), and cells were lysed with vated T cells (LAT; Y171) were from BD Biosciences (San Jose, CA). The detergent 1% lauryl maltoside on ice. The Ab–protein complexes were HRP-conjugated anti-phosphotyrosine mAb 4G10 was from Merck fished with protein A/G beads overnight. Beads were washed three times, Millipore (Billerica, MA). The anti-GAPDH mAb 14C10, anti–phospho- and proteins were eluted with Laemmli sample buffer. ZAP70 Ab (Y319)/Syk (Y352), anti-ZAP70 mAb 99F2 and L1E5, anti– The immunoprecipitates were analyzed by Western blotting. Briefly, phospho-p44/42 MAPK Ab ERK1/2 (Y202/Y204), anti-p44/42 MAPK Ab proteins were separated by SDS-PAGE, followed by transfer of the proteins to ERK1/2, anti–phospho-Src mAb (Y416), anti–non-phospho-Src mAb Immobilon polyvinylidene difluoride membranes (Merck Millipore). The (Y416), anti–phospho-Lck Ab (Y505), anti-Lck mAb 73A5, and the rabbit membranes were blocked with 5% nonfat milk (Maresi, Vienna, Austria) in mAb to early endosome Ag 1 (EEA1) C45B10 were from Cell Signaling TBS-Tween and incubated with specific Ab dilutions for detection of indicated Technology (Danvers, MA). The anti-Lck mAb H-95 was from Santa Cruz proteins. For visualization, blots were developed either with HRP secondary Biotechnology (Santa Cruz, CA). Biotinylation of unconjugated mAb was conjugates and a HRP substrate (Biozym, Hessisch Oldendorf, Germany) or performed in our laboratory. As secondary reagents we used HRP-con- with secondary fluorescence Abs, followed by detection at either the Fuji LAS- jugated goat anti-mouse and anti-rabbit IgG (Sigma-Aldrich, St. Louis, 400 imager (Fuji, Tokyo, Japan) or the Odyssey infrared imaging system MO), streptavidin-HRP conjugate (GE Healthcare, Buckinghamshire, (LiCor Biosciences, Lincoln, NE). Band intensities were quantified via Multi U.K.), and goat anti-mouse IgG+IgM (H+L)-FITC conjugate (An der Grub, Gauge Software Version 3.1 (Fuji), Image Studio Version 2.0, or ImageJ. Kaumberg, Austria). For mass spectrometric analysis, CD222 coimmunoprecipitates and cor- responding controls were digested in-solution as described previously (20), Cell culture and samples were prepared, according to the filter aided sample preparation procedure following analysis via liquid chromatography–mass spectrometry The T cell line Jurkat E6.1 and the line Raji were from the American (LC-MS). Lck coimmunoprecipitates were separated on 4–12% gradient Type Culture Collection. The Lck-deficient Jurkat T cell line JCaM 1.6 Bis-Tris NuPAGE gels (Invitrogen Life Technologies), the gels were washed (18) was from the European Collection of Cell Culture (Salisbury, U.K.). in distilled water, lightly stained with Colloidal Blue (Invitrogen Life

The immortalized CD222 negative mouse fibroblasts were provided by Downloaded from Technologies), and subjected to Gel-LC-MS as described previously (21). E. Wagner (Spanish National Cancer Research Centre, Madrid, Spain). The Jurkat IL-2 Luc T cells were stably transfected with a luciferase Gene knockdown via RNA interference and retroviral expressing IL-2 promoter reporter construct as described previously (19). transduction The Jurkat NFAT Luc cells containing an artificial promoter region with four NFAT binding sites were created in our laboratory. Human PBMC Stable knockdown cell lines were produced via lentiviral introduction of were isolated from purchased buffy coats of healthy adult volunteers (Rotes short hairpin (sh)RNA specific for the CD222 mRNA. The following CD222 Kreuz, Vienna, Austria) by density gradient centrifugation on Lymphoprep silencing sequences were used for cloning into the vector pLKOpuro1 (a gift (Nycomed). All primary human T cells as well as mouse and human from S. Steward, Washington University School of Medicine, St. Louis, MO): http://www.jimmunol.org/ T cell lines were maintained in RPMI 1640 medium, the HEK-293 cells shCD222/P1 at position 6588 with the sequence 59-GCCCAACGATCAG- in DMEM, all supplemented with 10% heat-inactivated FCS (Sigma- CACTTCttcaagagaGAAGTGCTGATCGTTGGGC-39 and shCD222/P2 at Aldrich), 2 mmol/L L-glutamine (Life Technologies, Carlsbad, CA), position 4525 with the sequence 59-GAGCAACGAGCATGATGACttcaaga- 100 mg/ml penicillin (Life Technologies), and 100 mg/ml streptomycin gaGTCATCATGCTCGTTGCTC-39. The MISSION nontargetshRNA control (Life Technologies). Cells were grown in a humidified atmosphere at 37˚C vector (pLKOpuro1; Sigma-Aldrich) was used as a negative control (shCTR). and 5% CO2 and passaged every 2–3 d. Virus particles were generated via transfection of HEK-293 cells with the si- lencing constructs and the lentiviral envelope coding plasmid pMD2.G and the T cell activation assay packaging vector psPAX 2 (both helping vectors were constructed by D. Trono, Primary human T cells isolated by CD14-depletion from PBMC were Ecole Polytechnique Federal de Lausanne, and obtained from Addgene, cultured for 2 d to separate non- from adherent cells. T cell lines and primary Cambridge, MA) as described previously (22). After 48 h, the virus particles by guest on October 6, 2021 human T cells (5 3 104) were seeded in 96-well plates and kept unstimu- were harvested, filtered, and used for the target cell line infections in the lated or stimulated by: 1) plate-bound CD3 mAb OKT3 (1 mg/ml) alone or presence of 5 mg/ml polybrene (Sigma-Aldrich). The next day, the cells were together with soluble CD28 mAb Leu-28 (0.5 mg/ml); 2) CD3 Ab-coupled washed with and maintained in medium containing 1 mg/ml puromycin beads; 3) staphylococcal enterotoxin E (SEE)–loaded Raji B cells; or 4) (Sigma-Aldrich) to select transduced cells. CD222 knockdown cells were PMA (5 ng/ml) plus ionomycin (1 mmol/l). After indicated time intervals, used from 10 d to 2 mo postinfection for all experiments. the cells were harvested and analyzed via flow cytometry, immunoblotting, Lck constructs were prepared in the retroviral expression vector pBMN-Z microscopy, or functional cellular assays. To measure proliferation, before for transfection of the Phoenix amphotropic virus producer cell line (both stimulation, the cells were resuspended in PBS, incubated with CFSE provided by G. Nolan, Stanford University School of Medicine, Stanford, (0.5 mmol/l; Molecular Probes, Eugene, OR) at 37˚C for 10 min, and CA) (15). The target cells were transduced one to three times with the viral thereafter washed twice with medium. supernatants depending on the efficacy of transduction and maintained afterward in normal culture medium. Immunoprecipitation, immunoblotting, and mass spectrometric Gene knockout via zinc finger nucleases analysis 3 7 Cys2His2-based zinc fingers were created via the Context Dependent For immunoprecipitation of CD222, 2–20 10 cells were lysed in lysis Assembly method provided by the free software tool ZiFiT to identify and buffer (20 mmol/l Tris and 140 mmol/l NaCl [pH 7.5]), containing 1% target the genomic sequence of CD222 specifically (23). The zinc finger lauryl maltoside or 1% Nonidet P-40 as detergent, complete protease in- constructs targeting exons 18 and 22 were cloned into pMLM290/ hibitor mixture (Roche, Basel, Switzerland), and benzonase (Novagen, pMLM292 and pMSM800/pMLM802 (both from Addgene) to generate Merck Millipore) for 30 min on ice. Lysates were incubated for 2 h at 4˚C zinc finger nuclease expression constructs. These constructs were used for with cyanogen bromide–activated beads coupled with CD222 mAb MEM- the transfection of Jurkat T cells via electroporation. For this, 2 3 106 cells 238 or isotype control mAb AFP-12. The beads were washed three times were resuspended in 100 ml Cytomix (120 mM KCl, 0.15 mM CaCl , with ice-cold lysis buffer, and the bound proteins were eluted with 1.23 2 5 mM MgCl2,10mMK2HPO4, 25 mM HEPES [pH 7.6], 2 mM EGTA, nonreducing Laemmli sample buffer at 95˚C for 5 min. 5 mM Glutathion, 1.25% DMSO [v/v], and 100 mM sucrose), mixed with For immunoprecipitation of Lck, the Lck-deficient Jurkat T cell clone the plasmids (each 0.5 mg), transferred in electroporation cuvettes, and JCaM 1.6 was transduced with a lentiviral expression construct for Lck transfected with the Amaxa System program S-18 (Amaxa, Lonza, Basel, fused to the One-Strep-tag epitope (Lck-OST). Lysates of resting or sodium Switzerland). Cells were immediately removed from the cuvettes, shortly pervanadate–stimulated cells were subjected to precipitation with Strep- spun down, and cultured in 12-well plates containing prewarmed medium. tactin–Sepharose beads. Biotin was used to elute bait proteins from the After cell recovery and expansion, CD222-negative cells were repeatedly capture beads. Biotin-blocked beads served as a negative control. sorted with a FACSAria (BD Biosciences), expanded in Jurkat T cell– For the analysis of the CD45–Lck interaction at the plasma membrane, JCaM conditioned medium, and cultured in RPMI 1640 medium until analysis. 1.6 T cells expressing Lck-GFP were silenced for CD222. Then, the silenced and control cells were surface labeled with CD45 mAb on ice, washed, and lysed Immunofluorescence staining and confocal laser scanning with lysis buffer containing detergent 1% Nonidet P-40. The mAb complexes microscopy were then precipitated by using protein A/G beads (Santa Cruz Biotechnology). For specific immunoprecipitation of cell surface CD222, unstimulated Primary T cells or T cell lines were let to adhere on adhesion slides PBMC were labeled with mAb against CD222, CD45 (positive control), or (Superior-Marienfeld Laboratory Glassware, Lauda-Ko¨ningshofen, Germany), 2720 CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck

fixed, and permeabilized with 4% formaldehyde and 0.1% saponin, re- PCR analysis spectively, and stained with the following Ab: the rabbit anti-Lck Ab H-95 and the rabbit anti-human mAbEEA1(C45B10),followedbya For real-time PCR analysis, primary human T cells, both resting and goat anti-rabbit AF488 Ab, the anti-Lck mouse Ab (73A5), followed by stimulated for 1 d with plate-bound CD3 mAb OKT3 (1 mg/ml) together anti-mouse AF488 Ab, the AF647-conjugated CD222 mAb MEM-238, with soluble CD28 mAb Leu-28 (0.5 mg/ml), were lysed in TRIzol and the Pacific Orange-conjugated CD45 mAb HI30 (all diluted 1:50) (Invitrogen Life Technologies), and RNA was extracted according to the in PBS containing 2% BSA. For stimulation of T cells before staining, manufacturer’s instructions. cDNA was synthesized from 500 ng total RNA with SuperScript VILO cDNA Synthesis Kit (Invitrogen Life Tech- cells were incubated with SEE-loaded Raji B cells at 37˚C for the indi- 2DDCT cated time points. Mouse fibroblasts were either cultured in 8-well chamber nologies). was measured by the 2 method (24), slides (Nunc, Thermo Scientific Fisher) until confluency or trypsinized based on quantitative real-time PCR using the CFX96 Real-Time PCR sys- and cytospun onto glass slides prior to fixation and staining as described tem (Bio-Rad) with TaqMan primer sets for human CD222, and YWHAZ as above. For each cell, one vertical and one horizontal cut was made through an endogenous control. For conventional PCR, 20 ng genomic DNA purified the cell, and the Lck distribution was analyzed using the ZEN 2011 blue by standard isopropanol precipitation was used as a template of PCR am- edition software (Zeiss, Jena, Germany). Nuclei were stained with DAPI. plification with Taq polymerase (40 cycles at 95˚C for 30 s, 61˚C for 30 s, and The slides were washed with 13 PBS and mounted with mounting me- 72˚C for 30 s) and with primer sets for human CD222 bridging an exon–exon dium for fluorescence analysis (Vectashield; Vector Laboratories, Burlingame, junction, and intronless gene endosialin, as an endogenous control (TaqMan CA). Isotype-matched controls were included. Pictures were captured with Gene Expression Assay; ABI, Life Technologies). a confocal laser scanning microscopy (CLSM) 700 (Zeiss). preparation Flow cytometry Cells (2–3 3 107) were lysed for 30 min on ice in lysis buffer containing For the analysis of cell surface Ags, cells were washed and resuspended in 1% detergent Brij-58 and protease inhibitors. Lysates were adjusted with 80% sucrose solution to 40% sucrose in 1 ml, placed on a 1 ml 60% su- staining buffer (13 PBS containing 1% BSA and 0.02% NaN3). Subse- crose layer and overlaid by 20, 10, and 5% sucrose fractions (all 1 ml quently, the cells were incubated for 20 min at 4˚C with a fluorochrome- Downloaded from conjugated mAb. For indirect immunofluorescence staining, the cells were containing 0.5% detergent Brij-58 and protease inhibitors). Gradients were 3 additionally incubated with secondary conjugates for 20 min at 4˚C. Be- ultracentrifuged for 16–18 h at 150,000 g at 4˚C. Fractions (500 ml) fore analysis, the cells were washed with staining buffer. Flow cytometry were taken from the top to the bottom and denatured at 95˚C with sample was performed using a LSR II (BD Biosciences) and analyzed with FlowJo buffer and analyzed by SDS-PAGE, followed by Western blot analysis. version 8.8.6 for Macintosh or FlowJo version 7.2.5 for Windows. Gel filtration analysis Luciferase assays The GE Healthcare AKTA FPLC system was used for the gel filtration http://www.jimmunol.org/ To assess the IL-2 promoter activity and NFAT binding to DNA, IL-2 lu- experiments. Cell lysates were prepared by solubilization with 1% detergent ciferase- and NFAT-reporter Jurkat T cells were assayed, respectively. Briefly, Triton X-100 (Promega). Samples were loaded at 4˚C in a volume of 500 ml the reporter cells (2 3 105) were either kept unstimulated or were stimulated onto a Superose 6 HR 10/300 GL column (GE Healthcare) equilibrated for 6 h in triplicates in CD3 mAb OKT-3–coated (1 mg/ml) 96-well plates with Triton X-100 (0.5%) in PBS. The absorbance at 280 nm was moni- with or without soluble CD28 mAb Leu-28. The cells were washed with PBS tored, and 500-ml fractions were collected and analyzed by immunoblot- and subsequently lysed in 100 ml luciferase lysis reagent (Promega, Madi- ting. The following standards of molecular mass (all from GE Healthcare son, WI) for 30 min on ice. Seventy-five microliters of lysate were transferred or Sigma-Aldrich) were used: Blue dextran (2000 kDa), thyroglobulin into white 96-well microplates, and luminescence was measured after ad- (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), BSA dition of the reaction reagent at a Mithras LB940 Elisa Reader (Berthold (66 kDa), and carbonic anhydrase (29 kDa). Technologies, Bad Wildbach, Germany). The protein concentration was Membrane–cytosol separation determined via Bradford assay (Bio-Rad, Hercules, CA), and the lumines- by guest on October 6, 2021 cence intensity was normalized to the protein content of each sample. 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, and measurement protease inhibitor mixture [pH 7.5]) for 30 min on ice and thereafter Culture supernatants (30 ml) of primary T cells were harvested after 24 h passaged nine times through a 30-g needle. Cell fragments were centri- 3 of stimulation and analyzed via the Luminex xMAP suspension array fuged twice at 300 g, and the resulting supernatant was centrifuged for 3 technology. Briefly, the cells were stimulated with plate-bound OKT3 40 min at 35,000 g at 4˚C. The cytosolic supernatant was collected and (1 or 5 mg/ml) and soluble Leu-28 (2 mg/ml) or kept unstimulated. Stan- the pellet containing membranous cellular parts was solubilized in ex- 3 dard curves were generated using recombinant (R&D Systems, traction buffer (1 lysis buffer containing 1% Triton X-100, 0.1% Minneapolis, MN). Experiments were performed at least twice with each NaDodSO4, 0.5% sodium deoxycholate, and the protease inhibitor mix- 3 sample in triplicates. Data are expressed as mean values of triplicates. ture) for 30 min on ice and centrifuged at 300 g to remove debris. The fractions were analyzed via Western blotting. mobilization assay Statistical analysis Cells (1 3 106) were washed, resuspended in 100 mlRPMI1640medium containing 1 mmol/l Indo-1 AM (Invitrogen Life Technologies) and incubated Data analysis was performed in GraphPad Prism5 (GraphPad Software, La for 30 min at 37˚C. The cells were washed with medium, incubated in 1 ml Jolla, CA). Different experiments were analyzed by one-sample or standard medium for next 30 min at 37˚C, and rested thereafter on ice until data ac- Student t test or two-way ANOVA, followed by the Bonferroni posttest. A quisition at a LSR II flow cytometer. For the analysis of intracellular calcium p value , 0.05 was considered as significant (*). Statistical analyses were flux, 300 ml Indo-1–loaded cells were prewarmed for 5 min at 37˚C, the base- performed in GraphPad Prism. In general, data are expressed as mean 6 line response was recorded for 30 s, before the cells were stimulated with a SEM (or mean 6 SD, when indicated). 1:300 dilution of the hybridoma supernatant of the anti-TCR mAb C305 for 3 min to analyze the increase in calcium mobilization. The indo-violet/ Results indo-blue ratio was calculated with the FlowJo software and plotted against the time. Ionomycin was used to check the overall responsiveness and cell viability. CD222 surface expression on human T cells increases upon TCR stimulation Analysis of T cell signaling molecules CD222 is upregulated on the cell surface of rodent T cells upon T cell 3 6 Jurkat T cells or primary T cells (1.5 10 ) were starved for 1 h in RPMI activation (17). Therefore, we hypothesized that this molecule might 1640 medium containing 1% FCS at 37˚C, 5% CO2 and 80% humidity. Cells were rested on ice for 10 min, stimulatory mAb were added, allowed play a role in T cell activation. We first investigated the expression to bind for 5 min, and then, the cells were incubated for several time points of CD222 on human . Isolated human PBMC were at 37˚C. The cells were quickly washed with ice-cold lysis buffer without labeled with CFSE and analyzed for surface expression of CD222 detergent and lysed in complete lysis buffer containing 1% detergent via flow cytometry using a specific CD222 mAb. Upon cross- Nonidet P-40 (Promega), a protease inhibitor mixture (Roche), sodium orthovanadate (Sigma-Aldrich), sodium fluoride, and benzonase for 30 min linking of the TCR complex with an activatory CD3 mAb, on ice. Lysates were sampled with 43 reducing or nonreducing sample CD222 was upregulated on the cell surface on days 2, 3, and 4 buffer and analyzed by Western blot analysis. (Fig. 1A). However, analysis of CD222 mRNA levels in resting The Journal of Immunology 2721 and CD3/CD28 mAb–stimulated T cells revealed no significant blasting T cells displayed more CD222 on the surface compared increase in CD222 expression upon activation (Fig. 1C). This sug- with unstimulated T cells (Fig. 1B). Notably, the majority of gests the regulation of CD222 via subcellular distribution and not proliferating cells was positive for CD222, which pointed toward via gene expression. Regardless of the cell type, ∼90% of CD222 a possible involvement of CD222 in T cell activation and/or dif- have been reported to be localized in the TGN and endosomal ferentiation. compartments at steady state (25). Therefore, the majority of CD222 is kept intracellular and only a small proportion is dis- CD222 knockdown dampens T cell effector functions played, dependent on the cellular condition, at the cell surface. To investigate the putative role of CD222 in T cell activation, pri- Investigation of the T cell proliferation kinetics showed that mary human PBMC were silenced for the expression of CD222 CD222 upregulation on the plasma membrane preceded T cell via the lentiviral-based introduction of shRNA. Total CD222 division cycles. Both stimulated, yet not blasting T cells and protein content was analyzed for shCTR and CD222-silenced Downloaded from http://www.jimmunol.org/ by guest on October 6, 2021

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. cDNA was 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. 2722 CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck

(shCD222/P1) primary T cells via Western blotting and revealed T cells (Fig. 2E) suggesting a direct correlation between the total a reduction by ∼75% (Fig. 1D). Subsequent flow cytometric amount of the CD222 protein and the T cell response to TCR analysis revealed that CD222 surface expression was downregu- stimuli. Furthermore, the artificial overexpression of CD222 led to lated by ∼50% on shCD222/P1 T cells compared with shCTR significantly higher intracellular Ca2+ flux in Jurkat T cells and T cells (Fig. 1E). To assess their effector functions, both shCTR CD222-overexpressing cells reacted faster than cells expressing and shCD222/P1 T cells were stimulated with low or high con- the control vector (Fig. 2F). However, it has to be mentioned that centrations of plate-bound CD3 mAb (1 and 5 mg/ml) in combi- cells highly overexpressing CD222 are more susceptible for ap- nation with costimulatory soluble CD28 mAb (2 mg/ml). Secreted optosis compared with control cells (28). Furthermore, high cytokine levels were measured via the Luminex system. Secretion amounts of CD222 lead to a decreased cellular growth rate (29). of TNF-a, IL-2, and IFN-g was reduced by .50% in shCD222/P1; Therefore, just cells slightly overexpressing CD222 survived and higher concentrations of CD3 mAb could not rescue the response in grew adequately. Finally, to verify the data obtained via RNA shCD222 cells (Fig. 1F). For further experiments, the wild-type (wt) interference, genomic CD222 was targeted and cut via sequence- Jurkat T cell line as well as the IL-2 and NFAT luciferase reporter specific zinc finger nucleases in Jurkat T cells (Supplemental Fig. Jurkat T cell lines were used because the overall CD222 silenc- 1A). Analysis of three independently sorted CD222 knockout ing efficiency was mostly moderate in primary T cells in our hands, populations (C20, C21, and C25; Supplemental Fig. 1B), revealed whereas in Jurkat T cells, the efficiency of silencing was much that each of them displayed a reduction in intracellular Ca2+ fluxes higher. Furthermore, CD222 is a very stable molecule. It takes ∼7– compared with CTR cells (Supplemental Fig. 1C). Importantly, 10 d upon silencing for the complete degradation of CD222 so that this phenotype was rescued in knockout cells that were stably the functional effects become observable. As primary T cells are transduced with recombinant human CD222 (C20 cells recon- very sensitive to prolonged in vitro culture, we used the T cell lines stituted with CD222; Supplemental Fig. 1D–F). Taken together, Downloaded from for further experiments. these data imply that CD222 is an essential regulator of the TCR To determine whether the cytokines were less synthesized or the signaling cascade. transport/secretion was hampered upon CD222 downregulation, we CD222 is crucial for early T cell signaling used human luciferase reporter T cell lines: first, CD222- and control-silenced Jurkat T cells that expressed luciferase under the To untangle the mode of operation of CD222 in TCR signaling, we control of the IL-2 promoter were stimulated via CD3 or CD3/ stimulated control- and CD222-silenced Jurkat T cells with the TCR- http://www.jimmunol.org/ CD28, and the lysates were analyzed for luciferase activity. Si- specific mAb C305 and evaluated the tyrosine phosphorylation in the lencing of CD222 at two distinct positions (shCD222/P1 and cell lysates via Western blot analysis: several molecules were less shCD222/P2) led to a reduction of the luciferase activity upon phosphorylated in CD222-silenced cells, particularly in molecular T cell stimulation via CD3 (Fig. 2A). Even activation of the co- mass regions corresponding to the TCR proximal signaling mole- stimulatory pathway via CD28 ligation, which quantitatively en- cules ZAP70, LAT, and CD3 (Fig. 3A), suggesting a very early hances TCR-induced signals (26), could not rescue the IL-2 pro- effect of CD222 on T cell signal transduction. To verify this, we moter activity in the reporter cells silenced at the first position used specific mAb in a multicolor fluorescence multiplexing ap- (P1) and resulted in a significant luciferase reduction. Although proach. Indeed, the earliest T cell signaling proteins CD3z and by guest on October 6, 2021 silencing at the second position (P2) did not reduce CD222 tran- ZAP70 displayed lower tyrosine phosphorylation upon CD222 scripts as effective as P1 (Fig. 2A, inset), the corresponding cells downregulation. Accordingly, all ensuing signaling molecules, such still displayed a reduced IL-2 promoter activity, although not as LAT and ERK, were less phosphorylated in shCD222 cells significant. Second, we used human reporter Jurkat T cells that (Fig. 3B). Statistical evaluation of multiple experiments analyzing expressed the luciferase gene driven by an artificially designed pCD3z and pERK1/2 revealed that the phosphorylation of both promoter region containing multiple binding sites for the tran- (i.e., early and late signaling molecules) was significantly reduced scription factor NFAT (27). The NFAT-driven luciferase expres- upon CD222 knockdown (Fig. 3C). In addition, Lck’s serine 59, sion was significantly reduced in shCD222/P1 cells compared a substrate of active ERK1/2 (30), was less phosphorylated in with shCTR cells upon stimulation with CD3/CD28 mAb (Fig. CD222 knockdown cells, which possibly could underlie an insuf- 2B). These data revealed that CD222 knockdown affected cyto- ficient signal amplification to sustain T cell activation (Supplemental kine production at the transcriptional level. Fig. 2A). Supporting evidence for the implication of CD222 in To analyze whether decreased Ca2+ mobilization was respon- TCR-proximal signaling was provided via CLSM: upon 15-min sible for reduced NFAT promoter activity, we measured intracel- stimulation of JCaM 1.6 T cells with SEE-loaded lular Ca2+ fluxes by flow cytometry with the Ca2+ sensitive dye Raji B cells, CD222 redistributed from the cell surface and/or Indo-1. To enable an equal Lck expression in control- and CD222- intracellular pericentrosomal compartments (31) to the interface silenced T cells, we used the Lck-deficient Jurkat T cells JCaM of the IS in ∼70% of JCaM 1.6 T cells that were in contact to Raji 1.6 that we reconstituted before silencing with a GFP-tagged Lck B cells (n = 24; Fig. 3D). followed by expressing gating. We found that silencing of CD222 at P1 and P2 led to a significant reduction in the cytosolic Ca2+ Lck and CD45 interact with CD222 mobilization capacity compared with control-silenced Lck-GFP+ To unravel possible interaction partners of CD222, we pulled down JCaM 1.6 T cells (Fig. 2C). Detailed analysis of Ca2+ flux CD222 with specific mAb-coupled beads after cell lysis of resting experiments showed that the peak values of the 390/495 ratio and Jurkat T cells with lauryl maltoside, and analyzed coprecipitated the overall mean values of shCD222/P1-silenced cells were sig- proteins via LC-MS. We found, along with known CD222 binding nificantly reduced, whereas the baseline values remained un- proteins, various T cell–specific proteins. Among these proteins changed (Fig. 2D). Silencing of CD222 at the less efficient P2 site we found Lck and CD45, molecules implicated in the regulation influenced the Ca2+ flux to a lesser extent, which might be of T cell signaling (Table I). To verify the protein–protein inter- explained via a dose-dependent mode of action of CD222. Indeed, actions, we precipitated CD222 from lysates of resting Jurkat this assumption was supported by other experiments: titration of T cells via CD222 mAb-coated beads and analyzed coimmuno- virus, carrying the silencing construct, correlated with lower ex- precipitated proteins by Western blotting. We detected both, Lck pression of CD222 and reduced Ca2+ mobilization in Jurkat and CD45, in the CD222 coimmunoprecipitates (Fig. 4A). The Journal of Immunology 2723 Downloaded from http://www.jimmunol.org/ by guest on October 6, 2021

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.

Moreover, we detected the Lck-CD222 interaction also by a re- Lck encompassing only the first 10 aa of Lck (LckN10) did not verse approach performed with resting JCaM 1.6 T cells, ex- coimmunoprecipitate with CD222 (Supplemental Fig. 2B), sug- pressing Lck fused to the One-Strep-Tag epitope (Lck-OST). gesting a specific protein–protein interaction. Furthermore, we Therein, CD222 was reproducibly found in the isolated Lck verified the interaction of CD222 and Lck in nonstimulated primary complexes via LC-MS analysis (Mascot protein score = 486.34). human T cells isolated from peripheral blood (Fig. 4C). The interaction of CD222 and Lck also was confirmed by coim- Immunofluorescence-based in situ localization via CLSM revealed munoprecipitation studies with JCaM 1.6 T cells, followed by that Lck colocalized with CD222 in resting Jurkat T cells (Fig. 4D). immunoblotting (Fig. 4B). A truncated nonfunctional mutant of However, the majority of interactions seemed to take place in in- 2724 CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck

tracellular compartments: Upon conditions allowing the specific immunoprecipitation of cell surface-bound CD222, no Lck was coimmunoprecipitated from lysates of primary T cells (Fig. 4E). Furthermore, CD222 and Lck colocalized with the EEA1 in resting Jurkat T cells (Supplemental Fig. 3). Moreover, when we stimulated JCaM 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 Lck prevails also upon T cell activation. Analysis of consecutive z-stacks of the depicted cells confirmed that CD222 colocalized with Lck almost exclusively within the cell with a Pearson’s co- efficient of ∼0.3, whereas the Pearson’s coefficient was practically zero at the plasma membrane edges (Fig. 4F, right panel). These findings suggest that the interaction between CD222 and Lck occurs preferentially in cytoplasmic vesicles and that CD222 is involved in the initiation of the early signaling cascade through acting on Lck. CD222 knockdown leads to an inhibitory phosphorylation

signature on Lck Downloaded from Lck has been reported to exist in resting T cells in four different pools, each constituting ∼25% of total Lck and constantly main- taining at this equilibrium (3, 4) with CD45 being an important regulator of the Lck phosphorylation status. Because we found CD222 to interact with both Lck and CD45, we hypothesized that

CD222 might be a mediator involved in the regulation of Lck http://www.jimmunol.org/ phosphorylation. 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). Corresponding CD222 surface expressions were analyzed via flow cytometry (Fig. 5B). Lck was significantly more phosphorylated at Y505 in shCD222/P1 cells compared with shCTR cells (Fig. 5A, 5C). Si- lencing with the second construct (shCD222/P2) as well increased Lck phosphorylation at Y505 (Fig. 5C), although not significantly, by guest on October 6, 2021 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 at Y394 (non-pY394) significantly increased upon CD222 knockdown (Fig. 5A, 5E). These findings suggest that silencing of CD222 causes a shift in Lck pools toward the less active form solely phosphorylated at the inhibitory Y505. To pinpoint whether the increased phosphorylation at Y505 was the cause of a blockade in signaling upon CD222 silencing, we used a mutated form of Lck that was made up of a phenylalanine instead of the tyrosine at the position 505 and consequently could not be phosphorylated at this site (32). Lckwt and the mutated form of Lck (LckY505mut) had been ectopically expressed in Lck-deficient

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 FIGURE 3. CD222 knockdown affects the phosphorylation of distal and ERK1/2. Data represent mean percentage of Max 6 SD. Significance was proximal TCR signaling molecules. (A) shCTR and shCD222/P1 Jurkat evaluated via the Bonferroni posttest following two-way ANOVA; *p , T cells were stimulated with CD3 mAb (MEM-92) for the indicated time 0.05. (D) Upper panels, JCaM 1.6 T cells were stimulated for 15 min with points, and lysates were analyzed via Western blotting for the presence of SEE-loaded Raji B cells, fixed, permeabilized, and stained for CD222 phosphotyrosines. GAPDH was used as a loading control after stripping of (red). Lower panels, JCaM 1.6 T cells without activated B cells were the membrane; kiloDalton ranges are indicated on the left. Arrows depict treated the same. Pictures were captured at a CLSM. Phase contrast pic- kiloDalton areas of 70, 38, and 22 kDa. (B) For the simultaneous detection tures were overlaid with the CD222-staining (right). Nuclei were visual- of total protein versus the phosphorylated form (green versus red), we ized with DAPI (blue). Shown is one of at least two representative ex- analyzed lysates from shCTR and shCD222/P1 Jurkat T cells with spe- periments. Scale bar, 5 mm. The Journal of Immunology 2725

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- 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 Downloaded from 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. The precipitated proteins were analyzed via LC-MS. The mAb AFP-12 to a-fetoprotein was used as a control (CTR mAb). Proteins with a MEM-238:CTR mAb ratio .3 were considered as specifically enriched. The table lists proteins reported to be relevant for T cell regulation sorted via the delta value (D) of MEM-238 and CTR Ab. http://www.jimmunol.org/ JCaM 1.6 T cells before the cells were silenced for CD222 and 1.6 T cells were retrovirally transduced to ectopically express intracellular calcium mobilization was measured via flow cytometry. GFP-tagged Lck (Lck-GFP) to visualize the molecule in situ JCaM 1.6 T cells expressing Lckwt showed a reduced calcium flux without staining (Fig. 6C, upper panel). CD222 silencing at P1 led upon CD222 compared with control silencing. However, silencing to a significant reduction of surface-localized Lck, as indicated by of CD222 did not affect the calcium flux in JCaM 1.6 T cells the decrease in the surface/ ratio (Fig. 6C). shCD222/P2 carrying the mutated form of Lck (Fig. 5F), suggesting that the cells also displayed a significantly decreased surface/cytoplasm altered phosphorylation of Lck upon CD222-silencing caused the ratio but again less pronounced than shCD222/P1. This phenom- less reactive T cell phenotype. enon was highly specific because the transport of the Lck mutant variant bearing only the first 10 aa (LckN10-GFP), encompassing by guest on October 6, 2021 CD222 regulates the intracellular distribution of Lck and sites indispensable for the cell To test whether CD222 was responsible for the efficient transport of membrane targeting (Fig. 6D, upper panel) and lipid raft parti- Lck, we analyzed the distribution and membrane localization of tioning, was unaffected (Fig. 6D). In addition, the GFP-tagged Lck via immunofluorescent staining and CLSM of resting control Lck lacking the first 10 aa (LckDN10-GFP), a mutant variant and CD222 knockdown Jurkat T cells. The typical homogenous that in contrast cannot be inserted into the inner leaflet of the surface distribution of Lck was disrupted in CD222 knockdown plasma membrane (Fig. 6E, upper panel) was distributed evenly cells (Fig. 6A). Approximately 60–70% of shCD222/P1 and within the cell of all cell types investigated, as can be seen by the shCD222/P2 Jurkat T cells showed a diminished Lck surface ratio around 1 (Fig. 6E). When human Lck-GFP was retrovirally distribution when counted manually. Surface plots of Lck staining introduced in CD222-negative mouse fibroblasts, we observed showed a punctuated distribution in CD222-silenced cells with similar patterns: Lck was not sufficiently transported to the sur- Lck-free gaps among Lck-rich membrane areas (Fig. 6B), al- face, whereas in cells coexpressing human recombinant CD222, though the total Lck content in crude cell lysates of unstimulated the surface localization of Lck-GFP increased (Fig. 6F). These Jurkat T cells was even higher in CD222-silenced cells as detected findings indicate that CD222 does not influence lipid raft parti- by immunoblotting (Supplemental Fig. 2C). Lck is posttransla- tioning of Lck but rather directs its cellular distribution. tionally modified by palmitoylation and myristoylation at its N terminus, which directs and anchors the molecule into lipid rafts CD222 knockdown impairs the interaction of CD45 with Lck (33, 34)—organizational platforms of cellular membranes im- Finally, we investigated the interaction of Lck and CD45. Unsti- portant for T cell signaling (35–38). We investigated total cellular mulated JCaM 1.6 T cells ectopically expressing Lck-GFP were lipid raft fractions in resting shCD222/P1 and shCTR Jurkat stained for CD45 and analyzed for the colocalization of CD45 with T cells by means of sucrose-based density gradient flotation Lck in shCTR and CD222-silenced cells (shCD222/P1 and /P2; analysis. The comparison of lipid raft- with nonraft fractions did Fig. 7A). The overall Pearson’s coefficient, a measure of coloc- not reveal any significant changes upon CD222 knockdown (Sup- alization, of the CD45-Lck interaction was low, as reported pre- plemental Fig. 4A). The size exclusion chromatography analysis viously (4), but markedly decreased upon silencing of CD222 on a Superose 6 column revealed in shCD222 cells a slight shift (Fig. 7B). In silenced cells, Lck was apparently retained in in- of both Lck and CD45 towards higher molecular mass fractions, tracellular membranes and/or accumulated freely in the cyto- which also contained EEA1 (Supplemental Fig. 4B) corresponding plasm. By biochemical separation of total cellular membranes to endosomal membranes (39). from cytosolic cell contents, we found a significant increase in To analyze the intracellular distribution of Lck in more detail, we cytosolic Lck upon CD222 silencing (Fig. 7C). To test whether the evaluated the surface/cytoplasm ratio of Lck. Lck-deficient JCaM CD45–Lck interaction occurred at the plasma membrane, we la- 2726 CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck Downloaded from http://www.jimmunol.org/ by guest on October 6, 2021

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). beled the surface of living cells with a CD45 mAb on ice, lysed surface CD45 (Fig. 7E). These data suggest that CD222 controls the cells, immunoprecipitated cell surface CD45, and analyzed the the transport of the kinase Lck to the plasma membrane and eluates for coimmunoprecipitated Lck (Fig. 7D). The interaction subsequently its interaction with CD45, the key activatory phos- of cell surface CD45 with Lck was reduced by ∼40 and 20% in phatase of Lck. shCD222/P1 and shCD222/P2 knockdown cells, respectively (Fig. 7D). In contrast, LckN10, the non-functional mutant variant of Discussion Lck consisting of the 10 N-terminal amino acids responsible for Lck is described to exist in four different phosphorylation states, plasma membrane anchorage, did not coimmunoprecipitate with constantly maintained at equilibrium, with ∼40–50% of consti- The Journal of Immunology 2727 Downloaded from http://www.jimmunol.org/

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- by guest on October 6, 2021 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. tutive active Lck within resting T cells (3, 40). As active Lck stuck in the TGN, without significant increase in cytosolic Lck. represents a high risk for a T cell to get permanently activated Furthermore, MAL was essential for lipid raft partitioning of Lck, resulting in hyperreactive adaptive immune reactions in an or- whereas the transport to nonraft membranes was unhampered (45). ganism, the spatial arrangement of Lck has to be accurately or- In contrast, knockdown of CD222 hampered the efficient transport ganized in time and space. Limiting the access of possible kinase of Lck to the plasma membrane with an apparent accumulation of substrates or orchestrating the interaction with regulatory proteins Lck within intracellular membranes and freely floating in the can prevent overwhelming or self-directed immune responses. On cytoplasm but without any significant effect on lipid raft parti- the basis of our data, we propose a novel function for the late tioning. It has been shown previously via single molecule mi- endosomal transporter CD222 in the early TCR signaling cascade. croscopy and bleaching experiments that single Lck molecules CD222 is essential for the efficient recruitment of Lck to the recolonize the cell membrane directly from the cytoplasm, rather plasma membrane, the place where it interacts with its phospha- than via two-dimensional transversal diffusion (49), which may tase CD45. The loss of CD222 leads to accumulation of Lck in its explain the high amount of non–membrane-bound cytosolic Lck. inactive form, which results in diminished T cell activation and Lck has been reported previously to be present in endosomes (47, effector functions. 50). Our confocal microscopy and also gel filtration fractionation Because Lck represents a very powerful kinase that is involved in experiments revealed an accumulation of Lck and CD222 in the various T cell signaling pathways (41–44), its transport and reg- EEA1-positive fractions corresponding to endosomal membranes. ulation has been investigated extensively (45–49). One important Furthermore, via mass spectrometry analysis we identified Rab11, molecule implicated in the transport of Lck is the myelin and a marker of endosomal membranes critically involved in protein lymphocyte protein (MAL). Upon knockdown of MAL, Lck was trafficking, to interact with CD222. This strongly suggests that 2728 CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck Downloaded from http://www.jimmunol.org/ by guest on October 6, 2021

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) The Journal of Immunology 2729

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 Downloaded from 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; http://www.jimmunol.org/ *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 by guest on October 6, 2021 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.

CD222 coordinates the endosome-dependent transport of Lck that CD222 can perform its action on human Lck even in mouse was shown to be essential for T cell activation (48). Uncoordi- fibroblasts (i.e., irrespective of other T cell–specific molecules). nated 119 protein has been shown to activate endosomal Rab11 for Moreover, the absence of MAL in fibroblasts (55) suggests that the plasma membrane targeting of Lck (48) and activation of Fyn CD222 operates upstream of MAL. (51). However, unlike MAL and uncoordinated 119 protein, To our knowledge, the role of CD222 in T cell signal trans- CD222 does not contribute to the IS, but rather builds an early duction has yet not been investigated in detail, but CD222 has been vesicular platform submembrane of the IS. Although several shown to enhance TCR-induced signaling via CD26 internalization consecutive devices obviously coordinate Lck transport (52–54), (56). However, we found no evidence for the contribution of CD26 the contribution of CD222 seems to be very specific as human to 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. 2730 CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck of CD222 knockdown on T cell activation in Jurkat T cells that that is both postnatally expressed in thymic epithelial cells (62) lack CD26 expression (57). Interestingly, ligation of the common and upregulated in diseases (28), namely via internalization and costimulatory molecule CD28, which also quantitatively enhances subsequent degradation in lysosomes (12, 63). 4) Furthermore, T cell responses to TCR stimuli (26), did not rescue, but even CD222’s surface upregulation might play a role in T cell differ- resulted in a more pronounced phenotype induced by silencing of entiation from the double negative to the double positive stage as CD222. CD222 Ab was reported to block T cell ontogeny (64). Notably, The loss of CD222 in resting T cells results in accumulation of Lck knockout mice also display a similar reduction in double- Lck single phosphorylated at residue Y505 that represents the positive thymocytes in T lymphocyte development (65) as the closed inactive form of Lck (7, 58). Consequently, the homeostasis one caused by the CD222 Ab treatment. However, because of the of the four proposed conformational forms of Lck (3) is shifted lethality of the CD222 knockout in mice (66–68), the function of toward the inhibitory closed conformation of Lck. Recently, it has CD222 in T cell differentiation has never been shown in vivo. 5) been proposed that the intracellular distribution of Lck is regu- An involvement of CD222 in T cell migration can also not be lated via its phosphorylation state and the corresponding con- excluded as it has been shown that CD222 negatively regulates formations (4, 6): the open active form (pY394) is thought to cell adhesion via uPAR and integrins (22). 6) Alternatively, it induce clustering, whereas the closed inactive form (pY505) could be the result of a better accessibility of the CD222 mAb prevents the formation of clusters, which has been shown to be caused by altered interactions that unmask the epitope. lipid independent (4). Putting these data together one might sug- Irrespective of the pathways that are amenable in the upregu- gest that the CD222 knockdown, which results in a 2-fold increase lation of cell surface CD222 upon T cell activation, we show in this of inhibitory Lck pY505, does prevent Lck from clustering and study a central function of the endosomal transporter CD222 in the T cells from getting activated. The finding that the hyperactive initiation of T cell signal transduction through controlling Lck Downloaded from mutated form LckY505mut triggers the activation of the secondary distribution: CD222 targets Lck to CD45 at the plasma membrane messenger Ca2+ also in the absence of CD222 supports this model. and thereby maintains the equilibrium of Lck phosphorylation at On the basis of our data, CD222 mediates the shift of Lck toward the steady state. the active dephosphorylated form by complexing it with CD45. We pinpoint the CD222-driven CD45–Lck interaction to the cell Acknowledgments http://www.jimmunol.org/ surface, which indicates, first, that dephosphorylation of Y505 via We thank Peter Steinberger (Institute of Immunology, Medical University CD45 takes place at the cell membrane, and second, that Csk- of Vienna) and Thomas Decker (Max F. Perutz Laboratories, Department induced phosphorylation of Y505 (59) can occur within the cell. of Genetics, Microbiology and Immunobiology, University of Vienna) for Because we found CD222 to interact with both the kinase Lck productive discussions and constructive ideas; Erwin Wagner (Spanish and its phosphatase CD45, the question aroused whether the National Cancer Research Centre, Madrid, Spain) for providing mouse transport of CD45 might be as well miss-regulated upon silencing fibroblasts, Va´clav Horejsı´ (Institute of Molecular Genetics, Academy of of CD222. However, several facts speak against the simultaneous Sciences of the Czech Republic, Prague, Czech Republic), Nicole Bou- cheron (Division of Immunobiology, Institute of Immunology, Center for transport deregulation of Lck and CD45 in the CD222 knockdown Pathophysiology, Infectiology and Immunology, Medical University of phenotype. First, we found just a minor downregulation of CD45 Vienna, Austria), and Arthur Weiss (Department of Medicine and De- by guest on October 6, 2021 at the cell surface. Second, CD45 is not only a positive but also partment of Microbiology and Immunology, Rosalind Russell-Ephraim a negative regulator of T cell activation and differentiation (59, P. Engleman Medical Research Center for Arthritis, and Howard Hughes 60). Intermediate levels of CD45 surprisingly produce hyperre- Medical Institute, University of California, San Francisco, San Francisco, active T cells (59), which we could not detect at all in our ex- CA) for providing Ab; Reinhard Fa¨ssler (Department of Molecular Medicine, perimental setup. Third, CD45 dephosphorylates not only Lck but Max Planck Institute of Biochemistry, Martinsried, Germany) for experimen- also CD3z (4, 59–61). However, the phosphorylation of CD3z was tal and intellectual help with mass spectrometric analysis; S. Steward (Wash- unaffected in resting, and notably, even significantly reduced in ington University School of Medicine, St. Louis, MO); G. Nolan (Baxter stimulated CD222 knockdown cells. Taking these facts into con- Laboratory in Stem Cell Biology, Department of Microbiology and Immu- nology, Stanford University, Stanford, California) for providing cloning sideration, we conclude that the primary function of CD222 in vectors and cell lines; and Cristiana Soldani, Anna Elisa Trovato and T cell activation is to act as intermediary in the interaction of Chiuhui Mary Wang (Humanitas Clinical and Research Center, Rozzano, CD45 with Lck resulting in the correct dephosphorylation of Milan, Italy) for practical help with immunofluorescence analysis. We are Lck at the inhibitory Y505. grateful to the imaging and flow cytometry core facilities of the Medical We show in this study that the interaction between CD222 and University of Vienna. Lck occurs preferentially inside the cell and disbands at the cell surface, where Lck is “handed over” to CD45. This indicates that Disclosures CD222 is involved in very early signaling events. 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