The Late Endosomal Transporter CD222
Directs the Spatial Distribution and Activity
of Lck
This information is current as
of June 18, 2017.
Karin Pfisterer, Florian Forster, Wolfgang Paster, Verena
Supper, Anna Ohradanova-Repic, Paul Eckerstorfer,
Alexander Zwirzitz, Clemens Donner, Cyril Boulegue,
Herbert B. Schiller, Gabriela Ondrovicová, Oreste Acuto,
Hannes Stockinger and Vladimir Leksa
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The Journal of Immunology is published twice each month by
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Copyright © 2014 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2014; 193:2718-2732; Prepublished online 15
August 2014;
doi: 10.4049/jimmunol.1303349
http://www.jimmunol.org/content/193/6/2718
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 Ondrovicová,x Oreste Acuto,†
Hannes Stockinger,* and Vladimir Leksa* ,x
T
cell signaling molecules have to be tightly controlled
spatially and temporally to ensure proper T cell activation
without hyperresponsiveness (1). A fundamental step
during the onset of T cell signaling is the recruitment of the
lymphocyte-specific protein tyrosine kinase (Lck) to the immunological synapse (IS) and the subsequent phosphorylation of
the ITAMs of the CD3 z-chain (CD3z). ZAP70 can thereafter bind
to the phosphorylated ITAMs of the TCR complex where it is
phosphorylated by Lck to initiate further downstream signaling
(2). For fine-tuning of T cell activation active and inactive Lck pools
must be kept at equilibrium. It has been shown that Lck activity is
not directly linked with TCR engagement (3, 4). Lck rather exists
in several conformational and activity states: 1) an unphosphorylated “primed” form; 2) an active form phosphorylated at the
*Molecular Immunology Unit, Institute for Hygiene and Applied Immunology,
Center for Pathophysiology, Infectiology and Immunology, Medical University of
Vienna, Vienna A-1090, Austria; †Sir William Dunn School of Pathology, University
of Oxford, Oxford OX1 3RE, United Kingdom; ‡Department of Molecular Medicine,
Max-Planck Institute of Biochemistry, Martinsried 82152, Germany; and xLaboratory
of Molecular Immunology, Institute of Molecular Biology, Slovak Academy of Sciences, 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 Förderung
der Wissenschaftlichen Forschung) P22908 (to V.L.), European Union Framework
Program 7 “NANOFOL” Grant NMP4-LA-2009-228827 (to H.S.), Wellcome Trust
Grant GR076558MA, and European Union Framework Program 7 Grant EC-FP7SYBILLA-201106 (to O.A.). W.P. was supported by the Erwin Schroedinger Fellowship from the Austrian Science Fund.
Address correspondence and reprint requests to Dr. Vladimir Leksa and Dr. Hannes
Stockinger, Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology, and Immunology, Medical University of Vienna, Lazarettgasse
19, 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, liquid
chromatography–mass spectrometry; Luc, luciferase; MAL, myelin and lymphocyte
protein; mut, mutant; OST, One-Strep-tag; pY, phospho-tyrosine; SEE, staphylococcal enterotoxin E; sh, short hairpin; TGN, trans-Golgi network; wt, wild-type.
Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303349
tyrosine 394 (pY394); 3) an inactive “closed“ form phosphorylated at the COOH-terminal tyrosine 505 (pY505); and 4) a
double phosphorylated form (pY394 + pY505) that is thought to
possess an equal kinase activity as single phosphorylated Lck at
Y394 (3). Hence, active Lck is constitutively present in resting
T cells, and it is its coordinated intracellular distribution that is
crucial to balance T cell activity and nonreactivity (5–7). The
antagonistic activities of the phosphatase CD45 and the kinase
Csk control Lck’s mode of action (2, 8, 9). CD45 plays a pivotal
role in the activation of Lck via dephosphorylation of Y505 (10,
11), but it is not clear where the interaction takes place and how it
is regulated in space and time.
The late endosomal transmembrane molecule CD222, also known
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 proteins
including acid hydrolases and TGF-b (13), the insulin-like growth
factor 2 (14) and plasminogen (15). On delivering its cargo, CD222
traffics between the trans-Golgi network (TGN), endosomes, and the
plasma membrane (16). CD222 is barely expressed on the surface of
resting T cell but has been reported to be upregulated at the plasma
membrane upon T cell activation in rodents (17). In this study, we show
that CD222 is also upregulated on the surface of human T cells
upon stimulation. Remarkably, we found that CD222 transports
Lck intracellularly and directs its distribution to CD45 at the plasma
membrane. The loss of CD222 causes a decrease in signal transduction and subsequently a profound blockade of T cell effector functions.
Materials and Methods
Abs
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. Horejsı́
(Institute of Molecular Genetics, Academy of Sciences of the Czech
Republic, Prague, Czech Republic). The anti–TCRb-chain mAb C305
was a gift from Dr. A. Weiss (University of California, San Francisco,
San Francisco, CA). The stimulatory CD3 mAb OKT3 was from Ortho
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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 CD222deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation 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.
The Journal of Immunology
Pharmaceuticals (Raritan, NJ). The CD28 mAb Leu-28, anti–phosphoCD3z/CD247 (Y142) mAb K25-407.69, and anti–phospho-linker of activated T cells (LAT; Y171) were from BD Biosciences (San Jose, CA). The
HRP-conjugated anti-phosphotyrosine mAb 4G10 was from Merck
Millipore (Billerica, MA). The anti-GAPDH mAb 14C10, anti–phosphoZAP70 Ab (Y319)/Syk (Y352), anti-ZAP70 mAb 99F2 and L1E5, anti–
phospho-p44/42 MAPK Ab ERK1/2 (Y202/Y204), anti-p44/42 MAPK Ab
ERK1/2, anti–phospho-Src mAb (Y416), anti–non-phospho-Src mAb
(Y416), anti–phospho-Lck Ab (Y505), anti-Lck mAb 73A5, and the rabbit
mAb to early endosome Ag 1 (EEA1) C45B10 were from Cell Signaling
Technology (Danvers, MA). The anti-Lck mAb H-95 was from Santa Cruz
Biotechnology (Santa Cruz, CA). Biotinylation of unconjugated mAb was
performed in our laboratory. As secondary reagents we used HRP-conjugated 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
T cell activation assay
Primary human T cells isolated by CD14-depletion from PBMC were
cultured for 2 d to separate non- from adherent cells. T cell lines and primary
human T cells (5 3 104) were seeded in 96-well plates and kept unstimulated or stimulated by: 1) plate-bound CD3 mAb OKT3 (1 mg/ml) alone or
together with soluble CD28 mAb Leu-28 (0.5 mg/ml); 2) CD3 Ab-coupled
beads; 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, before
stimulation, the cells were resuspended in PBS, incubated with CFSE
(0.5 mmol/l; Molecular Probes, Eugene, OR) at 37˚C for 10 min, and
thereafter washed twice with medium.
Immunoprecipitation, immunoblotting, and mass spectrometric
analysis
For immunoprecipitation of CD222, 2–20 3 107 cells were lysed in lysis
buffer (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 inhibitor mixture (Roche, Basel, Switzerland), and benzonase (Novagen,
Merck Millipore) for 30 min on ice. Lysates were incubated for 2 h at 4˚C
with cyanogen bromide–activated beads coupled with CD222 mAb MEM238 or isotype control mAb AFP-12. The beads were washed three times
with ice-cold lysis buffer, and the bound proteins were eluted with 1.23
nonreducing Laemmli sample buffer at 95˚C for 5 min.
For immunoprecipitation of Lck, the Lck-deficient Jurkat T cell clone
JCaM 1.6 was transduced with a lentiviral expression construct for Lck
fused to the One-Strep-tag epitope (Lck-OST). Lysates of resting or sodium
pervanadate–stimulated cells were subjected to precipitation with Streptactin–Sepharose beads. Biotin was used to elute bait proteins from the
capture beads. Biotin-blocked beads served as a negative control.
For the analysis of the CD45–Lck interaction at the plasma membrane, JCaM
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
with lysis buffer containing detergent 1% Nonidet P-40. The mAb complexes
were then precipitated by using protein A/G beads (Santa Cruz Biotechnology).
For specific immunoprecipitation of cell surface CD222, unstimulated
PBMC 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 with
detergent 1% lauryl maltoside on ice. The Ab–protein complexes were
fished 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 to
Immobilon polyvinylidene difluoride membranes (Merck Millipore). The
membranes were blocked with 5% nonfat milk (Maresi, Vienna, Austria) in
TBS-Tween and incubated with specific Ab dilutions for detection of indicated
proteins. For visualization, blots were developed either with HRP secondary
conjugates and a HRP substrate (Biozym, Hessisch Oldendorf, Germany) or
with secondary fluorescence Abs, followed by detection at either the Fuji LAS400 imager (Fuji, Tokyo, Japan) or the Odyssey infrared imaging system
(LiCor Biosciences, Lincoln, NE). Band intensities were quantified via Multi
Gauge Software Version 3.1 (Fuji), Image Studio Version 2.0, or ImageJ.
For mass spectrometric analysis, CD222 coimmunoprecipitates and corresponding controls were digested in-solution as described previously (20),
and samples were prepared, according to the filter aided sample preparation
procedure following analysis via liquid chromatography–mass spectrometry
(LC-MS). Lck coimmunoprecipitates were separated on 4–12% gradient
Bis-Tris NuPAGE gels (Invitrogen Life Technologies), the gels were washed
in distilled water, lightly stained with Colloidal Blue (Invitrogen Life
Technologies), and subjected to Gel-LC-MS as described previously (21).
Gene knockdown via RNA interference and retroviral
transduction
Stable knockdown cell lines were produced via lentiviral introduction of
short hairpin (sh)RNA specific for the CD222 mRNA. The following CD222
silencing sequences were used for cloning into the vector pLKOpuro1 (a gift
from S. Steward, Washington University School of Medicine, St. Louis, MO):
shCD222/P1 at position 6588 with the sequence 59-GCCCAACGATCAGCACTTCttcaagagaGAAGTGCTGATCGTTGGGC-39 and shCD222/P2 at
position 4525 with the sequence 59-GAGCAACGAGCATGATGACttcaagagaGTCATCATGCTCGTTGCTC-39. The MISSION nontarget shRNA control
vector (pLKOpuro1; Sigma-Aldrich) was used as a negative control (shCTR).
Virus particles were generated via transfection of HEK-293 cells with the silencing constructs and the lentiviral envelope coding plasmid pMD2.G and the
packaging vector psPAX 2 (both helping vectors were constructed by D. Trono,
Ecole Polytechnique Federal de Lausanne, and obtained from Addgene,
Cambridge, MA) as described previously (22). After 48 h, the virus particles
were harvested, filtered, and used for the target cell line infections in the
presence of 5 mg/ml polybrene (Sigma-Aldrich). The next day, the cells were
washed with and maintained in medium containing 1 mg/ml puromycin
(Sigma-Aldrich) to select transduced cells. CD222 knockdown cells were
used from 10 d to 2 mo postinfection for all experiments.
Lck constructs were prepared in the retroviral expression vector pBMN-Z
for transfection of the Phoenix amphotropic virus producer cell line (both
provided by G. Nolan, Stanford University School of Medicine, Stanford,
CA) (15). The target cells were transduced one to three times with the viral
supernatants depending on the efficacy of transduction and maintained
afterward in normal culture medium.
Gene knockout via zinc finger nucleases
Cys2His2-based zinc fingers were created via the Context Dependent
Assembly method provided by the free software tool ZiFiT to identify and
target the genomic sequence of CD222 specifically (23). The zinc finger
constructs targeting exons 18 and 22 were cloned into pMLM290/
pMLM292 and pMSM800/pMLM802 (both from Addgene) to generate
zinc finger nuclease expression constructs. These constructs were used for
the transfection of Jurkat T cells via electroporation. For this, 2 3 106 cells
were 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 with
the plasmids (each 0.5 mg), transferred in electroporation cuvettes, and
transfected with the Amaxa System program S-18 (Amaxa, Lonza, Basel,
Switzerland). Cells were immediately removed from the cuvettes, shortly
spun down, and cultured in 12-well plates containing prewarmed medium.
After cell recovery and expansion, CD222-negative cells were repeatedly
sorted 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 scanning
microscopy
Primary T cells or T cell lines were let to adhere on adhesion slides
(Superior-Marienfeld Laboratory Glassware, Lauda-Köningshofen, Germany),
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
The T cell line Jurkat E6.1 and the B cell line Raji were from the American
Type 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 by
E. Wagner (Spanish National Cancer Research Centre, Madrid, Spain).
The Jurkat IL-2 Luc T cells were stably transfected with a luciferase
expressing IL-2 promoter reporter construct as described previously (19).
The Jurkat NFAT Luc cells containing an artificial promoter region with
four NFAT binding sites were created in our laboratory. Human PBMC
were isolated from purchased buffy coats of healthy adult volunteers (Rotes
Kreuz, Vienna, Austria) by density gradient centrifugation on Lymphoprep
(Nycomed). All primary human T cells as well as mouse and human
T cell lines were maintained in RPMI 1640 medium, the HEK-293 cells
in DMEM, all supplemented with 10% heat-inactivated FCS (SigmaAldrich), 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˚C
and 5% CO2 and passaged every 2–3 d.
2719
2720
CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck
fixed, and permeabilized with 4% formaldehyde and 0.1% saponin, respectively, and stained with the following Ab: the rabbit anti-Lck Ab
H-95 and the rabbit anti-human mAb EEA1 (C45B10), followed by a
goat anti-rabbit AF488 Ab, the anti-Lck mouse Ab (73A5), followed by
anti-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 indicated time points. Mouse fibroblasts were either cultured in 8-well chamber
slides (Nunc, Thermo Scientific Fisher) until confluency or trypsinized
and cytospun onto glass slides prior to fixation and staining as described
above. For each cell, one vertical and one horizontal cut was made through
the cell, and the Lck distribution was analyzed using the ZEN 2011 blue
edition software (Zeiss, Jena, Germany). Nuclei were stained with DAPI.
The slides were washed with 13 PBS and mounted with mounting medium for fluorescence analysis (Vectashield; Vector Laboratories, Burlingame,
CA). Isotype-matched controls were included. Pictures were captured with
a confocal laser scanning microscopy (CLSM) 700 (Zeiss).
Flow cytometry
Luciferase assays
To assess the IL-2 promoter activity and NFAT binding to DNA, IL-2 luciferase- and NFAT-reporter Jurkat T cells were assayed, respectively. Briefly,
the reporter cells (2 3 105) were either kept unstimulated or were stimulated
for 6 h in triplicates in CD3 mAb OKT-3–coated (1 mg/ml) 96-well plates
with or without soluble CD28 mAb Leu-28. The cells were washed with PBS
and subsequently lysed in 100 ml luciferase lysis reagent (Promega, Madison, WI) for 30 min on ice. Seventy-five microliters of lysate were transferred
into white 96-well microplates, and luminescence was measured after addition of the reaction reagent at a Mithras LB940 Elisa Reader (Berthold
Technologies, Bad Wildbach, Germany). The protein concentration was
determined via Bradford assay (Bio-Rad, Hercules, CA), and the luminescence intensity was normalized to the protein content of each sample.
Cytokine measurement
Culture supernatants (30 ml) of primary T cells were harvested after 24 h
of stimulation and analyzed via the Luminex xMAP suspension array
technology. Briefly, the cells were stimulated with plate-bound OKT3
(1 or 5 mg/ml) and soluble Leu-28 (2 mg/ml) or kept unstimulated. Standard curves were generated using recombinant cytokines (R&D Systems,
Minneapolis, MN). Experiments were performed at least twice with each
sample 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 medium
containing 1 mmol/l Indo-1 AM (Invitrogen Life Technologies) and incubated
for 30 min at 37˚C. The cells were washed with medium, incubated in 1 ml
medium for next 30 min at 37˚C, and rested thereafter on ice until data acquisition at a LSR II flow cytometer. For the analysis of intracellular calcium
flux, 300 ml Indo-1–loaded cells were prewarmed for 5 min at 37˚C, the baseline response was recorded for 30 s, before the cells were stimulated with a
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/
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.
Analysis of T cell signaling molecules
Jurkat T cells or primary T cells (1.5 3 106) were starved for 1 h in RPMI
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
to bind for 5 min, and then, the cells were incubated for several time points
at 37˚C. The cells were quickly washed with ice-cold lysis buffer without
detergent and lysed in complete lysis buffer containing 1% detergent
Nonidet P-40 (Promega), a protease inhibitor mixture (Roche), sodium
orthovanadate (Sigma-Aldrich), sodium fluoride, and benzonase for 30 min
on ice. Lysates were sampled with 43 reducing or nonreducing sample
buffer and analyzed by Western blot analysis.
For real-time PCR analysis, primary human T cells, both resting and
stimulated for 1 d with plate-bound CD3 mAb OKT3 (1 mg/ml) together
with soluble CD28 mAb Leu-28 (0.5 mg/ml), were lysed in TRIzol
(Invitrogen Life Technologies), and RNA was extracted according to the
manufacturer’s instructions. cDNA was synthesized from 500 ng total
RNA with SuperScript VILO cDNA Synthesis Kit (Invitrogen Life Technologies). Gene expression was measured by the 22DDCT method (24),
based on quantitative real-time PCR using the CFX96 Real-Time PCR system (Bio-Rad) with TaqMan primer sets for human CD222, and YWHAZ as
an endogenous control. For conventional PCR, 20 ng genomic DNA purified
by standard isopropanol precipitation was used as a template of PCR amplification with Taq polymerase (40 cycles at 95˚C for 30 s, 61˚C for 30 s, and
72˚C for 30 s) and with primer sets for human CD222 bridging an exon–exon
junction, and intronless gene endosialin, as an endogenous control (TaqMan
Gene Expression Assay; ABI, Life Technologies).
Lipid raft preparation
Cells (2–3 3 107) were lysed for 30 min on ice in lysis buffer containing
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% sucrose layer and overlaid by 20, 10, and 5% sucrose fractions (all 1 ml
containing 0.5% detergent Brij-58 and protease inhibitors). Gradients were
ultracentrifuged 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 sample
buffer and analyzed by SDS-PAGE, followed by Western blot analysis.
Gel filtration analysis
The GE Healthcare AKTA FPLC system was used for the gel filtration
experiments. Cell lysates were prepared by solubilization with 1% detergent
Triton X-100 (Promega). Samples were loaded at 4˚C in a volume of 500 ml
onto a Superose 6 HR 10/300 GL column (GE Healthcare) equilibrated
with Triton X-100 (0.5%) in PBS. The absorbance at 280 nm was monitored, and 500-ml fractions were collected and analyzed by immunoblotting. The following standards of molecular mass (all from GE Healthcare
or 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 potassium chloride, 5 mmol/l magnesium chloride, 10 mmol/l HEPES, and
protease inhibitor mixture [pH 7.5]) for 30 min on ice and thereafter
passaged nine times through a 30-g needle. Cell fragments were centrifuged twice at 300 3 g, and the resulting supernatant was centrifuged for
40 min at 35,000 3 g at 4˚C. The cytosolic supernatant was collected and
the pellet containing membranous cellular parts was solubilized in extraction buffer (13 lysis buffer containing 1% Triton X-100, 0.1%
NaDodSO4, 0.5% sodium deoxycholate, and the protease inhibitor mixture) for 30 min on ice and centrifuged at 300 3 g to remove debris. The
fractions were analyzed via Western blotting.
Statistical analysis
Data analysis was performed in GraphPad Prism5 (GraphPad Software, La
Jolla, CA). Different experiments were analyzed by one-sample or standard
Student t test or two-way ANOVA, followed by the Bonferroni posttest. A
p value , 0.05 was considered as significant (*). Statistical analyses were
performed in GraphPad Prism. In general, data are expressed as mean 6
SEM (or mean 6 SD, when indicated).
Results
CD222 surface expression on human T cells increases upon
TCR stimulation
CD222 is upregulated on the cell surface of rodent T cells upon T cell
activation (17). Therefore, we hypothesized that this molecule might
play a role in T cell activation. We first investigated the expression
of CD222 on human lymphocytes. Isolated human PBMC were
labeled with CFSE and analyzed for surface expression of CD222
via flow cytometry using a specific CD222 mAb. Upon crosslinking 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
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For the analysis of cell surface Ags, cells were washed and resuspended in
staining buffer (13 PBS containing 1% BSA and 0.02% NaN3). Subsequently, the cells were incubated for 20 min at 4˚C with a fluorochromeconjugated mAb. For indirect immunofluorescence staining, the cells were
additionally incubated with secondary conjugates for 20 min at 4˚C. Before analysis, the cells were washed with staining buffer. Flow cytometry
was performed using a LSR II (BD Biosciences) and analyzed with FlowJo
version 8.8.6 for Macintosh or FlowJo version 7.2.5 for Windows.
PCR analysis
The Journal of Immunology
and CD3/CD28 mAb–stimulated T cells revealed no significant
increase in CD222 expression upon activation (Fig. 1C). This suggests the regulation of CD222 via subcellular distribution and not
via gene expression. Regardless of the cell type, ∼90% of CD222
have been reported to be localized in the TGN and endosomal
compartments at steady state (25). Therefore, the majority of
CD222 is kept intracellular and only a small proportion is displayed, 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 cell
division cycles. Both stimulated, yet not blasting T cells and
2721
blasting T cells displayed more CD222 on the surface compared
with unstimulated T cells (Fig. 1B). Notably, the majority of
proliferating cells was positive for CD222, which pointed toward
a possible involvement of CD222 in T cell activation and/or differentiation.
CD222 knockdown dampens T cell effector functions
To investigate the putative role of CD222 in T cell activation, primary human PBMC were silenced for the expression of CD222
via the lentiviral-based introduction of shRNA. Total CD222
protein content was analyzed for shCTR and CD222-silenced
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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
T cells (Fig. 2E) suggesting a direct correlation between the total
amount of the CD222 protein and the T cell response to TCR
stimuli. Furthermore, the artificial overexpression of CD222 led to
significantly higher intracellular Ca2+ flux in Jurkat T cells and
CD222-overexpressing cells reacted faster than cells expressing
the control vector (Fig. 2F). However, it has to be mentioned that
cells highly overexpressing CD222 are more susceptible for apoptosis compared with control cells (28). Furthermore, high
amounts of CD222 lead to a decreased cellular growth rate (29).
Therefore, just cells slightly overexpressing CD222 survived and
grew adequately. Finally, to verify the data obtained via RNA
interference, genomic CD222 was targeted and cut via sequencespecific zinc finger nucleases in Jurkat T cells (Supplemental Fig.
1A). Analysis of three independently sorted CD222 knockout
populations (C20, C21, and C25; Supplemental Fig. 1B), revealed
that each of them displayed a reduction in intracellular Ca2+ fluxes
compared with CTR cells (Supplemental Fig. 1C). Importantly,
this phenotype was rescued in knockout cells that were stably
transduced with recombinant human CD222 (C20 cells reconstituted with CD222; Supplemental Fig. 1D–F). Taken together,
these data imply that CD222 is an essential regulator of the TCR
signaling cascade.
CD222 is crucial for early T cell signaling
To untangle the mode of operation of CD222 in TCR signaling, we
stimulated control- and CD222-silenced Jurkat T cells with the TCRspecific mAb C305 and evaluated the tyrosine phosphorylation in the
cell lysates via Western blot analysis: several molecules were less
phosphorylated in CD222-silenced cells, particularly in molecular
mass regions corresponding to the TCR proximal signaling molecules ZAP70, LAT, and CD3 (Fig. 3A), suggesting a very early
effect of CD222 on T cell signal transduction. To verify this, we
used specific mAb in a multicolor fluorescence multiplexing approach. Indeed, the earliest T cell signaling proteins CD3z and
ZAP70 displayed lower tyrosine phosphorylation upon CD222
downregulation. Accordingly, all ensuing signaling molecules, such
as LAT and ERK, were less phosphorylated in shCD222 cells
(Fig. 3B). Statistical evaluation of multiple experiments analyzing
pCD3z and pERK1/2 revealed that the phosphorylation of both
(i.e., early and late signaling molecules) was significantly reduced
upon CD222 knockdown (Fig. 3C). In addition, Lck’s serine 59,
a substrate of active ERK1/2 (30), was less phosphorylated in
CD222 knockdown cells, which possibly could underlie an insufficient signal amplification to sustain T cell activation (Supplemental
Fig. 2A). Supporting evidence for the implication of CD222 in
TCR-proximal signaling was provided via CLSM: upon 15-min
stimulation of JCaM 1.6 T cells with superantigen SEE-loaded
Raji B cells, CD222 redistributed from the cell surface and/or
intracellular pericentrosomal compartments (31) to the interface
of the IS in ∼70% of JCaM 1.6 T cells that were in contact to Raji
B cells (n = 24; Fig. 3D).
Lck and CD45 interact with CD222
To unravel possible interaction partners of CD222, we pulled down
CD222 with specific mAb-coupled beads after cell lysis of resting
Jurkat T cells with lauryl maltoside, and analyzed coprecipitated
proteins via LC-MS. We found, along with known CD222 binding
proteins, various T cell–specific proteins. Among these proteins
we found Lck and CD45, molecules implicated in the regulation
of T cell signaling (Table I). To verify the protein–protein interactions, we precipitated CD222 from lysates of resting Jurkat
T cells via CD222 mAb-coated beads and analyzed coimmunoprecipitated proteins by Western blotting. We detected both, Lck
and CD45, in the CD222 coimmunoprecipitates (Fig. 4A).
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(shCD222/P1) primary T cells via Western blotting and revealed
a reduction by ∼75% (Fig. 1D). Subsequent flow cytometric
analysis revealed that CD222 surface expression was downregulated by ∼50% on shCD222/P1 T cells compared with shCTR
T cells (Fig. 1E). To assess their effector functions, both shCTR
and shCD222/P1 T cells were stimulated with low or high concentrations of plate-bound CD3 mAb (1 and 5 mg/ml) in combination with costimulatory soluble CD28 mAb (2 mg/ml). Secreted
cytokine levels were measured via the Luminex system. Secretion
of TNF-a, IL-2, and IFN-g was reduced by .50% in shCD222/P1;
higher concentrations of CD3 mAb could not rescue the response in
shCD222 cells (Fig. 1F). For further experiments, the wild-type (wt)
Jurkat T cell line as well as the IL-2 and NFAT luciferase reporter
Jurkat T cell lines were used because the overall CD222 silencing efficiency was mostly moderate in primary T cells in our hands,
whereas in Jurkat T cells, the efficiency of silencing was much
higher. Furthermore, CD222 is a very stable molecule. It takes ∼7–
10 d upon silencing for the complete degradation of CD222 so that
the functional effects become observable. As primary T cells are
very sensitive to prolonged in vitro culture, we used the T cell lines
for further experiments.
To determine whether the cytokines were less synthesized or the
transport/secretion was hampered upon CD222 downregulation, we
used human luciferase reporter T cell lines: first, CD222- and
control-silenced Jurkat T cells that expressed luciferase under the
control of the IL-2 promoter were stimulated via CD3 or CD3/
CD28, and the lysates were analyzed for luciferase activity. Silencing of CD222 at two distinct positions (shCD222/P1 and
shCD222/P2) led to a reduction of the luciferase activity upon
T cell stimulation via CD3 (Fig. 2A). Even activation of the costimulatory pathway via CD28 ligation, which quantitatively enhances TCR-induced signals (26), could not rescue the IL-2 promoter activity in the reporter cells silenced at the first position
(P1) and resulted in a significant luciferase reduction. Although
silencing at the second position (P2) did not reduce CD222 transcripts as effective as P1 (Fig. 2A, inset), the corresponding cells
still displayed a reduced IL-2 promoter activity, although not
significant. Second, we used human reporter Jurkat T cells that
expressed the luciferase gene driven by an artificially designed
promoter region containing multiple binding sites for the transcription factor NFAT (27). The NFAT-driven luciferase expression was significantly reduced in shCD222/P1 cells compared
with shCTR cells upon stimulation with CD3/CD28 mAb (Fig.
2B). These data revealed that CD222 knockdown affected cytokine production at the transcriptional level.
To analyze whether decreased Ca2+ mobilization was responsible for reduced NFAT promoter activity, we measured intracellular Ca2+ fluxes by flow cytometry with the Ca2+ sensitive dye
Indo-1. To enable an equal Lck expression in control- and CD222silenced T cells, we used the Lck-deficient Jurkat T cells JCaM
1.6 that we reconstituted before silencing with a GFP-tagged Lck
followed by expressing gating. We found that silencing of CD222
at 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+ flux
experiments showed that the peak values of the 390/495 ratio and
the overall mean values of shCD222/P1-silenced cells were significantly reduced, whereas the baseline values remained unchanged (Fig. 2D). Silencing of CD222 at the less efficient P2 site
influenced the Ca2+ flux to a lesser extent, which might be
explained via a dose-dependent mode of action of CD222. Indeed,
this assumption was supported by other experiments: titration of
virus, carrying the silencing construct, correlated with lower expression of CD222 and reduced Ca2+ mobilization in Jurkat
The Journal of Immunology
2723
Moreover, we detected the Lck-CD222 interaction also by a reverse approach performed with resting JCaM 1.6 T cells, expressing Lck fused to the One-Strep-Tag epitope (Lck-OST).
Therein, CD222 was reproducibly found in the isolated Lck
complexes via LC-MS analysis (Mascot protein score = 486.34).
The interaction of CD222 and Lck also was confirmed by coimmunoprecipitation studies with JCaM 1.6 T cells, followed by
immunoblotting (Fig. 4B). A truncated nonfunctional mutant of
Lck encompassing only the first 10 aa of Lck (LckN10) did not
coimmunoprecipitate with CD222 (Supplemental Fig. 2B), suggesting a specific protein–protein interaction. Furthermore, we
verified the interaction of CD222 and Lck in nonstimulated primary
human T cells isolated from peripheral blood (Fig. 4C).
Immunofluorescence-based in situ localization via CLSM revealed
that Lck colocalized with CD222 in resting Jurkat T cells (Fig. 4D).
However, the majority of interactions seemed to take place in in-
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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 experiment 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.
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 SEEloaded Raji B cells, we found a coordinated intracellular accumulation 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 coefficient 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
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 experiments 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 pictures were overlaid with the CD222-staining (right). Nuclei were visualized with DAPI (blue). Shown is one of at least two representative experiments. Scale bar, 5 mm.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Lck has been reported to exist in resting T cells in four different
pools, each constituting ∼25% of total Lck and constantly maintaining 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
phosphorylation. To test this, we investigated the tyrosine phosphorylation of Lck normalized to total Lck in shCD222/P1,
shCD222/P2, and shCTR Jurkat T cells via LiCor infrared fluorescence 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). Silencing with the second construct (shCD222/P2) as well increased
Lck phosphorylation at Y505 (Fig. 5C), although not significantly,
which correlated with the less effective silencing at this position (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
The Journal of Immunology
2725
Table I. Mass spectrometric identification of T cell–specific interaction partners of CD222
D
CD222
Dipeptidyl-peptidase 1
Voltage-gated potassium channel subunit b-2
Putative uncharacterized protein MAP4K4
14-3-3 protein
Calnexin
Tubulin b-2C chain; Tubulin b-2 chain
Rho guanine nucleotide exchange factor 2
Dedicator of cytokinesis protein 2
Proto-oncogene tyrosine-protein kinase LCK
Leukocyte common Ag (CD45)
Ras-related protein Rab-7a
MHC class I Ag
Serine/threonine-protein phosphatase 2A
Ras-related protein Rab-11B
Stomatin-like protein 2
Sodium/potassium-transporting ATPase subunit a-1
Ras-related protein Rab-1A
Integrin b-chain
Formin-like protein 1
Basigin
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1.22
6.43
2.14
8.88
3.16
2.59
1.53
1.47
1.37
1.34
1.16
1.15
1.02
9.27
7.97
5.46
5.28
4.45
3.83
3.63
3.29
CTR mAb
9
10
107
107
106
106
106
106
106
106
106
106
106
106
105
105
105
105
105
105
105
105
6.53
3.53
1.92
8.53
2.98
4.11
1.02
3.46
0.00
4.92
2.97
2.24
1.46
2.90
9.81
8.42
0.00
7.08
0.00
0.00
1.68
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
CD222 mAb
6
10
105
106
105
106
105
106
105
100
105
105
105
105
105
104
105
100
104
100
100
105
1.22
6.47
2.33
9.73
6.14
3.00
2.55
1.82
1.37
1.84
1.46
1.37
1.17
1.22
8.95
1.39
5.28
5.15
3.83
3.63
4.97
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
109
107
107
106
106
106
106
106
106
106
106
106
106
106
105
106
105
105
105
105
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.
JCaM 1.6 T cells before the cells were silenced for CD222 and
intracellular calcium mobilization was measured via flow cytometry.
JCaM 1.6 T cells expressing Lckwt showed a reduced calcium flux
upon CD222 compared with control silencing. However, silencing
of CD222 did not affect the calcium flux in JCaM 1.6 T cells
carrying the mutated form of Lck (Fig. 5F), suggesting that the
altered phosphorylation of Lck upon CD222-silencing caused the
less reactive T cell phenotype.
CD222 regulates the intracellular distribution of Lck
To test whether CD222 was responsible for the efficient transport of
Lck, we analyzed the distribution and membrane localization of
Lck via immunofluorescent staining and CLSM of resting control
and CD222 knockdown Jurkat T cells. The typical homogenous
surface distribution of Lck was disrupted in CD222 knockdown
cells (Fig. 6A). Approximately 60–70% of shCD222/P1 and
shCD222/P2 Jurkat T cells showed a diminished Lck surface
distribution when counted manually. Surface plots of Lck staining
showed a punctuated distribution in CD222-silenced cells with
Lck-free gaps among Lck-rich membrane areas (Fig. 6B), although the total Lck content in crude cell lysates of unstimulated
Jurkat T cells was even higher in CD222-silenced cells as detected
by immunoblotting (Supplemental Fig. 2C). Lck is posttranslationally modified by palmitoylation and myristoylation at its N
terminus, which directs and anchors the molecule into lipid rafts
(33, 34)—organizational platforms of cellular membranes important for T cell signaling (35–38). We investigated total cellular
lipid raft fractions in resting shCD222/P1 and shCTR Jurkat
T cells by means of sucrose-based density gradient flotation
analysis. The comparison of lipid raft- with nonraft fractions did
not reveal any significant changes upon CD222 knockdown (Supplemental Fig. 4A). The size exclusion chromatography analysis
on a Superose 6 column revealed in shCD222 cells a slight shift
of both Lck and CD45 towards higher molecular mass fractions,
which also contained EEA1 (Supplemental Fig. 4B) corresponding
to 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 express
GFP-tagged Lck (Lck-GFP) to visualize the molecule in situ
without staining (Fig. 6C, upper panel). CD222 silencing at P1 led
to a significant reduction of surface-localized Lck, as indicated by
the decrease in the surface/cytoplasm ratio (Fig. 6C). shCD222/P2
cells also displayed a significantly decreased surface/cytoplasm
ratio but again less pronounced than shCD222/P1. This phenomenon was highly specific because the transport of the Lck mutant
variant bearing only the first 10 aa (LckN10-GFP), encompassing
myristoylation and palmitoylation sites indispensable for the cell
membrane targeting (Fig. 6D, upper panel) and lipid raft partitioning, was unaffected (Fig. 6D). In addition, the GFP-tagged
Lck lacking the first 10 aa (LckDN10-GFP), a mutant variant
that in contrast cannot be inserted into the inner leaflet of the
plasma membrane (Fig. 6E, upper panel) was distributed evenly
within the cell of all cell types investigated, as can be seen by the
ratio around 1 (Fig. 6E). When human Lck-GFP was retrovirally
introduced in CD222-negative mouse fibroblasts, we observed
similar patterns: Lck was not sufficiently transported to the surface, whereas in cells coexpressing human recombinant CD222,
the surface localization of Lck-GFP increased (Fig. 6F). These
findings indicate that CD222 does not influence lipid raft partitioning 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. Unstimulated JCaM 1.6 T cells ectopically expressing Lck-GFP were
stained for CD45 and analyzed for the colocalization of CD45 with
Lck in shCTR and CD222-silenced cells (shCD222/P1 and /P2;
Fig. 7A). The overall Pearson’s coefficient, a measure of colocalization, of the CD45-Lck interaction was low, as reported previously (4), but markedly decreased upon silencing of CD222
(Fig. 7B). In silenced cells, Lck was apparently retained in intracellular membranes and/or accumulated freely in the cytoplasm. By biochemical separation of total cellular membranes
from cytosolic cell contents, we found a significant increase in
cytosolic Lck upon CD222 silencing (Fig. 7C). To test whether the
CD45–Lck interaction occurred at the plasma membrane, we la-
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Protein Names
2726
CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck
beled the surface of living cells with a CD45 mAb on ice, lysed
the cells, immunoprecipitated cell surface CD45, and analyzed the
eluates for coimmunoprecipitated Lck (Fig. 7D). The interaction
of cell surface CD45 with Lck was reduced by ∼40 and 20% in
shCD222/P1 and shCD222/P2 knockdown cells, respectively (Fig.
7D). In contrast, LckN10, the non-functional mutant variant of
Lck consisting of the 10 N-terminal amino acids responsible for
plasma membrane anchorage, did not coimmunoprecipitate with
surface CD45 (Fig. 7E). These data suggest that CD222 controls
the transport of the kinase Lck to the plasma membrane and
subsequently its interaction with CD45, the key activatory phosphatase of Lck.
Discussion
Lck is described to exist in four different phosphorylation states,
constantly maintained at equilibrium, with ∼40–50% of consti-
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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).
The Journal of Immunology
2727
tutive active Lck within resting T cells (3, 40). As active Lck
represents a high risk for a T cell to get permanently activated
resulting in hyperreactive adaptive immune reactions in an organism, the spatial arrangement of Lck has to be accurately organized in time and space. Limiting the access of possible kinase
substrates or orchestrating the interaction with regulatory proteins
can prevent overwhelming or self-directed immune responses. On
the basis of our data, we propose a novel function for the late
endosomal transporter CD222 in the early TCR signaling cascade.
CD222 is essential for the efficient recruitment of Lck to the
plasma membrane, the place where it interacts with its phosphatase CD45. The loss of CD222 leads to accumulation of Lck in its
inactive form, which results in diminished T cell activation and
effector functions.
Because Lck represents a very powerful kinase that is involved in
various T cell signaling pathways (41–44), its transport and regulation has been investigated extensively (45–49). One important
molecule implicated in the transport of Lck is the myelin and
lymphocyte 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 transport
of Lck to the plasma membrane with an apparent accumulation of
Lck within intracellular membranes and freely floating in the
cytoplasm but without any significant effect on lipid raft partitioning. It has been shown previously via single molecule microscopy and bleaching experiments that single Lck molecules
recolonize the cell membrane directly from the cytoplasm, rather
than via two-dimensional transversal diffusion (49), which may
explain 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 fractionation
experiments revealed an accumulation of Lck and CD222 in the
EEA1-positive fractions corresponding to endosomal membranes.
Furthermore, via mass spectrometry analysis we identified Rab11,
a marker of endosomal membranes critically involved in protein
trafficking, to interact with CD222. This strongly suggests that
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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, respectively. 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) Lckdeficient 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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CD222 DIRECTS SPATIAL DISTRIBUTION AND ACTIVITY OF Lck
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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, AF488coupled 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
CD222 coordinates the endosome-dependent transport of Lck that
was shown to be essential for T cell activation (48). Uncoordinated 119 protein has been shown to activate endosomal Rab11 for
the 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 early
vesicular platform submembrane of the IS. Although several
consecutive devices obviously coordinate Lck transport (52–54),
the contribution of CD222 seems to be very specific as human
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FIGURE 7. Silencing of CD222 results in decreased 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 separated 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 coimmunoprecipitated Lck by Western blot analysis. Statistical evaluation of the coimmunoprecipitated Lck
(Lck/CD45 ratios) was determined via ImageJ.
Mean ratios 6 SEM of two independent experiments 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 LckN10GFP 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 can perform its action on human Lck even in mouse
fibroblasts (i.e., irrespective of other T cell–specific molecules).
Moreover, the absence of MAL in fibroblasts (55) suggests that
CD222 operates upstream of MAL.
To our knowledge, the role of CD222 in T cell signal transduction has yet not been investigated in detail, but CD222 has been
shown to enhance TCR-induced signaling via CD26 internalization
(56). However, we found no evidence for the contribution of CD26
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
that is both postnatally expressed in thymic epithelial cells (62)
and upregulated in diseases (28), namely via internalization and
subsequent degradation in lysosomes (12, 63). 4) Furthermore,
CD222’s surface upregulation might play a role in T cell differentiation from the double negative to the double positive stage as
CD222 Ab was reported to block T cell ontogeny (64). Notably,
Lck knockout mice also display a similar reduction in doublepositive thymocytes in T lymphocyte development (65) as the
one caused by the CD222 Ab treatment. However, because of the
lethality of the CD222 knockout in mice (66–68), the function of
CD222 in T cell differentiation has never been shown in vivo. 5)
An involvement of CD222 in T cell migration can also not be
excluded as it has been shown that CD222 negatively regulates
cell adhesion via uPAR and integrins (22). 6) Alternatively, it
could be the result of a better accessibility of the CD222 mAb
caused by altered interactions that unmask the epitope.
Irrespective of the pathways that are amenable in the upregulation of cell surface CD222 upon T cell activation, we show in this
study a central function of the endosomal transporter CD222 in the
initiation of T cell signal transduction through controlling Lck
distribution: CD222 targets Lck to CD45 at the plasma membrane
and thereby maintains the equilibrium of Lck phosphorylation at
the steady state.
Acknowledgments
We 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, Václav Horejsı́ (Institute of Molecular Genetics, Academy of
Sciences of the Czech Republic, Prague, Czech Republic), Nicole Boucheron (Division of Immunobiology, Institute of Immunology, Center for
Pathophysiology, Infectiology and Immunology, Medical University of
Vienna, Austria), and Arthur Weiss (Department of Medicine and Department 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 Fässler (Department of Molecular Medicine,
Max Planck Institute of Biochemistry, Martinsried, Germany) for experimental and intellectual help with mass spectrometric analysis; S. Steward (Washington University School of Medicine, St. Louis, MO); G. Nolan (Baxter
Laboratory in Stem Cell Biology, Department of Microbiology and Immunology, 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.
Disclosures
The authors have no financial conflicts of interest.
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Supplemental Material
Supplemental Figure 1. Reconstitution of CD222 expression in knock-out T cells rescues
downregulated Ca2+ fluxes. (A) The genomic location of the targeted Exon 22 of CD222 on
chromosome 6 is denoted by the red line. Recognition sequences and genomic coordinates of
the zinc finger nuclease (ZFN) are shown below. (B) Total CD222 and Lck content in control
Jurkat T cells (CTR) and different CD222 knock-out cell populations (C20, C21, C25) were
analyzed via Western blot. GAPDH served as a loading control. (C) Ca2+ fluxes in control and
CD222 knock-out cells were measured upon stimulation with the anti-TCR mAb C305. (D)
CD222 surface expression of control cells (black), CD222 knock-out cells C20 (dashed, dark
1
gray) and C20 cells stably reconstituted with recombinant human CD222 (reconst., light
gray). The isotype control is illustrated as light gray filled histogram. (E) Genomic DNA
purified from control cells (CTR), CD222 knock-out cells (C20), and C20 cells reconstituted
with CD222 (reconst.) was used as a template for the PCR amplification with primer sets for
human CD222 and the intron-less short gene endosialin as an endogenous control. In contrast
to the control primers recognizing endosialin, CD222 primers bridge an exon-exon junction
and thus can only recognize the recombinant form of CD222. (F) Ca2+ flux was measured as
described in (C) in control cells (black), CD222 knock-out cells C20 (dark gray) and
reconstituted cells (light gray) Shown is one representative experiment out of two.
2
Supplemental Figure 2. (A) Serine 59 phosphorylation of Lck is decreased upon silencing of
CD222. Jurkat T cells were stimulated with the CD3 mAb MEM-92 for the indicated time
points and cell lysates were analyzed for total Lck expression by using mAb H-95 and
Western blotting. Phosphorylation of Lck serine 56 and Lck serine 59 were measured via the
LiCor multiplexing fluorescence immunoblotting system. The mean fluorescence intensity
was determined via ImageJ and the Lck serine 56/serine 59 ratios of four independent
experiments were evaluated and plotted via GraphPad Prism (mean ratio±SEM). Statistical
3
significant changes between shCTR (black) and shCD222/P1 cells (gray) were evaluated via
2-way ANOVA followed by the Bonferroni post test, * p<0.05, ** p<0.01, ns, not significant.
(B) CD222 interacts with Lck, but not with LckN10. Wild-type JCaM 1.6 T cells that lack
endogenous Lck and JCaM 1.6 T cells ectopically expressing either Lck-GFP (approx. 83
kDa) or truncated LckN10-GFP (approx. 28 kDa) were lysed using 1% detergent lauryl
maltoside. Then, CD222 was immunoprecipitated using mAb MEM-238. Lysates and
immunoprecipitates (IP) were analyzed by Western blotting. CD222 was detected via mAb
MEM-238, Lck and the truncated versions of Lck were detected by mAbs directed against the
GFP-tag (B-2). (C) Total Lck content in crude cell lysates of control- and CD222-silenced
Jurkat T cells. Whole cell lysates of shCTR and shCD222/P1 cells were probed for Lck and
LAT by Western blotting. Band intensities were calculated with ImageJ. Shown is one
representative experiment out of three.
4
Supplemental Figure 3. CD222 colocalizes with EEA1 and Lck. Jurkat T cells were let to
adhere on adhesion slides, fixed, permeabilized and stained for CD222 (red), EEA1 (blue) and
Lck (green). Triple staining of two representative Jurkat T cells are shown. Pictures were
captured with a CLSM. Scale bar = 5 µm.
5
6
Supplemental Figure 4. CD222 is influencing protein complex formation without affecting
lipid raft formation. (A) shCTR and shCD222/P1 Jurkat T cells were lysed using 1%
detergent Brij-58. Lipid raft proteins (light fractions, left) were separated from non-raft
proteins (heavy fractions, right) via sucrose density centrifugation and the indicated proteins
were detected via Western blotting using specific Abs. Data are representative of at least two
individual experiments. (B) Jurkat shCTR (upper panel) and shCD222/P1 (lower panel) T cell
lysates were prepared using the 1% detergent Triton X-100. The samples were loaded onto a
Superose 6 column and consecutive fractions were taken and analyzed by Western blotting.
mAbs to CD222 (MEM-238), CD45 (MEM-28), Lck (H-95), CD3ε (FL-207) and EEA1
(C45B10) were probed. kDa ranges are indicated at the right. Shown is one representative
experiment out of two.
7