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The Cytoplasmic Tail of the T Cell Receptor
CD3 ε Subunit Contains a
Phospholipid-Binding Motif that Regulates T
Cell Functions
Laura M. DeFord-Watts, Tara C. Tassin, Amy M. Becker,
Jennifer J. Medeiros, Joseph P. Albanesi, Paul E. Love,
Christoph Wülfing and Nicolai S. C. van Oers
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Material
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Copyright © 2009 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2009; 183:1055-1064; Prepublished online 19
June 2009;
doi: 10.4049/jimmunol.0900404
http://www.jimmunol.org/content/183/2/1055
The Journal of Immunology
The Cytoplasmic Tail of the T Cell Receptor CD3 ␧ Subunit
Contains a Phospholipid-Binding Motif that Regulates T Cell
Functions1
Laura M. DeFord-Watts,* Tara C. Tassin,† Amy M. Becker,* Jennifer J. Medeiros,*
Joseph P. Albanesi,† Paul E. Love,¶ Christoph Wülfing,*§ and Nicolai S. C. van Oers2*‡
T
cells recognize both self- and foreign-peptide molecules
through their Ag-specific TCRs. Interactions between the
␣␤ subunits of the TCR and peptide/MHC complexes are
relayed into intracellular signals through the associated CD3 invariant chains (CD3 ␥, ␦, ␧, and ␨). However, the mechanisms
whereby extracellular ligand-binding triggers intracellular signals
remain unknown. Several studies have shown that TCR engagement induces a conformational change within the cytoplasmic
tail of CD3 ␧, exposing a proline-rich sequence (PRS)3 that is
subsequently bound by the adaptor protein Nck (1, 2). Yet, the
PRS is not required for the conformational changes, as knock-in
mice with a deletion of the CD3 ␧ PRS retain TCR-mediated
signaling responses (3). Instead, these knock-in mice have increased cell surface TCR expression on immature thymocytes,
and increased TCR signaling processes following low avidity
TCR-ligand interactions (3).
In addition to initiating conformational changes in CD3 ␧, TCR
engagement also induces the phosphorylation of the ITAM motifs,
*Department of Immunology, †Department of Pharmacology, ‡Department of Microbiology, and §Department of Cell Biology, University of Texas Southwestern Medical
Center, Dallas, Texas 75390; and ¶Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes
of Health, Bethesda, Maryland 20892
Received for publication February 9, 2009. Accepted for publication May 12, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by grants from the National Institutes of Health T32
AI005284 (to A.B. and L.D.), AI42953, and AI71229 (to N.S.C.v.O.).
2
Address correspondence and reprint requests to Nicolai S. C. van Oers, Room
NA2.200, 6000 Harry Hines Boulevard, Department of Immunology, University of
Texas, Southwestern Medical Center, Dallas, TX 75390-9093. E-mail address:
[email protected]
3
Abbreviations used in this paper: PRS, proline-rich sequence; PI(3)P, phosphatidylinositol-3-phosphate; PI(4)P, phosphatidylinositol 4-monophosphate; PI(5)P, phosphatidylinositol 5-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; BRS,
basic-rich stretch; PC, phosphatidylcholine; DN, CD4⫺CD8⫺ double negative; DP,
CD4⫹CD8⫹ double positive; SP, CD4⫹ or CD8⫹ single positive; LAT, linker for
activation of T cell; MFI, mean fluorescent intensity.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900404
present within each of the CD3 subunits (4). Following their phosphorylation, the ITAMs are complexed by Syk/ZAP-70 family of
protein tyrosine kinases. This kinase family, in conjunction with
Src-family protein tyrosine kinases, phosphorylates and activates
multiple effector and adaptor proteins. Mutation of the CD3 ␧
ITAM reduces the efficiency of positive selection in low avidity
TCR transgenic lines (5). This is consistent with observations that
the number of TCR ITAMs available for phosphorylation directly
regulates both positive and negative selection processes in the thymus (6, 7). Moreover, a minimal number of functional ITAMs
within the TCR complex is needed to prevent autoimmunity, demonstrating that the regulation of ITAM phosphorylations are critical for maintaining effective T cell functions (8).
In addition to its PRS and ITAM, the CD3 ␧ subunit also contains a basic-rich stretch (BRS) of amino acids within the juxtamembrane portion of its cytoplasmic tail (Fig. 1A) (9). Although
all three subdomains can interact with proteins, the BRS has
uniquely been found to bind acidic phospholipids (1, 9 –12). Recent NMR studies have suggested that such lipid interactions may
embed the tyrosine residues of the CD3 ␧ ITAM in the inner leaflet
of the plasma membrane, thereby shielding them from phosphorylation (12). This notion is consistent with in vitro studies demonstrating that phospholipid binding by the CD3 chains can limit
the magnitude of their ITAM phosphorylation (12, 13). Based on
these findings, a new mechanism for regulating the accessibility of
the CD3 ␧ ITAM before TCR activation has been proposed (14).
However, it should be noted that these studies all used individual
CD3 ␧ molecules in the absence of the other TCR/CD3 subunits.
Consequently, the importance of the BRS in the context of an
intact TCR, as well as its role in T cell development and activation,
has yet to be established. Moreover, the complexity of the phospholipids that can be bound by CD3 ␧ remains unclear. Herein, we
report that the cluster of basic amino acids within the BRS selectively complexes a subset of charged phospholipids, including
PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and PI(4,5)P2. Elimination of
the BRS in transgenic mice resulted in a statistically significant
reduction in thymic cellularity. Furthermore, T cells from these
mice had reduced TCR expression and diminished TCR-induced
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The CD3 ␧ subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We
identified a third signaling sequence in CD3 ␧, termed the basic-rich stretch (BRS). Herein, we show that the positively charged
residues of the BRS enable this region of CD3 ␧ to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P,
PI(3,4,5)P3, and PI(4,5)P2. Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging
from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also
exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist
peptide-loaded APCs, and delayed CD3 ␧ localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 ␧ lipid-binding domain in T cell biology. The Journal of Immunology, 2009, 183: 1055–1064.
1056
CD3 ␧ COMPLEXES CHARGED PHOSPHOLIPIDS
tyrosine phosphorylation of several signaling intermediates. Relocation of the BRS to a membrane-distal section of the CD3 ␧ cytoplasmic tail (following the ITAM) blocked T cell development at the
CD4⫺CD8⫺ stage of thymopoiesis. Finally, the expression of a CD3
␧-GFP sensor in T cells revealed a role for the BRS in TCR relocalization to the immunological synapse. Taken together, these results
indicate that the cytoplasmic tail of the CD3 ␧ subunit contains a
phospholipid-binding motif that has diverse functions in T cells.
(UTSWMC). Transgenic founders were identified by Southern blotting and
PCR procedures. Transgenic founders (at least five per construct) were backcrossed onto a CD3 ␧⫺/⫺ background (16). HY and 5C.C7 TCR transgenic
mice have been described (17, 18). All mice were housed in the Specific
Pathogen Free Facility at UTSWMC. Mouse experimentation was performed
with Institutional Animal Care and Use Committee approved protocols.
Abs, cell lines, and peptides
The cDNA for murine CD3 ␧ was reverse transcribed using standard RTPCR cloning procedures (Invitrogen). Mutations, truncations, and relocation of the BRS of CD3 ␧ were generated by PCR-based mutagenesis
strategies, and confirmed by dsDNA sequencing. The various BRS-modified constructs were subcloned into pcDNA3.1, pEGFP-N1, or the VACD2 T cell specific transgenic cassette (15). GST-fusion proteins were
generated using pGEX-2TK vectors (GE Biosciences).
The 145-2C11 hybridoma and HEK293T cells were obtained from American Type Culture Collection (ATCC). The anti-CD28 mAb secreting hybridoma was generously provided by Dr. James Allison. Anti-CD3 ␧, -CD3
␨ (6B10.2), -ZAP-70 (1E7.2), and -CD28 mAbs were purified from culture
supernatants using standard affinity chromatography procedures. AntiGST, -ERK, and -TCR ␤ (H57-597) mAbs were from BD Biosciences.
Anti-phospho-linker for activation of T cells (LAT) (Y191), anti-phosphoERK (T202/Y204), and anti-phospho-ZAP (Y319) were from Cell Signaling Technology. Anti-CD3 ␥ (C-20), anti-CD3 ␦ (M-20), and anti-CD3 ␧
(M-20) polyclonal antisera were from Santa Cruz Biotechnology. The various fluorochrome-labeled Abs used for flow cytometry were described
elsewhere (19, 20). Peptides were synthesized by the UTSWMC Protein
Chemistry Technology Center.
Mice
Phospholipid-binding assays
For transgenic lines, the VA-CD2 plasmids containing the different CD3 ␧
constructs were injected into fertilized eggs from C57BL/6 mice by the Transgenic Core Facility at the University of Texas Southwestern Medical Center
PIP Strips (Echelon Biosciences) were blocked in 3% fatty acid-free BSA
prepared in TBST (25 mM Tris, 125 mM NaCl, 0.1% Tween 20 (pH 8.0)).
The membranes were then incubated with purified GST-tagged proteins (5
Materials and Methods
Constructs
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FIGURE 1. The CD3 ␧ subunit contains a basic-rich domain that complexes charged phospholipids. A, The amino acid sequence of the cytoplasmic tail
of CD3 ␧ and the individual subdomains, termed the BRS, PRS, and ITAM, are shown. Asterisks denote the positively charged lysine or arginine residues
in the BRS. In the lower section, PIP-Strips were probed with GST-fusion proteins consisting of the entire cytoplasmic tail of CD3 ␧, the individual
subdomains of CD3 ␧ (BRS, PRS, or ITAM), or with fusion proteins that contained mutations of the BRS (BRS-Substitute and BRS-Truncate). Binding
was detected by anti-GST western blotting. B, Sucrose-loaded liposomes, consisting of the indicated phospholipids, were incubated with GST (lanes 2–5)
or GST-BRS (lanes 7–10). Protein binding in the pellet fraction was assessed by anti-GST immunoblotting (upper panel). Lanes 1 and 6, GST or GST-BRS
were resolved as m.w. controls. Supernatants were also blotted to verify equal loading (lower panel). C, PC (⽧), or a 20:80 ratio of PC to PI(4)P (䡺) or
PI(4,5)P2 (‚), were coated onto 96-well plates and incubated with biotinylated peptides containing the BRS or the phosphorylated ITAM of CD3 ␨. Peptide
binding was assessed with streptavidin-HRP using ELISA-based assays. The assay was performed in triplicate.
The Journal of Immunology
␮g/ml) in 1% fatty acid-free BSA/TBST overnight at 4°C. The membranes
were subsequently washed and immunoblotted with anti-GST mAbs.
Lipids were purchased from Avanti Polar Lipids. Liposomes were prepared by sonicating the lipids in a HEPES buffer (50 mM HEPES (pH
7.0), 100 mM NaCl, 1 mM EDTA) containing 0.5 M sucrose. The
liposomes were then diluted in sucrose-free buffer. Three ␮M of GST
or GST-BRS was incubated with 100 ␮g of liposomes (20/80 ratio of
phospholipid to PC) for 10 min at 4°C. The liposomes were pelleted by
centrifugation, washed in HEPES buffer, resuspended in SDS-sample
buffer, and resolved by SDS-PAGE. For the solid-phase ELISA phospholipid binding assays, phospholipids were resuspended in methanol at
1 mg/ml (20/80 ratio of phospholipid to PC). One-hundred-microliter
aliquots were coated onto EIA/RIA 96-well plates (Costar) and air
dried. The wells were blocked with 10 mg/ml fatty acid-free BSA in
PBS. Biotinylated peptides were added for 2 h at room temperature.
After washing, peptide binding was detected with streptavidin-HRP colorimetric reactions (0.07 ␮g/ml).
Imaging
Cytometric analysis
Single cell suspensions were prepared from the thymus or lymph nodes of
the different mice. The cells were analyzed for the expression of various
cell surface and/or intracellular proteins by flow cytometry using FACSCalibur
cytometers as described previously (20). Analysis of cytometric data was performed using both FloJo (TreeStar) and Cell Quest Pro software (BD
Biosciences).
Transfections, stimulations, and immunoprecipitations
Transfections of HEK293T cells were performed using standard CaPO4/
DNA precipitation methods as described (22). Forty-eight hours post
transfection, cells were harvested and processed for Western blotting as
described below. In certain experiments, the cells were stimulated for
10 min at 37°C with pervanadate (11 ␮M sodium orthovanadate, 3.8%
H2O2).
Primary lymphocytes (107 cells) were stimulated with 10 ␮g/ml 1452C11 for the indicated times before lysis. The cells were then washed to
remove excess Ab, and lysed in a 1% Triton X-100 containing lysis buffer
(pH 7.6) (20 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1 mM NaF, and
protease inhibitors). For phospho-CD3 ␨ analysis, whole cell lysates were
incubated with 4 ␮g of anti-CD3 ␧ plus protein A-Sepharose beads at 4°C
for 1.5 h. Immunoprecipitates were washed and then processed for Western
blot analyses.
For stimulation of HY/BRS Tg/CD3 ␧⫺/⫺ lymphocytes, DC2.4 APCs
were left untreated or loaded with 100 nM SMCY peptide for 2 h at 37°C.
The APCs were then washed to remove unbound peptide. In brief, 107
lymphocytes were incubated with 3.5 ⫻ 106 APCs for 10 min at 37°C. The
cells were then pelleted and processed as above for Western blotting.
In vivo stimulations were performed by i.p. injecting 200 ␮g of antiCD3 ␧ in the indicated strains of mice. Seven days postinjection, the thymus was isolated, single cell suspensions were prepared, and aliquots were
analyzed by flow cytometry.
above. Immunoprecipitations of CD3 ␧ were performed as described
above. Detection of biotin-labeled proteins was performed using
streptavidin-HRP (Pierce).
Results
A BRS in the cytoplasmic tail of CD3 ␧ binds charged
phospholipids
The cytoplasmic tail of CD3 ␧ contains a cluster of positively
charged residues within its juxtamembrane position (Fig. 1A). This
BRS enables CD3 ␧ to associate with certain phospholipids, including phosphatidylglycerol (11, 12). However, a comprehensive
profiling of the phospholipids complexed by the BRS and the role
of these interactions in TCR-mediated functions remains unknown.
To identify which phospholipids are bound by CD3 ␧, membranes
embedded with an assortment of lipids (PIP strips) were probed
with GST-fusion proteins containing either the full-length cytoplasmic tail of CD3 ␧ (GST-␧) or the different subdomains of
CD3 ␧ (BRS, PRS, or ITAM). GST-␧ and GST-BRS bound to
several monophosphorylated lipids, including phosphatidylinositol 3-phosphate (PI(3)P), phosphatidylinositol 4-phosphate
(PI(4)P), phosphatidylinositol 5-phosphate (PI(5)P), and phosphatidic acid (PA) (Fig. 1A). Weaker binding occurred between the
BRS and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and phosphoinositol
3,4,5-trisphosphate (PI(3,4,5)P3). The PRS and the ITAM failed to
associate with any phospholipids, demonstrating that the BRS can
independently mediate lipid interactions.
To determine whether the phospholipid binding involved the
cluster of lysine and arginine residues in the BRS, PIP strips
were probed with GST-BRS fusion proteins that lacked five
positively charged residues by either amino acid substitution or
truncation (BRS-Substitute and BRS-Truncate) (Fig. 1A). Both
modifications eliminated phospholipid binding, demonstrating
the importance of the basic amino acids in these interactions
(Fig. 1A).
We next examined whether the BRS could complex phospholipids present in a lipid bilayer. Liposomes containing 100%
phosphatidylcholine (PC) or 80% PC and either 20% of PI,
PI(4)P, or phosphatidylserine were incubated with GST or
GST-BRS. Only the BRS-containing fusion protein precipitated
with charged phospholipids, including PI(4)P, PI(3)P, and
PI(4,5)P2 (Fig. 1B and supplemental Fig. 1).4 This binding was
specific because the BRS did not associate with liposomes containing PC or a mix of PC with phosphatidylserine. To exclude
the possibility that the GST portion of the fusion protein influenced lipid interactions, ELISA-based binding assays were performed using a biotinylated peptide consisting of just the BRS.
The BRS peptide associated with PI(4)P and PI(4,5)P2 in a
dose-dependent manner, while no binding was observed with
PC. A control peptide containing the phosphorylated ITAM of
CD3 ␨ did not bind any phospholipids (Fig. 1C). The affinity of
the BRS for PI(4)P and PI(4,5)P2 were similar in this assay,
with a calculated Kd in the range of 40 nM (data not shown).
Taken together, these experiments suggested that the BRS has
a high affinity for particular phospholipids.
The CD3 ␧ BRS regulates T cell development
Biotinylation assays
Primary thymocytes were harvested from the indicated mice and resuspended in PBS containing 0.5 mg of EZ-Link NHS-LC-Biotin (Pierce)
for every 1 ml of sample. The cells were incubated at 4°C for 20 min,
washed twice in PBS containing 5% FBS, washed twice with PBS
alone, and lysed in the 1% Triton X-100 lysis buffer described
The ability of the CD3 ␧ BRS to complex charged phospholipids
suggested an important role for this domain in T cells. To examine
4
The online version of this article contains supplemental material.
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CD3 ␧ sensors were generated by linking full-length CD3 ␧, CD3 ␧ BRSSubstitute, -Truncate, or -Displace to the N terminus of GFP. The sensors
were retrovirally transduced into primary, in vitro-primed 5C.C7 TCR
transgenic T cells. Sensor-transduced T cells were FACS-sorted for defined
sensor expression levels and live, sensor-expressing T cells were imaged in
three dimensions over time during restimulation with peptide-loaded
APCs. The frequencies of occurrence of specific spatiotemporal patterns of
CD3 ␧ accumulation were determined from the imaging data. The 5C.C7
T cell culture, retroviral transduction, FACS sorting, image acquisition,
and analysis procedures, including pattern definitions, are described in detail elsewhere (21).
1057
1058
CD3 ␧ COMPLEXES CHARGED PHOSPHOLIPIDS
the role of the BRS within the context of an intact TCR, we generated CD3 ␧ transgenic lines containing three distinct modifications of the BRS (Fig. 2A). Two mutants (BRS-Substitute and
-Truncate) were designed to eliminate phospholipid binding (Fig.
1A). In the third mutant, the BRS was relocated to the membranedistal portion of the cytoplasmic tail of CD3 ␧, following the
ITAM (BRS-Displace). Transgenic mice expressing a wild-type
version of CD3 ␧ were also generated to control for potential effects caused by forced expression of CD3 ␧ from a transgene
(BRS-Wild Type). All the transgenic lines were back-crossed with
CD3 ␧ knockout mice (which retain CD3 ␥ and ␦) to eliminate
endogenous protein expression, and several lines were selected for
similar levels of transgene-driven CD3 ␧ expression (supplemental
Fig. 2) (5, 16). Thymopoiesis in the BRS-Wild Type transgenic
lines was relatively normal (Fig. 2B). The BRS-Substitute and
-Truncate transgenic mice also exhibited normal percentages of
CD4⫹CD8⫹ double positive (DP) cells, with a slight increase in
the percentage of single positive (SP) thymocytes (Fig. 2B and
supplemental Fig. 3). In contrast, thymocytes from the BRS-Dis-
place mice were arrested at the CD4⫺CD8⫺ double negative (DN)
stage (Fig. 2B). Analysis of Thy1.2⫹ DN thymocytes in the BRSSubstitute and BRS-Truncate lines did not reveal any statistically
significant differences in the percentages of cells progressing to the
DN4 (CD25⫺CD44⫺) stage relative to BRS-Wild Type controls
(supplemental Fig. 4). Conversely, the BRS-Displace transgenic
lines had an almost complete block at the DN3 (CD25⫹CD44⫺)
stage of thymopoiesis (four independent founders) (supplemental
Fig. 4).
All the BRS-modified transgenic lines had a statistically significant reduction in thymic cellularity, with the numbers in the
BRS-Displace lines resembling CD3 ␧-deficient mice (Fig. 2C).
Staining these thymocytes for markers of apoptosis revealed a
1.5- to 2-fold increase in the number of apoptotic cells, indicating that the reduced thymic cellularity in these mice may be
partially due to an increase in cell death (Fig. 2D). On occasion,
a small percentage (20 –50%) of the BRS-Displace thymocytes
progressed to the DP stage (supplemental Fig. 5; example ⫽
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FIGURE 2. The CD3 ␧ BRS regulates thymic cellularity. A, Schematic representation of the CD3 ␧ subunit with the various BRS modifications
(EXTRA, extracellular domain; TM, transmembrane region). B, Thymocytes were isolated from the indicated strains of mice (4 to 6 wk of age), stained
with fluorochrome-labeled Abs directed against CD4 and CD8, and analyzed by flow cytometry. The percentage of cells in each quadrant is indicated.
Results are representative of four independent assays using at least two distinct transgenic founder lines. C, Thymic cellularity was enumerated for mice
from the indicated transgenic lines. F, Cell count from an individual mouse. Bars represent the average thymic cellularity for each genotype. CD3 ␧
BRS-Wild Type (n ⫽ 19); CD3 ␧ BRS-Substitute (n ⫽ 26); CD3 ␧ BRS-Truncate (n ⫽ 12); CD3 ␧ BRS-Displace (n ⫽ 22); CD3 ␧⫺/⫺ (n ⫽ 8). D,
Thymocytes from the indicated mice were stained with fluorochrome-labeled Abs against apoptotic (Annexin 5) or necrotic (7-aminoactinomycin D) cells
(n ⫽ six mice per group).
The Journal of Immunology
1059
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FIGURE 3. The BRS regulates TCR expression on developing and peripheral T cells. A and B, Thymocytes (A) or lymphocytes (B) from the
indicated mice were stained with fluorochrome-labeled anti-CD3 ␧ or hamster Ig control mAbs in combination with different fluorescently labeled
anti-CD4, -CD8, and -B200 mAbs. Histograms showing CD3 ␧ overlaid with control Ig staining are shown. The associated graph demonstrates the
average MFI of CD3 ␧ on the indicated T cell subpopulations, which were identified by electronic gating (n ⫽ minimum of eight mice/group). C,
Lymphocytes from the indicated mice were isolated, aliquots were stained with fluorescently labeled anti-CD4 and -CD8 mAbs, and the samples
were analyzed by flow cytometry. The average percentage of CD4⫹ or CD8⫹ lymphocytes for each strain is graphed (n ⫽ minimum of 12
mice/group). (ⴱ, p ⱕ 0.05; ⴱⴱ, p ⱕ 0.01; ⴱⴱⴱ, p ⱕ 0.001).
mouse 1). However, this did not restore normal thymic cellularity (Fig. 2C).
To more carefully define a role for the BRS in thymocyte selection, the BRS transgenic lines were mated with HY TCR transgenic mice. This transgenic line expresses an ␣␤ TCR that recognizes the male specific SMCY peptide (17, 20). Both positive and
negative selection processes were intact and relatively equivalent
in the various HY/BRS-Wild Type, -Substitute and -Truncate
female and male mice, respectively (supplemental Fig. 6, A and B).
Interestingly, the thymic cellularity of female HY/BRS-Substitute
and -Truncate mice was normal when compared with the HY/
BRS-Wild Type controls (supplemental Fig. 6C). Taken together,
these findings suggest that the BRS regulates an early step in T cell
development as thymocytes expand in number.
1060
CD3 ␧ COMPLEXES CHARGED PHOSPHOLIPIDS
The BRS regulates cell surface TCR expression
The PRS of CD3 ␧ has been shown to regulate TCR expression,
but only on immature DP thymocytes (3). When comparing thymocytes from the BRS-Wild Type, -Substitute, and -Truncate
lines, we noted a significant reduction in the mean fluorescence
intensity (MFI) of CD3 ␧ not only on developing DP thymocytes,
but also on more mature CD4⫹CD8⫺ and CD4⫺CD8⫹ SP thymocytes (Fig. 3A). The BRS-Displace lines exhibited the most
severe reduction in CD3 ␧ MFIs, primarily due to the limited thymopoiesis in these mice.
Examination of peripheral T cells revealed that all the BRS-mutant
lines maintained their statistically significant decrease in the MFI of
CD3 ␧ after they exited the thymus (Fig. 3B). This was also true for
the MFI of surface TCR ␤ (data not shown). Importantly, this reduction in TCR surface expression was not dependent on protein levels,
as these mice possessed similar CD3 ␧ protein expression as BRSWild Type controls (supplemental Fig. 2). Consistent with the requirement for the BRS in maintaining normal TCR expression levels,
peripheral T cells from the HY/BRS-Substitute and HY/BRS-Truncate double transgenic female mice also exhibited a significant reduction in surface TCR expression (supplemental Fig. 6B).
A comparison of peripheral T cell subpopulations revealed that
the BRS-Substitute and -Truncate lines had a slight increase and
decrease in the percentage of CD8⫹ and CD4⫹ T cell subsets,
respectively (Fig. 3C). Conversely, the BRS-Displace transgenic
lines possessed very few CD4⫹ or CD8⫹ T lymphocytes in their
peripheral lymphoid organs (average ⬍5%) (Fig. 3C and supplemental Fig. 5). Taken together, our results suggest that the presence and/or location of the BRS within the cytoplasmic tail of CD3
␧ is necessary for maintaining normal TCR expression levels.
The CD3 ␧ BRS is required for efficient recruitment to the
immunological synapse
Upon T cell/APC contact, the TCR rapidly localizes to a structure
at the center of the T cell/APC interface known as the central
supramolecular activation cluster (cSMAC) (Fig. 4) (23). In
primed 5C.C7 TCR transgenic T cells interacting with peptideloaded APCs, the cSMAC is enriched in proximal signaling intermediates, such as ZAP-70, LAT, Itk, and PLC ␥, as well as in more
distal signaling intermediates, including PKC ⍜, Rac, and Rho
(21). Thus, in these T cell/APC couples, the cSMAC is a site of
active signaling. To explore whether mutagenesis of the BRS affected CD3 ␧ relocation to the immunological synapse, we performed live cell imaging studies using GFP-fusion proteins containing the BRS-Wild Type, -Substitute, -Truncate, or -Displace
versions of CD3 ␧. These constructs were retrovirally transfected
into primed 5C.C7 TCR transgenic T cells. The cells were then
subjected to immunofluorescent imaging with APCs bearing their
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FIGURE 4. The spatiotemporal accumulation of CD3 ␧ at the immunological synapse is partially regulated by the BRS. Primed 5C.C7 TCR transgenic
T cells were retrovirally transduced with constructs encoding the indicated GFP-labeled CD3 ␧-chains (BRS-Wild Type, -Substitute, -Truncate, or
-Displace). The cells were then imaged with APCs (CH27 B cell lymphoma) that had been loaded with 10 ␮M MCC agonist peptide. Time ⫽ 0 s is defined
as the time point at which the T cell and APC formed a tight, wide interface. The graphs display the percentage of T cells with the indicated patterns of
CD3 ␧ accumulation (n ⫽ 47–53 cell couples analyzed per construct). Significant differences in CD3 ␧ accumulation were as follows: CD3 ␧ BRS-Wild
Type vs -Substitute: Difference in Any accumulation of CD3 ␧ at the interface at 0, 20, 80, and 120 – 420 s time points (p ⱕ 0.05/0.005); CD3 ␧ BRS-Wild
Type vs -Substitute: Difference in Central accumulation at 0, 40, 80, 180 –300, and 420 s time points (p ⱕ 0.05/0.005); CD3 ␧ BRS-Wild Type vs -Truncate:
Difference in Any and Central accumulation at 0 and 20 s time points (p ⱕ 0.05); CD3 ␧ BRS-Wild Type vs -Displace: Difference in Any accumulation
at all time points (p ⱕ 0.05/0.005); CD3 ␧ BRS-Wild Type vs -Displace: Difference in Central accumulation at 0 – 40, 60, 80, 100 –180, 300, and 420 s
time points (p ⱕ 0.05/0.005). Representative movies are shown as supplemental movies 1– 4. The patterns are named using criteria described elsewhere
(Ref. 21, 33).
The Journal of Immunology
1061
FIGURE 5. Eliminating the positively charged residues of the BRS augments CD3 ␧ phosphorylation
when expressed as a monomer but not when present in
the TCR complex. A, HEK293T cells were cotransfected with Lck and either CD3 ␨ or the various CD3 ␧
BRS constructs. CD3 ␨ (lane 1) or CD3 ␧ (lanes 2–5)
were immunoprecipitated and Western immunoblotted
with anti-PO4-Y (upper panel) or anti-CD3 ␧ Abs
(lower panel). B, Experiment was performed as in A
except before lysis the cells were treated with the phosphatase inhibitor pervanadate. C, Primary thymocytes
from the indicated strains of mice were treated with (⫹)
or without (⫺) anti-CD3 ␧. The cells were then lysed,
the TCR was immunoprecipitated, and Western blotting
was performed using anti-PO4-Y (upper panel). The
blots were subsequently stripped and reprobed using antiCD3 ␧ (lower panel).
TCR-mediated phosphoprotein induction following peptide-MHC
stimulation is regulated by the BRS
Recent findings have shown that the cytoplasmic tail of CD3 ␧ is
latterly sequestered along the inner leaflet of the plasma membrane
via phospholipid interactions (12). Based on these findings, a signaling model has been proposed wherein TCR engagement induces a conformational change through CD3 ␧, disrupting the CD3
␧-phospholipid interactions at the plasma membrane so that the
ITAM of CD3 ␧ becomes tyrosine phosphorylated (14). In the
context of this model, eliminating the positively charged residues
of the CD3 ␧ BRS should enhance the tyrosine phosphorylation of
the ITAM because it would be constitutively exposed to Src kinases. To test this hypothesis, we coexpressed the full-length BRSwild type, -substitute, -truncate, or displace versions of CD3 ␧ into
HEK293T cells with the tyrosine kinase, Lck (Fig. 5A) (24). CD3
␨ was coexpressed with Lck as a positive control (lane 1). Phosphotyrosine immunoblotting revealed that CD3 ␨ and all the CD3
␧ constructs lacking the basic amino acids (CD3 ␧ BRS-Substitute
and BRS-Truncate) were easily detected as tyrosine phosphorylated proteins (lanes 1, 3, and 4). Yet, the two CD3 ␧ constructs
that retained the BRS (CD3 ␧ BRS-Wild type and BRS-Displace)
were not tyrosine phosphorylated (lanes 2 and 5). When the transfected cells were treated with pervanadate to inhibit intracellular
phosphatases, all of the BRS constructs were tyrosine phosphorylated (Fig. 5B). Taken together, these findings fully support the
model that electrostatic interactions between basic amino acids in
the CD3 ␧ BRS and acidic phospholipids limit the ITAM phosphorylation, at least until the TCR is engaged (14).
To examine the validity of this model using cells expressing an
intact TCR, lymphocytes from the BRS-Wild Type, -Substitute, and
-Truncate lines were analyzed for phosphoprotein content both before
and after TCR cross-linking. Because the BRS-Displace mice generally lacked T cells, these mice were not included in the study. There
was no consistent evidence of hyperphosphorylation of the CD3 ␧
ITAM either before or after TCR engagement in the BRS-mutant
transgenic lines (Fig. 5C). These findings are very different compared
with the phosphorylation state of monomeric CD3 ␧ expressed in
HEK cells. Furthermore, similar levels and kinetics of tyrosine phosphorylation of other signaling molecules was also noted when comparing the various BRS-modified lines (CD3 ␨, ZAP-70, and ERK)
(supplemental Fig. 7). Consequently, no statistical differences were
observed in the up-regulation of activation markers (CD69 and
CD25), production of several cytokines, and T cell proliferation following anti-CD3 treatment (data not shown).
Because subtle differences in TCR signaling pathways in T cells
containing the BRS-modifications could have been masked by the
use of anti-TCR cross-linking mAbs, we next examined BRS-mutant TCR signaling responses to peptide-loaded APCs. Experimentally, peripheral T cells from the HY/BRS double-transgenic females were incubated for 10 min with dendritic cells that were
either untreated or had been loaded with the SMCY peptide. Analysis of phospho-␨ and phospho-LAT revealed a marked decrease
in the phosphorylation of these proteins in HY/BRS-Substitute and
-Truncate cells relative to the HY/BRS-Wild Type controls (Fig.
6). These findings suggest that the BRS may contribute to early
TCR signaling events. Analysis of later time points (30 and 60
min) revealed variations in the level of phosphoprotein induction,
with the HY/BRS-Substitute and -Truncate lines exhibiting both
increases and decreases in the levels of phospho-CD3 ␨ in comparison to wild-type controls in different assays (data not shown).
Yet, the 10-min time point was consistently reduced in the BRSmodified lines compared with the wild-type controls in these additional experiments. The inconsistent differences noted at the later
time points might reflect alterations in TCR internalization, degradation, and/or recycling that might be regulated by the BRS. The
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cognate ligand to determine the spatiotemporal pattern of the CD3
␧-chains. Upon T cell/APC couple formation (t ⫽ 0 s), the BRSWild Type CD3 ␧-chain exhibited a rapid translocation to the T
cell/APC interface in up to 80% of cells analyzed (Fig. 4; Any
Accumulation at Interface). The majority of CD3 ␧-GFP localized
to the center of the interface and persisted there for up to 7 min
(Fig. 4 and supplemental Movie 1). All of the BRS mutants
showed distinct defects in the recruitment of CD3 ␧ to the T cell/
APC interface, particularly to its center (Fig. 4 and supplemental
Movies 2– 4). The CD3 ␧ BRS-Substitute and -Truncate proteins
exhibited a 20 – 40 s delay in central accumulation, as well as a
significant reduction in the overall percentage of T cells exhibiting
this accumulation pattern (29 and 35%, respectively) (Fig. 4). The
BRS-Displace CD3 ␧-chain exhibited reduced interface recruitment at all time points. These results established that the BRS
contributes to the recruitment of CD3 ␧ to the T cell/APC interface, particularly to its center where numerous downstream signaling intermediates are located.
1062
CD3 ␧ COMPLEXES CHARGED PHOSPHOLIPIDS
FIGURE 6. Initial TCR-mediated tyrosine phosphorylation events are reduced by modifications to the BRS. Primary lymphocytes from the indicated
strains of mice were stimulated for 10
min with unloaded (⫺) or peptide-loaded
(⫹) APCs. The cells were then lysed,
and whole cell lysates (WCLs) (upper
panel) or CD3 ␧ immunoprecipitates
(middle panel) were processed for immunoblotting. Anti-phospho-LAT blots
were subsequently stripped and reprobed
using CD3 ␧ antisera. Graphs represent
the average integrated density value
(IDV) for the indicated phosphoproteins
obtained in three independent assays.
(ⴱ, p ⱕ 0.05; ⴱⴱⴱ, p ⱕ 0.001).
Effective pre-TCR signaling requires a BRS in proximity to the
transmembrane domain
The inability of the BRS-Displace transgenic lines to support early T
cell development suggested that these mutants had a severe signaling
defect. This could be accounted for by the delayed and extremely poor
recruitment of the BRS-Displace CD3 ␧ protein to the cSMAC. Alternatively, inefficient pre-TCR assembly in the BRS-Displace lines
could block the progression of thymocytes from the DN3 to DN4
stage. To examine these possibilities, CD3 ␧ pairing with the other
TCR/CD3 subunits was analyzed in BRS-Displace thymocytes. Immunoprecipitation experiments demonstrated that the BRS-Displace
CD3 ␧-chain paired with both CD3 ␥ and CD3 ␦ in whole cell lysates,
as well as with TCR ␤ that was isolated from the cell surface (Fig. 7,
A and B). To further verify that the BRS-Displace CD3 ␧-chain was
present at the cell surface, thymocytes from RAG⫺/⫺, BRS-Displace,
and CD3 ␧ knockout mice were biotinylated and lysed. Both CD3 ␧
and CD3 ␥ were biotinylated when extracted from RAG-deficient and
FIGURE 7. The membrane-proximal location of the BRS is essential for thymocyte development. A, CD3 ␧ was immunoprecipitated from C57BL/6
(lane 1), BRS-Displace (lane 2), or CD3 ␧-deficient (lane 3) thymocytes. Precipitates were first immunoblotted using anti-CD3 ␦ (upper panel) or CD3
␥ (middle panel) polyclonal antisera, then reprobed using anti-CD3 ␧ (lower panel and data not shown). B, Thymocytes were stained using biotinylated
anti-TCR ␤. The cells were then lysed, and TCR ␤ was immunoprecipitated using immobilized streptavidin beads. Whole cell lysates (lower panel) or TCR
␤ precipitates (upper panel) were resolved and probed with CD3 ␧ antisera. C, Cell surface proteins on thymocytes were biotinylated. CD3 ␧ was
immunoprecipitated, and the precipitates were immunoblotted with streptavidin-HRP to detect surface-biotinylated proteins. D, RAG-deficient or BRSDisplace mice were injected i.p. with PBS or anti-CD3 ␧ mAbs. Seven days postinjection, the thymocytes were enumerated and analyzed for the expression
of CD4 and CD8 by flow cytometry. RAG⫺/⫺: n ⫽ 2 mice per group. BRS-Displace: n ⫽ 3 mice per group.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
defects in early signaling events did not translate into altered T cell
responses, such as proliferation (data not shown).
The Journal of Immunology
BRS-Displace mice, but not from CD3 ␧⫺/⫺ mice (Fig. 7C). These
results indicate that the BRS-Displace CD3 ␧-chain can form a cell
surface expressed pre-TCR.
Finally, we analyzed whether the surface expressed CD3
␧-chain within the BRS-Displace lines could transduce intracellular signals. Experimentally, BRS-Displace mice were given intraperitoneal injections of anti-CD3 ␧. Using such procedures, thymocytes from the RAG-deficient mice underwent a 10- to 50-fold
expansion and up-regulated both CD4 and CD8 (Fig. 7D) (25).
The BRS-Displace mice exhibited no increase in thymic cellularity
and no differentiation of developing thymocytes to the DP stage
(Fig. 7D). The findings confirmed that the BRS-Displace CD3
␧-chain is signaling deficient.
Discussion
Golgi network) (27). Such disruptions could lead to a failure of the
TCR to recycle properly between the plasma membrane and endosomes, or in an inability of the TCR to translocate to the cell
surface following ER assembly. This may reflect a role for the
BRS in binding select phospholipids to properly localize within a
T cell. Consistent with this hypothesis, BRS-modified CD3
␧-chains showed a diminished capacity to properly translocate to
the immunological synapse following APC interactions. This phenotype is consistent with the fact that TCR-engagement causes
transient increases in the levels of PI(4)P and PI(4,5)P2 at the
plasma membrane (29, 30). Such increases could contribute to redistribution and stabilization of the TCR at the cSMAC.
In terms of proximal signaling events, BRS-mutant lymphocytes
only exhibited consistent defects in their signaling capacity when
stimulated for short time periods with peptide-loaded APCs (10
min). There was much more variability at later time point. Such
findings may reflect discrepancies in the ability of the TCR to
undergo proper recycling in the BRS-modified lines following ligand binding. Thus, while the TCR signals were weaker at early
time points, inefficient TCR turnover, recycling, or degradation at
later time points could have contributed to prolonged or equivalent
signaling after 30 – 60 min of stimulation. This could arise from the
absence of BRS interactions with selected phospholipids located
on endosomal compartments, such as PI(3)P.
Relocating the BRS to a membrane-distal position had the most
pronounced effect on T cell development. The simplest interpretation for this effect is that displacement of the BRS prevented the
regulated binding of CD3 ␧ to phospholipids at diverse intracellular locations, thereby significantly interfering with pre-TCR signaling. Alternatively, the topology of the CD3 ␧ subunit could
have been affected by displacement of the BRS. Positively charged
residues located downstream of a hydrophobic transmembrane
segment regulate the proper orientation of transmembrane proteins
as they pass through the endoplasmic reticulum (31). Displacing
the basic residues of a transmembrane protein up to 30 amino acids
away from its hydrophobic segment can invert the NH2-domain of
a protein (31, 32). Although the BRS-Displace CD3 ␧-chain associated with CD3 ␥, CD3 ␦, and TCR ␤, the topology of CD3
␧-chain could have been inverted, preventing effective pre-TCR
signaling. Furthermore, the BRS-Displace construct resulted in the
repositioning of the PRS and ITAM, which may have also contributed to a more severe phenotype.
In summary, we have demonstrated that the polybasic cluster of
amino acids in the cytoplasmic tail of CD3 ␧ complexes acidic
phospholipids. Interestingly, there are a number of additional
ITAM (CD3 ␨ and Fc␧RI␥) and non-ITAM containing transmembrane proteins (CD43, CD44, and ICAM-1/2) that also contain
polybasic motifs in their cytoplasmic tails. These findings suggest
that the function and/or assembly of many transmembrane receptors could be dynamically regulated by changes in protein/phospholipid interactions.
Acknowledgments
We thank John Ritter and his staff from the UTSWMC Transgenic and
Knock-out Facility. In addition, we appreciate scientific discussions and
insights from Drs. Helen Yin, Hans Deisenhofer, Sandra Hayes, and Melanie Cobb. We thank Angela Mobley for assistance with flow cytometry.
Disclosures
The authors have no financial conflict of interest.
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