From the Institute for Biochemistry, University of
Lausanne, Epalinges 1066, Switzerland, the ¶ Ludwig Institute for
Cancer Research, Lausanne Branch, University of Lausanne, Epalinges
1066, Switzerland, and the
Laboratory of Transplantation
Immunology and Nephrology, University Hospital Basel, Basel 4031, Switzerland
Received for publication, August 8, 2002, and in revised form, September 4, 2002
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ABSTRACT |
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T cells expressing T cell receptor (TCR)
complexes that lack CD3 The differentiation of CD4 CD8 double-positive
(DP)1 thymocytes into CD8
single-positive (SP) T cells requires appropriate signals from the TCR
and the coreceptor CD8 (1, 2). DP thymocytes and CD8 SP peripheral T
cells express TCR A conserved motif in the TCR On the other hand, the To investigate whether and how CD3 Antibodies--
The following monoclonal antibodies were
purchased from BD Pharmingen (San Diego, CA): anti-CD8 Cell Cultures and T1 TCR Transgenic Mice--
The
CD8 FACS and FRET Analyses--
T cell hybridomas and T1 thymocytes
were washed and resuspended at 2 × 106 cells/ml in
serum-free Opti-MEM medium (Invitrogen, Merebeke, Belgium) containing
1% BSA and 0.02% NaN3. For FACS analysis aliquots of
25-µl cell suspension were incubated in a 96-well plates with fluorescence-labeled antibodies (5 µg/ml) for 20 min at 4 °C. After two washes in the same medium, fluorescence associated with live
cells was measured on a FACSCalibur (BD Biosciences, Erembodegen, Belgium). For FRET analysis, 50-µl aliquots of cells
(2 × 106 cells/ml) were incubated in the same medium
in 96-well plates with 5 µg/ml Cy5-labeled anti-CD8 Soluble Kd-peptide
Complexes--
Kd-125"IASA"-YIPSAEK(ABA)I
complexes (about 2000 Ci/mmol) were prepared as described previously
(31). Non-radioactive Kd-PbCS(ABA) complexes were obtained
by refolding of Kd heavy chain and human Intracellular Calcium Mobilization--
P815 mastocytoma cells
(1 × 106 cells/ml) were pulsed with graded amounts of
IASA-YIPSAEK(ABA)I for 2 h at 37 °C and UV-irradiated at TCR Photoaffinity Labeling--
T1 TCR hybridomas or T1 TCR
thymocytes (7 × 106 cells/ml) were incubated in a
12-well plate with
Kd-125"IASA"-YIPSAEK(ABA)I (0.5-1.5 × 107 cpm/3 × 106 cells). After 1 h
of incubation at 26 °C and UV irradiation at 312 ± 40 nm for
30 s with 90 watts, the cells were washed twice and lysed for Isolation of Lipid Rafts--
T1 TCR hybridomas or T1 TCR
thymocytes (5 × 107 cells) photoaffinity-labeled with
Kd-125"IASA"-YIPSAEK(ABA)I were lysed in 1 ml of MN buffer (25 mM MES, pH 6.5, 150 mM
NaCl) containing 0.5% Brij96 (Sigma) and protein inhibitors (Roche
Molecular Biochemicals) for 30 min on ice. The lysates were homogenized
with a Dounce homogenizer (10 strokes) and fractionated on sucrose
density gradients as described (29, 38). The sucrose gradients were
fractionated from the top in ten fractions of 500 µl. Aliquots of the
fractions were resolved on SDS-PAGE (10%, reducing) and either
analyzed by phosphorimaging or Western blotted using antibodies
specific for Thy-1 or CD45.
Confocal Microscopy--
T1 TCR hybridomas or T1 thymocytes were
washed with DMEM and incubated with 10 mM
methyl- Co-immunoprecipitation and Western Blotting--
T1 TCR
hybridomas or T1 thymocytes (1.5 × 107 cells) were
lysed on ice for 2 h in Tris (20 mM, pH 8.0)
containing 0.3% Triton X-100 and protease inhibitors (Roche Molecular
Biochemicals). The lysates were spun at 10,000 × g for
10 min, and the supernatants were immunoprecipitated using monoclonal
antibodies specific for CD8 Modeling of CD3 CD8
Using the same method we next examined thymocytes from T1 TCR
transgenic mice. These mainly DP cells exhibited strong and stable
intracellular calcium mobilization upon incubation with IASA-YIPSAEK(ABA)I-pulsed P815 cells (Fig. 1C). Maximal
response was observed at 10 CD8 Increases MHC-peptide Binding on Cells Expressing Wild Type but
Not
Remarkably, on hybridomas expressing T1 TCR Wild Type but Not
To investigate which chain of CD8 was important for coupling of CD8
with TCR·CD3, we performed FRET experiments on hybridomas expressing
CD8
Strong FRET was observed on T1 thymocytes following staining with
PE-labeled Kd-PbCS(ABA) multimers and Cy5-labeled
anti-CD8 Wild Type but Not
In T1 thymocytes an appreciable fraction of
photoaffinity-labeled TCR was found in the light fractions (Fig.
4C). This fraction was greatly diminished when TCR
photoaffinity labeling was performed in the presence of the
CD8 Cross-linking of TCR·CD8 Adducts Results in the Formation of
Large TCR·CD8 Aggregates--
We next investigated what consequences
TCR·CD3 cross-linking has on TCR aggregation. As assessed by confocal
microscopy, incubation of T cell hybridomas expressing CD8 CD3
To obtain further information on the association of CD3
In CD8 A key finding of the present study is that CD3 Moreover, because CD8 How does CD3 Our co-immunoprecipitation experiments indicate that the association of
CD8, either due to deletion of the CD3
gene, or by replacement of the connecting peptide of the
TCR
chain, exhibit severely impaired positive selection and
TCR-mediated activation of CD8 single-positive T cells. Because the
same defects have been observed in mice expressing no CD8
or
tailless CD8
, we examined whether CD3
serves to couple TCR·CD3
with CD8. To this end we used T cell hybridomas and transgenic
mice expressing the T1 TCR, which recognizes a photoreactive
derivative of the PbCS 252-260 peptide in the context of
H-2Kd. We report that, in thymocytes and hybridomas
expressing the T1 TCR·CD3 complex, CD8
associates with the TCR.
This association was not observed on T1 hybridomas expressing only
CD8
or a CD3
variant of the T1 TCR. CD3
was
selectively co-immunoprecipitated with anti-CD8 antibodies, indicating
an avid association of CD8 with CD3
. Because CD8
is a raft
constituent, due to this association a fraction of TCR·CD3 is
raft-associated. Cross-linking of these TCR-CD8 adducts results in
extensive TCR aggregate formation and intracellular calcium
mobilization. Thus, CD3
couples TCR·CD3 with raft-associated CD8,
which is required for effective activation and positive selection of
CD8+ T cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
that are associated with three
signal-transducing units, namely CD3
and CD3
heterodimers and a disulfide-linked
chain homodimer (3-5). The CD3
and
subunits contain in their cytoplasmic tail a single immunoreceptor tyrosine-based activation motif, whereas the tail of the
chain harbors three immunoreceptor tyrosine-based activation motifs. For
surface expression of TCR
, their association with CD3
,
,
and
but not with CD3
is required (6-9). Accordingly, knockout of CD3
,
, and
chain arrests T cell development at early
stages (6-11). By contrast, in CD3
knockout mice T cell development proceeds to the DP stage, but positive selection of CD8 (and CD4) SP T
cells is severely compromised (9, 12). During TCR
assembly the
TCR
chain first associates with CD3
and the TCR
chain with
CD3
, and the resulting trimers then associate and the TCR
disulfide bond is formed (13). Although CD3
is physically associated
with the pre-TCR complex, it is not required for pre-TCR signaling,
which is essential for the transition of double-negative (DN) to DP
thymocytes (10, 14, 15).
chain-connecting peptide domain,
which connects the transmembrane and the Ig domains, referred to as
CPM, plays a crucial role in positive selection of CD8 and CD4 SP T
cells (16-18). The
CPM consists of seven highly conserved amino
acids (FETDXNLN) and is present in TCR
but not
in TCR
(16). In mice expressing TCR in which the
CPM is
replaced by the corresponding sequence of the TCR
chain, positive
selection of SP T cells is greatly impaired, whereas
negative selection is normal (16-18). These variant TCRs, referred to
as
IV/
III (16) TCR, exhibit impaired association with CD3
,
-chain phosphorylation, defective activation of p59Fyn
and extracellular signal-regulated kinase, impaired
phosphorylation and recruitment of ZAP-70, p56lck
(Lck), and LAT to lipid rafts (17-19). Very similar findings
were obtained in CD3
knockout mice (12, 15), arguing that the defects observed for the
CPM variant TCR are mainly accounted for by
their impaired association with CD3
.
chain of CD8 plays a key role for positive
selection of CD8 SP T cells. Although CD8 can be readily expressed as
the CD8
homodimer, the number of CD8 SP T cells in CD8
knockout mice is greatly reduced (20, 21). In a milder form, positive
selection is also compromised in mice overexpressing tailless CD8
,
and activation is impaired in CD8+ T cells expressing
tailless CD8
(22, 23). Given the similarity in impaired activation
and positive selection of CD8 SP T cells in mice lacking CD8
or
CD3
, we examined here whether this is accounted for by the same
mechanism, i.e. whether CD3
couples TCR with CD8
.
Several studies indicated that CD8 (and CD4) associates with
TCR·CD3. For example, the proximity between CD8 (and CD4) and
the TCR has been demonstrated on cells by using fluorescence resonance
energy transfer (FRET) (24, 25). In other studies, CD8 was
co-immunoprecipitated with anti-TCR·CD3 antibodies (26-29). According to one of these studies the tail of CD8
is involved in the
association of CD8 with TCR·CD3 (29), and another suggested that
CD3
can be selectively co-immunoprecipitated with CD8 (and CD4)
(26).
establishes a functional link
between the TCR·CD3 and CD8, we used thymocytes and T cell hybridomas
expressing the T1 TCR. This TCR recognizes the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 (SYIPSAEKI)
containing photoreactive 4-azidobenzoic acid on Lys-259
(PbCS(ABA)) in the context of Kd (30). Photoactivation of
the ABA group results in cross-linking of the T1 TCR with
Kd-PbCS(ABA), which permits direct assessment of TCR-ligand
interactions by TCR photoaffinity labeling (30-32). Using TCR
photoaffinity labeling, FRET, co-immunoprecipitation, and confocal
microscopy, we find that CD8
, but not CD8
, associates with
the TCR via CD3
and that this is required for efficient T cell activation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
53.6.72 (FITC
or Cy5), anti-CD8
H35-17 (PE), anti-TCR
H57
(allophycocyanide), anti-CD3
17A2 (PE), anti-CD4 GK 1.5 (FITC
or Cy5), and anti-Thy-1.2 III/5 (Cy5). The anti-CD8
KT112 mAb (32)
was purified and labeled with Cy5. For Western blotting
antibodies specific for CD3
(M-20, Santa Cruz Biotechnology, Santa
Cruz, CA), CD3
(17), CD8
tail (33),
chain (H146) (American
Tissue Culture Collection, Manassas, CA), Thy-1 (rabbit IgG; gift from
Dr. C. Bron, University of Lausanne, Switzerland), and CD45
(Transduction Laboratories, San Diego, CA) were used. FITC-labeled
rabbit anti-rat antibody was from BD Biosciences. For blocking of CD8
binding to Kd Fab' fragments of anti-Kd mAb
SF1-1.1.1 (SF') or anti-CD8
mAb H35 were used (32).
, CD8
+, and CD8
+ T
cell hybridomas were obtained and cultured as described previously
(29). Hybridomas expressing the
CPM variant TCR (
IV/
III) were
obtained by transfection of CD8
or CD8
+
58 T cell hybridomas as described previously (34) with wild type T1
TCR
and
chains or T1 TCRIV
chain, in which the sequence 225-270 of the TCR
chain was replaced with the corresponding sequence of the TCR
chain and T1 TCRIII
chain in which the
sequence 270-300 of the TCR
chain was replaced with the
corresponding sequence of the TCR
chain (16). Transfected hybridomas
were FACS-sorted for high TCR expression and cultured in DMEM
containing 2% fetal calf serum, 100 units/ml penicillin, 100 µg/ml
streptomycin, 50 µM
-mercaptoethanol, 10 µg/ml G418
(Calbiochem, San Diego, CA), 4 µg/ml puromycin (Calbiochem), and 0.5 mg/ml hygromycin (Calbiochem). To generate T1 TCR transgenic mice
(H-2d/d) cDNA encoding the T1 TCR
and
chains
were separately cloned into the transgenic expression vector pHSE3'
using standard methods. XhoI fragments encoding the T1
TCR
and
chains were co-injected into BDF2 zygotes. T1 TCR
transgenic BDF2 mice were backcrossed to Balb/c, RAG knockout
mice. Backcrossing of the T1 TCR transgene to Balb/c, RAG knockout mice
was repeated for over six generations. Surface expressions of TCR and
CD8 of the cells under study were determined by FACS by using
anti-CD8
mAb 53.6.72 (FITC), anti-CD8
mAb H35 (PE), and anti-TCR
mAb H57 (allophycocyanide), respectively. The mean fluorescence
intensities (in parentheses) were as follows: for the previously
described CD8
T cell hybridomas (3, 5, 134), for
CD8aa+ hybridomas (492, 8, 82), and for CD8ab+
hybridomas (453, 427, 72). These cells were used only in FRET analysis.
For the hybridomas obtained from transfection of 58 cells, the
following mean fluorescence intensities were measured as follows: for
CD8
+ T1 TCR+ hybridomas (342, 68, 22),
for CD8
T1 TCR+ hybridomas (4, 5, 28), for
CD8
+ T1 TCR
IV/
III+ cells (305, 61, 19), for CD8
T1 TCR
IV/
III+ hybridomas
(4, 5, 25), and for T1 thymocytes (494, 1283, 128).
53.6.72, anti-CD8
KT112, anti-CD4 GK 1.5, or anti-Thy-1 antibody; PE-labeled
anti-CD3
17A2 mAb; or 50 nM of PE-coupled
Kd-PbCS(ABA) multimers. After 45 min of incubation at
4 °C, the cells were washed and fixed with paraformaldehyde (3% w/v
in PBS) for 10 min at room temperature, and cell-associated
fluorescence was assessed on a FACSCalibur at 580 nm upon excitation at
488 nm (E1), at 670 nm after excitation at 630 nm (E2), and at 670 nm after excitation at 488 nm
(E3). The transfer of fluorescence was calculated as FRET
units as follows: FRET unit = [E3both
E3none]
[(E3Cy5
E3none) × (E2both/E2Cy5)]
[(E3PE
E3none) × (E1both/E1PE)].
The different fluorescence values (E) were measured on
unlabeled cells (Enone), or cells labeled with
PE (EPE), Cy5 (ECy5), or
Cy5 and PE (Eboth).
2
microglobulin, produced in Escherichia coli using the
dilution method (35, 36). The refolded monomers were biotinylated,
purified, and reacted with PE-labeled extravidin (Sigma) as described
previously (35, 36).
350
nm for 90 s to cross-link the peptide to Kd. T1
TCR hybridomas or T1 thymocytes (1 × 106 cells/ml)
were incubated with 5 µM Indo-1 (Sigma, Buchs,
Switzerland) at 37 °C for 45 min, washed in DMEM, and incubated with
P815 cells for 1 min at 37 °C at an E/T ratio
of 1/3. Calcium dependent Indo-1 fluorescence was measured on a
FACStarTM as described (37).
60
min on ice in 1 ml of PBS containing 50 mM
n-octylglucoside and a mixture of protease inhibitors (Roche
Molecular Biochemicals, Rotkreuz, Switzerland). TCR was
immunoprecipitated using Sepharose-conjugated mAb H57. The
immunoprecipitates were resolved on SDS-PAGE (10% reducing), and
radioactivity was quantified by phosphorimaging analysis using a Fuji
BAS1000 (29-32).
-D-cyclodextrin at room temperature, washed again
with medium, and incubated in PBS containing 1% BSA for 30 min at room
temperature or for 40 min at 4 °C with PE-labeled Kd-PbCS(ABA) multimers (50 nM). Following two
washes with PBS, cells were analyzed using an LSM510 Zeiss confocal
microscope. Median sections of cells were recorded. Alternatively, TCR
hybridomas were incubated for 20 min at room temperature with
anti-CD8
mAb KT112 and anti-TCR mAb H57 mAb in PBS containing 1%
BSA, washed, and incubated with FITC-labeled anti-rat IgG antibody, and
the distribution of CD8
was examined.
(KT112), CD8
(53.6.72), or TCR (H57).
The immunoprecipitates were washed twice with Tris buffer, pH 8.0, containing n-octylglucoside (50 mM) (Sigma) or
as indicated with Tris buffer, pH 8.0, containing Triton X-100
(0.15%), supplemented with EDTA (5 mM),
ethylmaleimide (5 mM) (Sigma), or NaCl (0.5 M). Immunoprecipitates were resolved on SDS-PAGE (15%,
reducing), transferred onto a nitrocellulose membrane, and Western
blotted using anti-CD3
, anti-CD3
, or anti-CD8
antibodies. For
detection the enhance chemiluminescence (ECL) (Amersham Biosciences)
was used as described (29).
--
An homology model of the CD3
complex was built based on the CD3
3D structure and CD3
sequence alignment (5), using the MODELLER program (39). The
conformations of the connecting loops of the immunoglobulin fold were
refined using an ab initio method based on simulated
annealing (40).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and Wild Type TCR
CPM Are Required for Efficient
Intracellular Calcium Mobilization--
To examine the role of CD8 and
the TCR
CPM for T cell activation we first assessed intracellular
calcium mobilization in T cell hybridomas expressing CD8 and wild type
T1 TCR or T1 TCR in which the
CPM was replaced with the
corresponding sequence of TCR
(T1 TCR
IV/
III). As shown in
Fig. 1A, hybridomas expressing the wt TCR and CD8
exhibited strong calcium mobilization upon incubation with P815 cells pulsed with 10
6 to
10
5 M IASA-YIPSAEK(ABA)I. This calcium flux
was stable over the assayed period of 15 min. By contrast, no
significant calcium mobilization was observed in the presence of the
CD8
blocking antibody H35. Similarly, on hybridomas expressing the
T1 TCR
IV/
III, only scant calcium mobilization was observed,
which was reduced to background levels in the presence of mAb H35 (Fig.
1B).
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Fig. 1.
Blocking of CD8 or replacement of the
CPM inhibits antigen recognition.
Indo-1-labeled CD8+ T cell hybridomas expressing wild type
(wt) (A), the
CPM variant T1 TCR
IV/
III
(B) T1 TCR, or thymocytes from T1 TCR transgenic mice
(C) were incubated in the presence (circles) or
in the absence (squares) of anti-CD8
mAb H35 with P815
cells sensitized with graded concentrations of IASA-YIPSAEK(ABA)I, and
calcium-dependent Indo-1 fluorescence was measured by FACS.
Mean values and S.D. were calculated from three experiments.
7 M
IASA-YIPSAEK(ABA)I. The stronger calcium responses observed on T1
thymocytes, as compared with CD8+ T1 T cell hybridomas, is
explained, at least in part, by the higher surface expression of TCR
and CD8 (see "Experimental Procedures"). In the presence of mAb
H35, no marked calcium mobilization was observed, indicating that CD8
was required for this response (Fig. 1C). Taken together
these results indicate that, for efficient calcium mobilization,
CD8
and CD3
+ TCR are required, which is in
accordance with previously studies showing that CD8 and the
CPM are
crucial for efficient TCR signaling and positive selection of CD8 SP T
cells (16-22).
CPM Variant TCR--
TCR photoaffinity labeling with soluble
monomeric Kd-125"IASA"-YIPSAEK(ABA)I
complexes allows direct assessment of TCR-ligand binding and its
dependence on CD8 (30-32). Using this technique we compared TCR-ligand
binding on T cell hybridomas expressing the wild type T1 TCR or the T1
TCR
IV/
III. As shown in Fig. 2A, TCR photoaffinity labeling
was reduced by over 6-fold in the presence of Fab' fragments of
anti-Kd
3 mAb SF1-1.1.1, which block CD8 binding to
Kd (32). A slightly larger inhibition was observed on T1
thymocytes and on cloned T1 CTL (Fig. 2B and Ref. 32). The
same reductions were observed upon blocking of CD8 with anti-CD8
mAb
H35 (data not shown).
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Fig. 2.
CD8 strengthens TCR-ligand binding on cells
expressing wild type but not CPM variant
TCR. T cell hybridomas expressing CD8
and wild type
(wt) or
CPM variant T1 TCR
IV/
III (A) or
T1 thymocytes (B) were incubated at 26 °C for 30 min in
the absence (
) or presence of Fab' fragments of
anti-Kd
3 mAb SF1-1.1.1 (SF') (20 µg/ml) or
anti-Kd
1 mAb 20-8-4S (10 µg/ml) with
Kd-125"IASA"-YIPSAEK(ABA)I. After UV
irradiation, the cells were lysed and TCR was immunoprecipitated and
analyzed by SDS-PAGE (10%, reducing) and phosphorimaging. The TCR
labeling observed in the absence of SF' was defined as 100%. Mean
values and S.D. were calculated from two experiments.
IV/
III,
Kd-PbCS(ABA) binding was over 4-fold lower than on cells
expressing wild type T1 TCR and blocking of CD8 binding to
Kd caused only a small reduction. The nonspecific labeling,
as seen in the presence of anti-Kd
1 mAb 20-8-4S, which
blocks binding of Kd to TCR (32), was in the range of 3%
on the hybridomas and below 1% on thymocytes. Upon blocking of CD8
binding to Kd, TCR photoaffinity labeling was slightly
lower on hybridomas expressing T1 TCR
IV/
III, as compared with
hybridomas expressing the wild type T1 TCR (Fig. 2A).
Because the TCR expression is slightly lower on the former as compared
with the latter cells (see "Experimental Procedures"), it seems
that both TCR bind Kd-PbCS(ABA) with very comparable
efficiency. This is consistent with the fact that both TCR have the
same variable and constant domains and argues that the poor TCR
photoaffinity labeling observed on T1 TCR
IV/
III is accounted for
by inefficient participation of CD8 in TCR ligand binding. Because
CD8-mediated increase in TCR-ligand binding relies on association of
CD8 with TCR·CD3 (29), this argues that the T1 TCR
IV/
III
associates poorly with CD8.
CPM Variant TCR Associates with CD8 on Intact
Cells--
To validate this conclusion, we assessed the proximity of
TCR·CD3 and CD8 by FRET. To this end we stained hybridomas expressing CD8 and wild type T1 TCR in the cold with Cy5-labeled anti-CD8
mAb
KT112 and PE-labeled Kd-PbCS(ABA) multimers and measured
the FRET value from PE to Cy5 by FACS. As shown in Fig.
3A, on CD8+
hybridomas expressing the wild type T1 TCR, FRET was 2.2 units but only
0.4 unit on hybridomas expressing the T1 TCR
IV/
III. The
nonspecific signal, as recorded on the corresponding CD8
hybridomas, was about 0.03 unit. When using PE-labeled anti-CD3
mAb
17A2, slightly less efficient FRET was observed (1.5 units, Fig.
3B). Remarkably, this FRET value was enhanced very little when soluble Kd-PbCS(ABA) monomers were present in the
incubation at saturating concentration, indicating that the proximity
of CD8 and TCR·CD3 on these hybridomas was not induced by
MHC-peptide, i.e. it was constitutive.
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Fig. 3.
Wild type, but not
CPM variant TCR, associates with CD8.
A, T cell hybridomas expressing wild type (wt) or
CPM variant T1 TCR (
IV/
III) and CD8
(+) or not (
) were
stained with PE-labeled Kd-PbCS(ABA) multimer and
Cy5-labeled anti-CD8
mAb KT112. B, alternatively,
staining was performed with PE-labeled anti-CD3
mAb 17A2 and
Cy5-labeled mAb KT112 in the absence (
) or presence (+) of soluble
Kd-PbCS(ABA) monomer (1 µM). C, T
cell hybridomas expressing CD8
, CD8
, or no CD8 were stained
with PE-labeled Kd-PbCS(ABA) multimer and Cy5-labeled
anti-CD8
KT112 or anti-CD8
mAb 53.6.72. D, thymocytes
from T1 TCR transgenic mice were stained in the absence (
) or
presence (+) of anti-CD8
mAb H35 (15 µg/ml) with PE-labeled
Kd-PbCS(ABA) multimers and Cy5-labeled mAb KT11, 53.6.72, anti-CD4 mAb GK1.5 or anti-Thy-1 mAb III/5. Cells were analyzed by FACS
using excitation at 488 and 630 nm. FRET units were calculated from the
fluorescence emissions at 580 and 670 nm (see "Experimental
Procedures"). Mean values and S.D. were calculated from two to four
experiments, each performed in triplicate.
or CD8
. Upon staining of CD8
+
hybridomas with PE-labeled Kd-PbCS(ABA) multimers and
Cy5-labeled anti-CD8
KT112, a 4.3-fold stronger FRET was observed
than on CD8
+ hybridomas stained with Cy5-labeled
anti-CD8
mAb 53.6.72 (Fig. 3C). The nonspecific signal in
this experiment, as assessed on CD8
hybridomas, was 0.04 unit.
mAb KT112 (37 units, Fig. 3D). About one-third
lower FRET (25 units) was recorded when using Cy5-labeled anti-CD8
mAb 53.6.72. Because thymocytes express only CD8
, but
CD8-transfected T cell hybridomas always express high levels of
CD8
(see "Experimental Procedures" and Refs. 29 and 33), the
over 4-fold reduced FRET observed on CD8
+ T cell
hybridomas indicates that CD8
couples with TCR·CD3 more extensively than does CD8
(Fig. 3C). This is
consistent with the finding that CD8
is co-immunoprecipitated
with the TCR more efficiently as compared with CD8
(29). In the
presence of anti-CD8
mAb H35, this FRET was reduced to 6.2 units,
indicating that mAb H35 impedes the association of TCR and CD8.
Moreover, faint FRET (5 units) was observed when using Cy5-labeled
anti-CD4 mAb GK1.5 as acceptor, which was about 2-fold above
background, as recorded when using Cy5-labeled anti-Thy-1 antibody
(Fig. 3D). This is consistent with the observation that CD4
also associates with TCR·CD3 (25, 26). This FRET was not reduced in
the presence of mAb H35, indicating that this antibody does not impair
the PE Kd-PbCS(ABA) multimer staining. The differences in
FRET values observed in the different experiments in Fig. 3 are
accounted for in part by variations in TCR and CD8 expression of the
different cells (see "Experimental Procedures"). Together these
results indicate that CD8 and TCR·CD3 are in close proximity in T1
thymocytes and in T1 T cell hybridomas, given they express
CD3
+ TCR and CD8
.
CPM Mutant T1 TCR Docks to Raft-associated
CD8--
Because wild type T1 TCR, but not variant T1 TCR
IV/
III, associates with CD8
(Figs. 2 and 3) and CD8
is a raft constituent (33, 36), we examined the raft association of
these two TCR. To this end we TCR photoaffinity-labeled
CD8
+ T cell hybridomas expressing wild type or T1 TCR
IV/
III, lysed the cells in Brij96, and fractionated the
detergent-soluble and -insoluble components on sucrose gradients. A
significantly larger fraction of
Kd-125"IASA"-YIPSAEK(ABA)I-labeled T1 TCR
was found in the detergent-insoluble light fractions 2-4 on hybridomas
expressing the wild type T1 TCR as compared with hybridomas
expressing the T1 TCR
IV/
III (Fig.
4, A and B). To
verify that our fractionation procedure was correct, we assessed the
distribution of CD45 and Thy-1, which are known markers for the
detergent-soluble and detergent-insoluble raft fractions, respectively
(41, 42). Indeed CD45 was found exclusively in the detergent-soluble
dense gradient fractions (fractions 6-10), whereas
glycosylphosphatidylinositol-linked Thy-1 was detected only in the
detergent-insoluble light fractions (fractions 2-4) (Fig. 4,
E and F).
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Fig. 4.
Wild type, but not
CPM variant TCR, docks to raft-associated CD8.
T cell hybridomas expressing CD8
and wild type (A,
E, and F) or
CPM variant T1 TCR
IV/
III
(B) or thymocytes from T1 TCR transgenic mice (C
and D) were incubated in the absence (A-C,
E, and F) or presence (D) of
anti-CD8
mAb H35 (15 µg/ml) at 26 °C with
Kd-125IASA-YIPSAEK(ABA)I complexes. After UV
irradiation the washed cells were lysed in Brij96 (0.5%) and the
lysates were fractionated on sucrose gradients. Ten fractions were
collected from the top, aliquots were immunoprecipitated with anti-TCR
mAb H57, and the precipitates were resolved on SDS-PAGE (10%,
reducing) and evaluated by phosphorimaging (A-D). Aliquots
of the fractions from A were analyzed by SDS-PAGE (10%,
reducing) and Western blotted with antibodies specific for CD45
(E) or Thy-1 (F). Detergent-insoluble components
were mainly found in fractions 2-4, and detergent-soluble components
were in fractions 6-10. Each experiment was repeated at least
once.
-blocking mAb H35 (Fig. 4D). These findings indicate
that a fraction of TCR is raft-associated due to association of
TCR·CD3 with raft-resident CD8. This is consistent with the findings
that
CPM variant TCR (18) or TCR from CD3
knockout mice (12)
exhibit impaired raft-association and argues that CD3
couples
TCR·CD3 with CD8
and hence with lipid rafts.
and
wild type T1 TCR with PE-labeled Kd-PbCS(ABA) multimers at
room temperature resulted in extensive patch formation and
internalization (Fig. 5A). The
aggregate formation, but not the internalization, also took place when
the incubation was performed in the cold (Fig. 5B),
suggesting that it does not require cell activation. Strong TCR·CD8
aggregate formation was also seen on T1 thymocytes upon incubation with
Kd-PbCS(ABA) multimers and on CD8
+, T1
TCR+ hybridomas after incubation with anti-TCR, and
anti-CD8
antibodies (Fig. 5, A and C). By
contrast, on hybridomas expressing T1 TCR
IV/
III, aggregate
formation and internalization were greatly reduced. The same diffuse,
mainly surface staining, was also observed when CD8
+,
T1 TCR+ hybridomas, or T1 thymocytes were pretreated with
methyl-
-cyclodextrin, which destabilizes lipid rafts (Fig.
5A). Taken collectively, these findings demonstrate that
co-cross-linking of TCR·CD3 and CD8 results in formation of large
TCR·CD8 aggregates and that for this to occur CD8
, lipid rafts,
and CD3
+ TCR are required.
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Fig. 5.
CD3 and
CD8
are required for
cross-linking-induced formation of large TCR·CD8 aggregates.
A, T cell hybridomas expressing CD8
and wild type
(wt), T1 TCR (
IV/
III), or thymocytes from T1 TCR
transgenic mice, pretreated or not with
methyl-
-D-cyclodextrin (MCD), were incubated
at room temperature for 20 min with PE-labeled Kd-PbCS(ABA)
multimers and examined using confocal microscopy. B,
alternatively, the staining of the hybridomas was performed for 40 min
at 4 °C. C, the hybridomas were incubated for 20 min at
room temperature with anti-CD8
mAb KT112, anti-TCR mAb H57, and
FITC-labeled rabbit anti-rat IgG antibody and analyzed by confocal
microscopy. Representative images are shown from over ten cells
analyzed per condition and from at least two different
experiments.
Associates with CD8--
We next examined whether CD3
co-immunoprecipitates with CD8. To this end, we lysed T1 thymocytes in
Triton X-100 and analyzed their lysate as well as TCR and CD8
immunoprecipitates by SDS-PAGE and Western blotting. In the CD8
immunoprecipitate, CD3
, but neither CD3
nor
chains, was
highly enriched; especially when compared with the TCR
immunoprecipitates, the preferential co-precipitation of CD3
was
striking (Fig. 6A and data not
shown).
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Fig. 6.
CD3 associates with
CD8 on CD8ab+, T1 TCR+
cells. A, thymocytes from
T1 TCR transgenic mice were lysed in cold Tris, pH 8, containing 0.3%
Triton X-100, and the total lysate or immunoprecipitates with
anti-CD8
mAb KT112 or anti-TCR mAb H57 was resolved on SDS-PAGE
(15%, reducing) and Western blotted with antibodies specific for
CD3
, CD3
, and CD8
. B, the immunoprecipitates
with anti-CD8
mAb 53.6.72 were either (i) washed twice with
in Tris, pH 8, containing 0.15% Triton X-100 (lane 1) or 50 mM octylglucoside (lane 2) or (ii) washed twice
with Tris, pH 8, containing 0.15% Triton X-100 and either 5 mM EDTA (lane 3), 5 mM
ethylmaleinimide (lane 4), or 0.5 M NaCl
(lane 5) and then Western blotted using antibodies specific
for CD3
, CD3
, and CD8
, respectively. The Western blots with
anti-CD3
and -CD3
from three experiments were quantified by
densitometry and expressed in percentages, with 100% being the value
recorded for lane 1. C, hybridomas expressing T1
TCR wild type and CD8
were analyzed as described for
A.
with CD3
and CD8, we washed the CD8 immunoprecipitates twice with different
buffers and assessed the amount of co-precipitated CD3
and CD3
by
Western blotting. The association of CD3
with CD8 was substantially
reduced (to 36%) upon washing with ethylmaleinimide, which alkylates
free cysteines (Fig. 6B). A smaller reduction (to 55%) was
observed upon washing with EDTA-containing buffer, which chelates
divalent cations. About 40% reduction was noted upon washing with
n-octylglucoside, which disrupts association of
transmembrane proteins (41). Strikingly, washing with 0.5 M
NaCl had no marked effect, suggesting that the association of CD3
with CD8 is not ionic in nature. By contrast, washing with this buffer
removed most of the CD3
from the immunoprecipitates. The other
buffers affected the co-precipitation of CD3
in the same way as the
co-precipitation of CD3
.
+, T1 TCR+ T cell
hybridomas, similar as with T1 thymocytes (Fig. 6A),
anti-CD8
antibody efficiently co-immunoprecipitated CD3
but
little CD3
(Fig. 6C). In contrast to the T1 thymocytes, there was less CD8 co-immunoprecipitated with the TCR, especially when
compared with the amount of CD8 in the lysate. However, although thymocytes express only CD8
, the hybridomas express CD8
heterodimers and CD8
homodimers (29, 33), which accounts for the
large amount of CD8
detected in the lysate. Taken together these
results indicate that association of CD3
with CD8 is remarkably
strong and resists washing with n-octylglucoside and
high salt, which combined effectively disrupt the association of CD3
with CD3
. On the other hand, the association of CD8 with CD3
(and
CD3
) is sensitive to alkylation or chelating of divalent cations,
suggesting that it involves free cysteines and chelate complexes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mediates a
functional link between TCR·CD3 and CD8 and that this is crucial for
efficient TCR triggering and activation of CD8+ T cells.
CD3
knockout mice or mice expressing an
CPM variant TCR, which
lacks the
chain in their CD3 complex, exhibit strongly impaired
positive selection and TCR-mediated activation of CD8 SP T cells (Fig.
1 and Refs. 12, 15, 18). The same findings were obtained on CD8
knockout mice (20, 21) and in a milder form, on mice expressing
tailless CD8
(22, 23, 29). The present study shows that these
signaling defects are explained by the same molecular mechanism, namely
that CD3
couples the TCR with the coreceptor CD8. Several
observations support this conclusion. Using TCR photoaffinity labeling
with soluble monomeric Kd-PbCS(ABA) complexes, we find that
on cells expressing the
CPM variant T1 TCR, CD8 fails to markedly
increase TCR-ligand binding (Fig. 2). It is known that CD8 increases
the avidity of TCR-ligand interactions by binding to TCR-associated MHC
complexes and that this coordinate binding requires association of TCR
and CD8 (29, 32, 43). For example, CD8
or CD8
lacking the
tail of CD8
(CD8
'), poorly associate with TCR·CD3 and
therefore inefficiently increase TCR-ligand binding (29). Because T1
TCR
IV/
III lack the
chain of their CD3 complexes (17), our
TCR photoaffinity labeling experiments argue that CD3
mediates
association of TCR·CD3 with CD8. Consistent with this are our FRET
data showing that in T cell hybridomas wild type T1 TCR is in close
proximity to CD8 whereas T1 TCR
IV/
III is not (Fig. 3).
is palmitoylated and partitions in lipid
rafts, a fraction of T1 TCR·CD3 is raft-associated, due to its
association with CD8 (Fig. 4 and Ref. 29). Several observations indicate that raft association of TCR·CD3 is mediated by CD3
and
CD8
. First, TCR lacking CD3
, due either to disruption of the
CD3
gene (12) or to
CPM replacement, exhibit no or little raft
association (Fig. 4 and Ref. 18). Second, no significant TCR raft
association was observed on T cell hybridomas lacking CD8
or on
thymocytes upon blocking of CD8 (Fig. 4 and Ref. 29). Third, CD3
was
selectively co-immunoprecipitated with CD8 (and CD4) (Fig. 6 and Ref.
26). Because CD3
is known to associate with CD3
(4, 5), this
implies that the association of CD3
with the coreceptor is stronger
than with CD3
, i.e. is remarkably avid.
associate with the coreceptor ? Because CD8
poorly associates with TCR·CD3 (Fig. 3 and Ref. 29), CD8
is important for this interaction. It has been shown that the tail of
CD8
is involved in coupling CD8 with TCR·CD3 (29), but it is
unclear what other portions of CD8 are involved in this interaction and
in what way. Moreover, CD3
can also be selectively
immunoprecipitated with CD4 (26), which is surprising, given the
striking structural differences between CD4 and CD8. Because CD4 and
CD8 have in common that they associate with Lck and LAT (44), the
question arises whether these may be involved in coupling the TCR with
the coreceptor. This possibility, however, seems unlikely because mice
expressing tailless CD3
exhibit nearly normal positive selective of
SP T cells (12), arguing that the tail of CD3
is not required for its interaction with the coreceptor. Because Lck is intracellular and
LAT has only nine extracellular residues, it seem inconceivable that
they could interact with tailless CD3
. Moreover, D3 and D4 of CD4
have been shown to be critical for coupling the TCR with CD4 (25).
with CD3
is sensitive to EDTA and even more to alkylation
of cysteines by ethylmaleinimide (Fig. 6B). It is interesting to note that CD3
,
, and
and CD8
and
all
have two free cysteines in their extracellular membrane proximal
regions (Fig. 7A). It has been
shown that those of the CD3 chains are engaged in chelate complexes
with Zn2+, which contribute to their dimer formation (4,
5). Although for CD8 two of these cysteines form an interchain
disulfide bond, the other two may participate in such chelate complexes
and thus strengthen its association with CD3
(Fig. 7A).
The transmembrane portions of CD8 and CD3
may also contribute to
their association, as suggested by the destabilizing effect of
n-octylglucoside (Fig. 6B), which disrupts the
association of spanning proteins (41). Further studies are clearly
needed to elucidate how CD3
associates with the coreceptor, in
particular what role its Ig domain plays. Based on the 3D structure of
CD3
(5), straightforward homology modeling of the Ig domain of
CD3
is possible. Such modeling suggests that the outer surfaces of
CD3
and CD3
(opposite the interface with CD3
), which are most
likely to interact with the coreceptor, are strikingly different. The
outer surface of CD3
is flatter and much less polar than the
one for CD3
, which is consistent with the finding that the
association of CD3
with CD8 is only slightly ionic in nature (Figs.
6B and 7B).
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Fig. 7.
Concepts of CD3
association with CD8. A, transmembrane
and adjacent extracellular and cytoplasmic sequences of CD8
, CD8
,
CD3
, CD3
, CD3
, CD3
, and CD4. The sequences and definition
of the transmembrane regions were taken from Swiss-Prot (available at
www.expasy.ch/sprot). The spanning regions are shown in gray
boxes, and the numbers indicate their N- and C-terminal
residues. Cysteines are shown in boldface, basic residues in
black boxes, and acidic ones are in ovals.
B, the electrostatic potential of the outer surface
(opposite to CD3
) of the CD3
structure (left) and the
CD3
model (right) shown from top to
bottom. Acidic domains are shown in red, and
basic ones are in blue. The images were produced using the
software GRASP (49).
What are the implications of CD3-mediated TCR·CD8 coupling for TCR
signaling? TCR·CD3, lacking CD3
, exhibits defective signaling such
as impaired activation of kinases like Lck, p59fyn, ZAP-70, and Erk and reduced phosphorylation of LAT (12, 18, 19). This
has been attributed to their poor association with lipid rafts, which
by concentrating kinases and their substrates and by excluding
phosphatases are privileged sites for the induction of TCR signaling
(12, 18, 36, 41, 42). Our results demonstrate that raft association of
TCR is mediated by binding of CD3
to raft-resident CD8 or, more
precisely, with CD8/Lck, because CD8 associates with Lck in rafts (29,
33). Although they are small in resting cells rafts, they
dramatically increase in size upon TCR triggering, which greatly
increases the separation of kinases and phosphatases and hence the
efficiency of TCR signaling (41, 42). Our confocal studies show that
co-cross-linking of TCR and CD8 by soluble MHC-peptide multimers or
anti-TCR·CD3 and CD8 antibodies results in the formation of large
aggregates of TCR and CD8 (Fig. 5). This was also observed under
conditions where cell activation is prevented, e.g. in the
cold or in the presence of Src kinase inhibitors (Fig. 5 and Ref.
45).2 This aggregate
formation was also inhibited by methyl-
-cyclodextrin or similar
agents, which disrupt lipid rafts, and was not observed on
cells expressing the CD3
CPM variant TCR (Fig. 5).
Taken together these findings argue that cross-linking of
raft-associated TCR·CD3 adducts with CD8/Lck results in strong TCR
aggregation and the formation of large rafts and that this is essential
for efficient TCR signal induction. Consistent with this is the
observation that disruption of rafts greatly diminishes multimer
staining of CD8+ CTL, because TCR and CD8 aggregate
formation increases the binding of soluble MHC-peptide complexes (45).
Furthermore, in cells lacking CD8
or upon blocking of CD8, raft
association of TCR is diminished and TCR cross-linking-mediated TCR
aggregation is strongly impaired, just as it is in cells expressing
CD3
TCR·CD3 or upon disruption of rafts (Fig. 5 and
Refs. 12, 18, 29, 36, and 45).
In conclusion, the present study shows that CD3 serves to establish
a functional link between the TCR and the coreceptor CD8 and that this
is essential for efficient TCR signaling, which in turn is needed for
activation and positive selection of CD8 SP T cells. A similar
conclusion was reached in a related study using a different approach
(50). Even though strikingly different in structure, CD4 seems also to
associate with CD3
, thus forming a similar link with the TCR (25,
26). Indeed, mice expressing CD3
TCR
IV/
III also
exhibit impaired positive selection of CD4 SP T cells (16, 17). In
accordance with this is the finding that Lck plays a crucial role in T
cell development and that for positive selection of CD4 and CD8 SP T
cells, Lck must be associated with the coreceptors CD4 and CD8,
respectively (1, 2, 46). Finally, it has been shown that the negative
selection of CD8 (and CD4) SP T cells is normal in mice expressing
CD3
TCR (16-18). It is interesting to note that
TCR-mediated apoptosis of CD8+ cells is CD8-independent,
i.e. in contrast to cell activation and positive selection
it is not impaired by the lack of CD8 co-engagement (47, 48).
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful for helpful discussion with Drs. Jean-Charles Cerottini and Pascal Batard and for expert technical assistance by Sandra Levrand.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Swiss National Foundation (Grant 31-61946.00) and the Sandoz Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to the work.
** To whom correspondence should be addressed. Tel.: 41-21-692-5988; Fax: 41-21-653-4474; E-mail: iluesche@eliot.unil.ch.
Published, JBC Papers in Press, September 4, 2002, DOI 10.1074/jbc.M208119200
2 M.-A. Doucey, L. Goffin, D. Naeher, O. Michielin, P. Baumgärtner, P. Guillaume, E. Palmer, and I. F. Luescher, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
DP, CD4+CD8+ double-positive thymocytes;
SP, single-positive;
ABA, 4-azidobenzoic acid;
CPM, TCR
chain
connecting peptide motif;
DN, CD4
CD8
double-negative thymocytes;
FRET, fluorescence resonance energy
transfer;
IASA, iodo-4-azidosalicylic acid;
LAT, linker for activation
of T cells;
PbCS, Plasmodium berghei circumsporozoite;
TCR, T cell receptor;
TCR
IV/
III, TCR in which residues 225-270 of
the
chain and residues 270-300 of the
chain are replaced with
the corresponding sequences of the TCR
and
chain, respectively;
mAb, monoclonal antibody;
FACS, fluorescence-activated cell sorting;
DMEM, Dulbecco's modified Eagle's medium;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
MES, 4-morpholineethanesulfonic acid;
MHC, major histocompatibility complex.
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