By
From the Basel Institute for Immunology, CH-4005 Basel, Switzerland
A single amino acid residue, Gln136, located within the connecting peptide domain of C controls the ability of the
/
TCR to transmit a full signal. TCRs in which this C
residue is
mutated to Phe, the residue found in TCR-
, are unresponsive to antigenic ligands. Interestingly, this C
residue is either polar or charged in every species studied thus far, including the
trout and the skate. In contrast, the analogous residue in C
is always hydrophobic. In spite of
their compromised antigen responsiveness, the mutant TCR complex contains the CD3-
, -
,
-
, and -
chains, and undergoes
chain phosphorylation and ZAP-70 recruitment. However,
the biological response of the mutant TCR could be rescued with a calcium ionophore, implying that mutant TCRs are defective in generating a calcium-mediated signal. The implications
of the differences between C
and C
are considered.
One of the primary goals of immunologists over the last
10 yr has been to understand how the TCR transduces a signal (1) . The TCR complex consists of an antigen-binding Considered together, the cytoplasmic tails of the CD3
and Although homologous to its We wondered whether there exists a region within the
TCR- Generation of DNA Constructs.
The V
Production of Retroviral Supernatants and Infection of T Cell Hybridomas.
Retroviruses encoding wt or chimeric TCR chains were
generated and used to infect the Cell Lines.
The 58hCD4 cell line has been previously described (15, 17) and was provided by O. Acuto (Pasteur Institute,
Paris, France). The murine fibroblast cell line DAP.3 transfected
with human HLA class II DR1 molecules (18) or the human,
DR1-expressing, B lymphoblastoid cell line .221 (19) was used to
present SEB. The use of the cytokine indicator cell lines HT-2
and FDC-P1 have been previously described (15).
Antibodies.
The use of anti-V Flow Cytometry, T Cell Stimulation, and IL-2 and IL-3 Assays.
The levels of cell-surface antigens were detected by immunofluorescence and analyzed on a FACScan® using the CELLQuest
analysis software (Becton Dickinson, Rutherford, NJ). T cell
stimulation and IL-2 and IL-3 assays were performed as previously described (15).
Immunoprecipitation and Western Blotting.
T hybridoma cells
(2 × 107/sample) were lysed in buffers containing 1% digitonin
and the TCR complex immunoprecipitated with 2 µg of B20.1
(anti V To better understand the functional relationships between
the To test this idea, we constructed two additional chimeras,
TCRs from these hybridomas were immunoprecipitated, and the associated polypeptides were analyzed by
Western blotting (data not shown). The CD3- The tyrosine phosphorylation of the
Binding of an appropriate ligand to the TCR eventually
leads to the activation of the protein kinase C/ras and the
calcium intracellular signaling pathways (1, 7). Deficient
activation of the PKC/ras pathway can be compensated for
by the addition of the phorbol ester, PMA, whereas deficient activation of the calcium pathway can be compensated for by the addition of a calcium ionophore, such as
ionomycin. In Fig. 4, hybridomas expressing the wt or the
defective
The functional chimera,
We took advantage of the differences between There is evidence that the Examining the CP domain of the There is little structural information available for the CP
domains since this region has been truncated from the
TCR heterodimers used in crystallographic analyses (25,
26). Nevertheless, the CP domains of the Functionally, the CP domains may mediate an interaction with the /
heterodimer supported by noncovalently
associated CD3-
, -
, and -
as well as
chains (2). The
and
constant regions are presumably required to propagate a signal to the CD3 and
components of the complex.
chains contain 10 immunoreceptor tyrosine-based
activation motifs (ITAMs), which are rapidly phosphorylated upon TCR engagement (3, 4). Subsequently, the
protein tyrosine kinase ZAP-70 is recruited to the TCR
complex and activated (5, 6). Thereafter, large multimolecular complexes of proteins participating in the signaling
cascades assemble around the cytoplasmic domains of activated TCRs (7), which leads to the initiation of intracellular signaling pathways.
/
relative, the
/
TCR
is expressed in a separate lineage of T lymphocytes (8).
While it has been generally assumed that both classes of
TCR use similar mechanisms to generate a signal, this may
not be the case, as there are several features that distinguish
these two types of T cell receptors. The CD3-
chains contained within human
/
receptors express an epitope
which is masked in
/
receptors (9, 10). Furthermore, unlike
/
receptors,
/
TCRs can be expressed in the absence of CD3-
, can develop in MHC deficient mice, and
can recognize unprocessed antigens in the absence of MHC
presentation (11). Finally, the TCR-
chain contains a
motif within its connecting peptide (CP) domain that is
required for the transduction of antigenic signals (15).
/
TCRs lacking this
chain-connecting peptide motif
(
-CPM) do not properly interact with the CD3 complex
and fail to generate Ca2+-mediated signals. The
-CPM is
specific for the
/
TCR and seems to have evolved after
the divergence of TCR-
and -
chains. That this motif is
found within
/
but not
/
TCRs implies that these two types of TCRs may use subtly different signaling
mechanisms.
chain, analogous to the
-CPM, which is required
to produce a functional
/
receptor. The experiments reported here identify a single amino acid within the
chain-
CP domain that controls TCR signaling efficiency. Mutation of this
chain amino acid to a residue commonly seen
in TCR-
chains generates an
/
receptor which is particularly inefficient in transducing signals from antigenic
ligands.
2.1 and V
8.1 TCR
cDNAs were isolated from the T cell hybridoma, 3BBM74, and
confer reactivity to the I-Abm12 alloantigen and the staphylococcal
enterotoxin B (SEB) superantigen (16). The wild-type (wt) and
mutated TCR constructs were generated using overlapping oligo
nucleotides and PCR as previously described (15). All constructs
were verified by DNA sequencing using the SequiThermTM cycle
sequencing kit (Epicentre Technologies Corp., Madison, WI)
and the deduced amino acid sequences of the mutant
chains are
shown in Fig. 1.
Fig. 1.
Amino acid sequences and surface expression
of chimeric TCR- chains. (A)
The sequences of the wt TCR-
chain (C
), wt TCR-
chain
(C
), and the 3 chimeric TCR-
chains (
V-
VII) are shown using the single letter amino acid
code. The boxes indicate the TCR-
chain-derived amino acids. Only the CP, transmembrane,
and Cyto domains of the TCR
constant regions are shown. The
complete
and
chain cDNAs
have been previously described
(16). The NH2-terminal amino
acid in A represents the interchain Cys127 of the TCR-
constant region. The dotted lines indicate the approximate boundary
of the TM domain, defined using
the Lasergene Navigator Protean
Software program (DNASTAR,
Inc., Madison, WI). (B) 58hCD4 (
/
)T cell hybridomas expressing wt
chains and wt
or
chimeric TCR-
chains were stained with the biotinylated anti-V
8 mAb, F23.1, and SAPE, and then analyzed by flow cytometry. Dashed lines represent fluorescence of the same cells stained with streptavidin-phycoerythrin alone. The solid vertical lines indicate the mean fluorescence intensity of
58hCD4 cells expressing the wt
/
TCR. Similar results were obtained using anti-V
2, anti-CD3-
, or anti-C
specific mAbs (data not shown).
[View Larger Version of this Image (32K GIF file)]
/
T cell hybridoma,
58hCD4, as previously described (15).
2 (B20.1), anti-V
8.1 (F23.1),
and anti-CD3-
(145-2c11) mAbs, as well as anti-CD3-
, anti-
CD3-
, and anti-CD3-
rabbit antisera have been previously described (20). The anti-
chain mAb, H146-968 (21), was purified
from culture supernatants using protein G-Sepharose beads
(Pharmacia Biotech AB, Uppsala, Sweden). The rabbit anti-
ZAP-70 antiserum was provided by Dr. L. Samelson (National
Institutes of Health, Bethesda, MD). The antiphosphotyrosine
mAb 4G10 was purchased from Upstate Biotechnology Inc.
(Lake Placid, NY).
2). Immunoprecipitations, Western blotting, and detection of CD3 chains were carried out as previously described (20).
Chain Tyrosine Phosphorylation Analysis.
For superantigen stimulation, 106 .221 APCs were incubated with or without SEB (10 µg/ml) for 2 h in 1 well of a 24-well plate. 107 T hybridoma cells
were added to the APCs, centrifuged at 1,000 rpm for 30 s, and
then incubated at 37°C for 20 min. Cells were washed twice with
ice cold PBS containing 0.4 mM Na3VO4 and 0.4 mM EDTA,
and were then lysed in 0.5 ml lysis buffer (10 mM Tris/HCl, pH
7.5, 150 mM NaCl, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) containing 2 mM Na3VO4, 25 mM NaF, and
1% Triton X-100. Lysates were centrifuged at 12,000 g for 10 min, and the relevant proteins in the supernatant were immunoprecipitated with 1.5 µg of H146-968 (anti-
) mAb or 3 µl of a
rabbit polyclonal anti-ZAP-70 antiserum. Immunoprecipitates
were recovered using protein G-Sepharose beads. The tyrosine
phosphorylated proteins present in these immunoprecipitates were analyzed by Western blotting and detected using the antiphosphotyrosine mAb 4G10.
/
heterodimer and the CD3 and
components, we
made a number of chimeric TCR-
chains by replacing
domains from the
chain constant region with homologous domains from the TCR-
chain. We first generated a
chimeric
chain,
VII, which contained TCR-
-derived V, D, J, and C region sequences up to and including the
interchain Cys, followed by C
sequences encoding the
COOH-terminal part of the CP domain, the transmembrane domain, and the cytoplasmic tail (Fig. 1 A). As seen
in Fig. 1 B, this chimeric
VII chain was not expressed at
the cell surface when paired with a wt
chain. However,
C
and C
differ in the length of their CP domains. There
are 13 amino acids between the interchains Cys127 and
Ala141 in all
chains and 16 amino acids between Cys117
and Ala134 in all mammalian
chains (Fig. 1 A and reference 22). Thus, the fact that the
VII chimera could not be
expressed at the cell surface might have been due to the
possibility that the C
length in this region (16 amino acids) was incompatible with surface expression in the context
of an
/
TCR.
V and
VI, which respected the conserved
chain
length (13 amino acids) in this region (
wt; Fig. 1 A). Both
the
V and the
VI chimeras were expressed at the cell surface in conjunction with a wt
chain (
wt; Fig. 1 B). In fact,
the
wt/
V and the
wt/
VI chimeric TCRs were expressed at two- to threefold higher levels on the cell surface
than were the
wt/
wt receptor. Hybridomas expressing
these chimeric TCRs were stimulated with the superantigen, SEB bound to APCs. As seen in Fig. 2 A, the
wt/
V
TCR responded to SEB about as well as the wt TCR,
while the
wt/
VI TCR was clearly less sensitive (~100-fold) to this superantigen. This signaling deficit was seen
even more clearly when the SEB response of a hybridoma
expressing the
wt/
VI mutant receptor was compared to
that of an
wt/
wt hybridoma sorted for an equivalently
high level of TCR expression (Fig. 2 B). Furthermore,
transgenic mice expressing the
wt/
VI TCR were defective in responding to SEB and the I-Abm12 alloantigen (data
not shown). Thus, the signaling defect of this mutant TCR
was not limited to superantigens. On the other hand, these
chimeric TCRs could be activated by plate-bound anti-TCR mAbs (Fig. 2 C and data not shown), indicating that
the hybridomas expressing the
wt/
VI TCR were not
intrinsically defective. Therefore, TCRs comprised of the
chimeric
VI chain seemed to be specifically deficient in
transducing signals from antigenic ligands.
Fig. 2.
Response of chimeric TCRs to SEB and anti-TCR mAbs.
In each experiment, 5 × 104 58hCD4 cells/well expressing wt or chimeric TCRs were stimulated with 2 × 104 DAP.3-DR1 cells and increasing doses of SEB (A and B) or plate-bound anti-V8 (C), and the
culture supernatants were assayed for IL-2. Similar results were obtained
using plate-bound anti-CD3-
and anti-V
2 mAbs (data not shown).
Results shown are representative of two or more experiments.
[View Larger Version of this Image (14K GIF file)]
, -
, and -
chains as well as the
polypeptides were coprecipitated from
hybridomas expressing either the wt TCR or the signaling-defective,
wt/
VI TCR (data not shown). In spite of a
pronounced signaling defect (Fig. 2), the interactions with the CD3 and
chains were preserved in complexes containing the functionally defective,
wt/
VI heterodimer.
chain was examined in these hybridomas as well. From the data in Fig. 3
A, it was apparent that the
chains in the
wt/
wt and
wt/
VI TCRs could be tyrosine phosphorylated and that
both forms (p21 and p23) of the phosphorylated
chain
could be generated. Although the tyrosine phosphorylation of ZAP-70 could not be demonstrated even in hybridomas
expressing the
wt/
wt receptor (data not shown), an
anti-ZAP-70 antiserum was used to evaluate ZAP-70 recruitment (Fig. 3 B). In superantigen-stimulated cells expressing either wt or mutant TCRs, the p21 and p23 forms of the
chain as well as the p34 phosphoprotein were coprecipitated with ZAP-70. These experiments indicated
that even in hybridomas expressing the signaling-defective
wt/
VI TCR, ZAP-70 was nevertheless recruited to the
phosphorylated
chains. Thus, the signaling defect in hybridomas expressing this mutant TCR was likely downstream from ZAP-70 recruitment.
Fig. 3.
chain phosphorylation of SEB-stimulated hybridomas expressing chimeric TCRs. Hybridomas (58hCD4) expressing wt or chimeric TCRs were stimulated using .221 cells with (10 µg/ml) or without SEB. Cells were lysed in 1% Triton X-100, and the relevant chains were
immunoprecipitated with an anti-
mAb (A) or with an anti-ZAP-70 antiserum (B). After Western blotting, the presence of tyrosine phosphorylated proteins was detected with the antiphosphotyrosine mAb 4G10 as
described in Materials and Methods. The positions of phosphorylated
chains, p34, unphosphorylated
chains, and ZAP-70 are indicated.
[View Larger Versions of these Images (39 + 49K GIF file)]
wt/
VI TCR were stimulated with SEB in the
presence of PMA or ionomycin. The impaired response of
the
wt/
VI TCR was only marginally affected by several
different concentrations of PMA, but was rescued in the
presence of ionomycin used at several different concentrations (Fig. 4 and data not shown). These findings suggested
that the mutant TCR was defective or inefficient in activating the calcium pathway.
Fig. 4.
Effects of calcium ionophore and PMA on superantigen
stimulation. The 58hCD4 hybridoma (5 × 104 cells/well) expressing wt
or chimeric TCRs was stimulated with 2 × 104 DAP.3-DR1 cells and
the indicated combinations of 3 µg/ml SEB, 100 ng/ml ionomycin, or 30 ng/ml PMA. Culture supernatants were assayed for IL-2 (A) or IL-3 (B).
[View Larger Version of this Image (22K GIF file)]
V, contains the
chain sequence ASYQQ, which has been replaced with the
chain sequence, LQFQF, in the nonfunctional chimeric
chain,
VI (Fig. 1 A). The most striking amino acids in this
region are Tyr134 and Gln136, both of which are encoded by
Phe in murine C
1. Point mutations were introduced into
the wt
chain cDNA, and the resulting mutant
chains
were paired with an
wt chain and were tested for their reactivity to SEB. As seen in Fig. 5, A and B, the Tyr134
Phe mutation had a negligible effect on the response to
SEB, whereas TCRs carrying the Gln136
Phe mutation
had a marked and reproducible effect on SEB responsiveness. Considering the pronounced effect of the Gln136
Phe exchange on superantigen responsiveness, we examined the amino acid residues present at this position in all C
and C
sequences, some of which are shown in Fig. 5
C. Strikingly, the amino acids at this position in the
chain are always polar or charged. In contrast,
chains always contain hydrophobic amino acids at this position (Fig.
5 C and reference 23).
Fig. 5.
Point mutation of Gln136 affects antigen responsiveness. Hybridomas expressing the wt TCR- chain and wt or mutant
chains were stimulated with DAP.3-DR1-presenting cells and SEB as described in Fig. 2. IL-2 and IL-3 responses are shown in A and B, respectively. Sequences of the CP domains from different species (22, 23) are shown in
C, using the single letter amino acid codes. Sequences were aligned using
the Lasergene Navigator MegAlign Software program (Clustal alignment
method with the PAM250 residue weight table). The conserved amino
acids present in the TCR-
and
chain CP domains are indicated in
boldface.
[View Larger Version of this Image (42K GIF file)]
/
and
/
TCRs to generate signaling-defective receptor mutants.
Since the TCR-
and -
constant regions are structurally
homologous, we could replace domains of C
with similar
domains of C
. Most of the resulting chimeric chains
paired with a wt
chain and were expressed on the cell
surface (Fig. 1 B). Hybridomas expressing
wt/
VI TCRs
are 100-fold less sensitive to antigenic signals than hybridomas expressing wt receptors (Fig. 2, A and B). The signaling defect seems specific for antigenic ligands, since
wt/
VI TCRs respond fully to mAbs capable of receptor
cross-linking (Fig. 2 C and data not shown). What distinguishes the functional
V chimera from the defective
VI
chimera are the amino acids present from position 132 to
136 that are derived from C
and C
, respectively (Fig. 1
A). Site-directed mutagenesis defined Gln136 as a critical
residue in this region. This position has been conserved in
the
chains of all known species from the skate to humans
(23) and is either polar or charged (Fig. 5 C). In contrast,
the analogous residue in the
chain has been conserved as
a hydrophobic amino acid (Fig. 5 C). This conservation is
functionally significant since changing this residue from a
hydrophilic to a hydrophobic amino acid is accompanied
by a significant loss in signaling efficiency (Figs. 2 and 5).
/
and
/
heterodimers do
not interact with the CD3 complex in the same way. For
example,
/
TCRs can be expressed in the absence of
CD3-
(11). Furthermore, a CD3-
epitope, contained
within human
/
receptors and recognized by the mAb
WT31 is masked in
/
receptors (9, 10, 24). Many of
these differences might be attributed to the CP (membrane proximal) domains of the different TCR chains. We have
previously identified a motif within the
CP domain which
is strikingly absent from TCR-
chains. This motif controls
the proper association of CD3-
and -
to the TCR complex
and is required for antigen-driven signal transduction (15).
chain, we have
found a region within C
which controls the efficacy of
antigen-driven signal transduction. Although alteration of
this region does not lead to changes in the subunit composition of the TCR complex (data not shown), a clear biological defect was observed (Fig. 2). Mutation of this region
had no observable effect on the amount of
chain phosphorylation nor on the recruitment of ZAP-70 to the
homodimer (Fig. 3). Thus, the signaling defect in this mutant
is likely downstream from ZAP-70 recruitment. The signaling defect can be corrected or at least compensated in
the presence of a calcium ionophore (Fig. 4), suggesting
that the mutation within the
chain may lead to the inefficient induction of Ca2+ mobilization.
and
chains
may function as a unit since amino acids within these domains have been conserved in
/
TCRs during the last
500 million yr (15, 22, 23). The length of the CP domains
has been conserved as well. Considering the region between the interchain Cys and the transmembrane domain (defined by the beginning of the CART motif [22]), all
known
chains contain 13 amino acids (22, 23). On the
other hand,
chains contain from 17 to 20 amino acids in
this region. Thus, in
/
TCRs, a length disparity between the
and
chain CP domains has been conserved
throughout vertebrate evolution. In contrast, the lengths of
the TCR-
and -
chain CP domains (between the interchain Cys and the transmembrane domain) are comparable
in each species examined thus far (22, 23). Therefore, a
length similarity between the
and
chains has been conserved in this region in
/
receptors during a similar period of vertebrate evolution.
homodimer. In this regard,
/
TCRs
compete more avidly for the
chain compared to a
/
TCR (27). Furthermore, mutation of the
-CPM leads to
a TCR complex that is unstably associated with the
chains (15). Nevertheless, why the CP domains have diverged between
/
and
/
TCRs is puzzling. It is conceivable that the CP domains in the
/
TCR have a specialized function such as interacting with the CD4 and
CD8 coreceptors or that they have evolved to optimize the
efficiency of signal transduction from MHC encoded
ligands.
Address correspondence to Dr. Ed Palmer, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland. Phone: 41-61-605-1277; FAX: 41-61-605-1364; E-mail: Palmer{at}bii.ch B. Thomas Bäckström's current address is Malaghan Institute of Medical Research, PO Box 7060, Wellington South, New Zealand.
Received for publication 16 July 1997 and in revised form 26 September 1997.
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche LTD, Basel, Switzerland.The authors thank Drs. Luca Bolliger, Thomas Göbel, Wayne Hein, Michel Record, and Sabine Stotz for careful reading of the manuscript, Beatrice Pfeiffer for photography, Dr. L. Samelson for the rabbit anti- ZAP-70 antiserum, and O. Acuto for the 58hCD4 cell line.
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