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-Chain Connecting Peptide Motif in Mediating TCR-CD8 Cooperation
* Laboratory of Transplantation Immunology and Nephrology, University Hospital, Basel, Switzerland; and
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain connecting
peptide motif (-CPM), which allows the TCR to deliver positive
selection signals. Thymocytes expressing -CPM-deficient receptors do
not undergo positive selection, whereas their negative selection is not
impaired. In this work we studied the ligand binding and receptor
function of -CPM-deficient TCRs by generating T cell hybridomas
expressing wild-type or -CPM-deficient forms of the T1 TCR. This
Kd-restricted TCR is specific for a photoreactive
derivative of the Plasmodium berghei
circumsporozoite peptide252–260 IASA-YIPSAEK(ABA)I and is
therefore amenable to TCR photoaffinity labeling. The experiments
presented in this work show that -CPM-deficient TCRs fail to
cooperate with CD8 to enhance ligand binding and functional
responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TCR constant regions couple the 
heterodimer to the CD3
complex. TCR constant regions are composed of the cytosolic
(Cyto),2 the
transmembrane (TM), the connecting peptide (CP), and the Ig-like
domains. In terms of positive selection, a particularly important role
is played by a motif located in the membrane proximal CP domain of the
TCR chain (23, 24). This -chain connecting peptide
motif (-CPM) consists of seven highly conserved amino acids
(FETDxNLN), is only found in TCR, and is absent from TCR
.
Thymocytes from mice expressing -CPM mutant TCRs fail to undergo
positive selection, while negative selection is not impaired.
Characterization of thymocytes or hybridomas expressing -CPM mutant
TCRs revealed an impaired CD3
phosphorylation (23, 25),
a defective activation of the p56Fyn protein
kinase (26), and an impaired CD3 association
(24). CD3 is also important for thymic selection
signals because thymocytes from CD3 knockout mice fail to undergo
positive selection as well (27, 28). The defect observed
with -CPM mutant receptors is selective for low-affinity
(positive-selecting) ligands. Low-affinity ligands fail to recruit
tyrosine-phosphorylated isoforms of the signaling components
lck, CD3-, ZAP-70, and linker for activation of T cells
into detergent-insoluble, membrane rafts in -CPM mutant thymocytes
(25). Why these defects are specific for
positive-selecting ligands is still unclear.
To investigate whether the defects observed with -CPM-deficient TCRs
are mediated by altered ligand binding, we studied the T1 TCR, a
receptor that was developed to investigate TCR-ligand interactions by
photoaffinity labeling (29). In the T1 TCR system,
photoaffinity labeling is used to cross-link soluble, monomeric, pMHC
ligands to surface TCRs on living cells. This system is based on the
Plasmodium berghei circumsporozoite (PbCS)
peptide derivative, IASA-YIPSAEK(ABA)I, with two photoreactive groups,
IASA and ABA, which can be selectively activated by UV light of
different wavelengths. This bireactive peptide derivative can be
covalently cross-linked to the Kd molecule by
selective photoactivation of the IASA group, leaving the ABA group
intact. After specific binding of these pMHC complexes to T1 TCRs,
photoactivation of the ABA group allows covalent cross-linking of the
pMHC ligand to the TCR.
In this study we generated the wild-type and two chimeric forms of the
T1 TCR, in which the -CPM was either present or absent. All
receptors were expressed in CD8- and
CD8+ hybridomas. We were able to show that
replacement of the -CPM does not alter the binding of the T1 TCR to
its pMHC ligand per se. However, these studies demonstrate that the
-CPM is required for mediating TCR-CD8 cooperation to increase
ligand binding. Furthermore, we show that the functional defects
observed in hybridomas expressing -CPM-deficient TCRs are CD8
mediated and most prominent with low-affinity ligands.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The T1 TCR is derived from the
Kd-restricted, PbCS derivative
peptide253–260 (IASA)-YIPSAEK(ABA)I
(Ag11.3)-specific CTL clone, T1 (30). The cDNA encoding
the TCR chain was cloned into the G418-resistant retroviral vector,
LXSN (31, 32, 33). Similarly, the T1 TCR chain was cloned
into the puromycin-resistant retroviral vector, LXSP (23).
The cDNAs encoding the T1 TCR chimeras were constructed using
the previously described II and IV chimeric cDNAs
(23) specific for the 3BBM74 TCR (34)
by replacing the VJC-containing EcoRI-SpeI
fragment by T1 TCR VJC -chain sequences. Similarly, the T1 TCR
chimera III was constructed by replacing the VDJC-containing
EcoRI-XbaI fragment of the 3BBM74 chimeric
-chains by T1 TCR VDJC -chain-containing sequences.
Cells
The TCR-CD8- T
hybridoma, 58, and its
TCR-CD8+ derivative,
58CD8, have been described previously (35, 36).
Retroviral infection was used to introduce the wild-type or chimeric T1
TCR chains and the wild-type or chimeric T1 TCR chains into the
58 (TCR-CD8-) and the
58CD8 (TCR-CD8+)
hybridomas. Transduced hybridomas were subsequently FACS-sorted for
high TCR expression. All cells were grown in IMDM supplemented with 2%
FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 50 µM 2-ME. The T2-Kd cell
line (generously provided by T. Potter, National Jewish Medical and
Research Center, Denver, CO) used for peptide presentation was
grown in IMDM with 10% FCS. The indicator cell line HT-2
(37) was grown in IMDM containing 10% FCS and 250 U/ml
rIL-2. The ectotropic packaging cell line BOSC23 was purchased from
American Type Culture Collection (Manassas, VA) and grown in IMDM
containing 10% FCS.
Transduction of cDNAs
The BOSC23 packaging cell line was transfected as previously
described (38). The supernatant containing retroviral
particles was used to infect the
TCR-CD8- cell line, 58,
and its CD8+ derivative, 58CD8. In short,
5 x 105 58 or 58CD8 hybridomas were
resuspended in 500 µl IMDM containing 4 µg/ml polybrene
(Sigma-Aldrich, St. Louis, MO). After 24 h, 5 ml of fresh
IMDM and the appropriate selective drugs were added (1 mg/ml G418 (Life
Technologies, Rockville, MD), 3 µg/ml puromycin (Sigma-Aldrich), 2 mM
histidinol (Sigma-Aldrich), or 0.5 mg/ml hygromycin B (Calbiochem, La
Jolla, CA)). Surviving cells were analyzed after 4 days and sorted for
high TCR surface expression by FACS. Transfected cells were
continuously maintained in medium containing selective drugs.
Antibodies
The anti-TCR C mAb H57-597 (39), the
anti-CD3
mAb 145-2C11 (40), and the anti-CD8
mAb H35-17 (41) were purified from culture supernatants
using protein G (Amersham Pharmacia Biotech, Piscataway, NJ). The
anti-CD8 mAb 53-6.7 and the anti-CD8 mAb 53-5.8 were
purchased from BD PharMingen (San Diego, CA).
Quantitation of TCR surface expression
To calculate the relative amounts of the three different TCRs
expressed on CD8+ and CD8-
hybridomas, the expression of the TCR, measured with the anti-TCR
C mAb H57-597, was normalized to the expression of the wild-type TCR
measured on CD8+ hybridomas. The following
equation was used: relative TCR expression = MCF of TCR staining
expressing variant TCR/MCF of TCR staining on
CD8+ hybridomas expressing the wild-type
TCR.
Kd and TCR photoaffinity labeling
Soluble Kd molecules were produced and
loaded with the 125IASA-YIPSAEK(ABA)I peptide or
the P255A derivative as previously described (29). TCR
photoaffinity labeling was also performed as previously described
(5). Briefly, 5 x 106 cpm of
Kd-125IASA-YIPSAEK(ABA)I
were incubated with 107 T hybridoma cells either
at 0°C for 2 h or at 37°C for 10 min, followed by UV
irradiation at 312 ± 40 nm. After UV irradiation, cells
(107 cells/ml) were washed twice and lysed in 1
ml lysis buffer containing 1% Triton X-100, 1% Nonidet P-40, 150 mM
NaCl, 0.2 mM EDTA, 50 mM HEPES, 1 mM PMSF, 10 µg/ml leupeptin, 10
µg/ml aprotinin, and 10 µg/ml iodoacetamide for 2 h at 4°C.
Postnuclear supernatants were subjected to immunoprecipitation with
anti-TCR C mAb H57-597. The immunoprecipitates were analyzed by
reducing SDS-PAGE and quantified using a PhosphorImager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA). To compare the
photoaffinity labeling of the various receptors, the values were
normalized for TCR expression and compared with the labeling value
obtained on CD8+ hybridomas expressing the
wild-type T1 TCR. The following equation was used: relative
binding = (CPM bound by variant TCR/CPM bound by
CD8+ hybridoma expressing wild-type TCR)/relative
TCR expression.
IL-2 assays
A total of 80 µl IMDM containing 6 x
104 T2-Kd cells was plated
in flat-bottom 96-well plates and incubated with the indicated amounts
of peptide (see Fig. 4
) for 2 h at 37°C. T hybridoma cells
(6 x 104 in 100 µl IMDM) were
subsequently added. After a further 24 h of incubation at 37°C,
the supernatant was harvested and assayed for IL-2 using the indicator
line HT-2 as previously described (23).
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Relative receptor sensitivities were defined by determining the peptide concentration required for half-maximal IL-2 production from each hybridoma and peptide tested. These values were normalized to the concentration of P255S peptide required for half-maximal IL-2 production from the CD8+ hybridomas expressing the wild-type T1 TCR. The following equation was used: relative receptor sensitivity = [P255S] required for 1/2 max IL-2 production of CD8+ hybridomas expressing wild-type TCR/[pep] required for 1/2 max IL-2 production of hybridomas expressing a variant TCR
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test whether altered ligand binding was responsible for the
functional defects previously observed with mutant TCRs lacking the
-CPM domain (23, 24, 25, 26), we analyzed the ligand binding
properties of several chimeric T1 receptors. The T1 receptors are
specific for photoreactive derivatives of a PbCS
peptide253–260(IASA)YIPSAEK(ABA)I
(29), which can be photo-cross-linked to
Kd and the TCR. The CP, TM, and Cyto domains of
the wild type and the two chimeric TCRs used in this study are
schematically shown in Fig. 1A. Because a simple deletion
or replacement of the -CPM prevented TCR expression (data not
shown), we generated chimeric TCRs in which the CP, TM, and Cyto
domains were replaced with the corresponding domains from a TCR.
In Fig. 1A, TCR sequences, which replaced -sequences,
are shown in black and TCR- sequences, which replaced the TCR
sequences, are shown in gray. The -CPM mutant receptor is comprised
of IV and III chains. The IV chain encodes V, D, J, and parts
of the C region sequences from the T1 TCR chain followed by C
sequences; this chain lacks the -CPM (Fig. 1B). In the
III chain, the TM and the Cyto domains of the -chain were
replaced by homologous C sequences (Fig. 1B, boxed
sequences); the III chain is required for expression of the IV
chain, which lacks the -CPM. The control for the -CPM mutant
receptor is the TM control receptor, which expresses the III chain
and the II chain. The II chain is identical to the IV chain,
except that it includes the -CPM (Fig. 1B). Thus, the
only difference between the -CPM mutant and the TM control receptors
is the absence of the -CPM domain from the -CPM mutant
receptor.
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The expression of chimeric TCRs was examined on 58
(CD8-) and 58CD8
(CD8+) T cell hybridomas (Fig. 2 and data not shown). The wild-type and
-CPM mutant receptors were expressed at comparable, low levels while
the TM control (-CPM intact) receptor was expressed at higher
levels. TCR expression was similar on CD8- and
CD8+ hybridomas (data not shown) and expression
of a particular TCR did not significantly affect CD8 expression (Fig. 2). Finally, CD8 expression was similar in all
CD8+ cell lines used in this study (Fig. 2).
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To test whether replacement of the -CPM affects ligand binding,
we performed photoaffinity labeling of wild-type and chimeric T1 TCRs
using photoreactive ligands. To this end, hybridomas expressing the
different T1 TCRs were incubated with monomeric
Kd-peptide complexes. Two different ligands were
used: the high-affinity Ag11.3 and the variant, P255A, which has a
lower relative affinity for the T1 TCR (42). After
incubating hybridomas expressing the different T1 TCRs with equal
amounts of Kd-peptide complexes, TCR-bound
Kd-peptide complexes were cross-linked by UV
irradiation. TCRs were immunoprecipitated and analyzed by SDS-PAGE and
phosphor imaging. Fig. 3A
shows a representative experiment in which TCR photoaffinity labeling
was performed on CD8- and
CD8+ hybridomas expressing the various T1 TCRs.
The labeled material at 90 kDa represents the trimolecular complex
consisting of the TCR, the peptide, and Kd. The
material at 45 kDa represents a minor fraction of
peptide-Kd complexes that were not cross-linked
to but were coprecipitated with the T1 TCR. When
TCR- and CD8- hybridomas
were incubated with peptide-Kd complexes under
the same conditions, no signal could be detected, demonstrating that
the binding of the peptide-Kd complexes was
specific for the T1 TCR (Fig. 3A).
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, B–D, shows a quantification of the
signal intensities measured by phosphor imaging after correction for
the different TCR expression levels and normalization to the signal
observed on hybridomas expressing the wild-type T1 TCR on
CD8+ hybridomas at 0°C (see Materials and
Methods for calculations).
Fig. 3B shows relative ligand binding observed for the
different T1 TCRs upon incubation with
125IASA-YIPSAEK(ABA)I-Kd
(Ag11.3) complexes at 37°C. Importantly, ligand binding at 37°C was
equivalent on all TCRs in the absence of CD8 (Fig. 3B, open
bars), demonstrating that the -CPM-deficient TCR binds ligand as
well as the wild-type and TM control TCRs. In contrast, a significant
increase in ligand binding was observed only on
CD8+ hybridomas (Fig. 3B, filled bars)
expressing the wild-type or the TM control receptor, which contain an
intact -CPM. No increase in ligand binding was observed for
CD8+ hybridomas expressing the -CPM mutant
receptor, which lacks the -CPM. Therefore, at 37°C only hybridomas
expressing receptors with an intact -CPM showed a CD8-mediated
increase in ligand binding. We also performed affinity labeling of T1
TCRs using the weak
125IASA-YIASAEK(ABA)I-Kd
(P255A) ligand. Because the signals obtained with the weak ligand at
37°C were too weak to be accurately quantified, we repeated the
experiments with the strong (Ag11.3) and the weak (P255A) ligands at
0°C where the signal intensities were higher, which is in accordance
with previous observations (5).
As shown in Fig. 3, C and D, binding on
CD8- hybridomas (open bars) was similar for all
TCRs tested, irrespective of whether the strong (Ag11.3) ligand (Fig. 3C) or the weak (P255A) ligand (Fig. 3D) was
used. In the presence of CD8, a significant increase in ligand binding
was observed only on hybridomas expressing the wild-type and TM control
receptors, while the -CPM mutant TCR exhibited only a small increase
in ligand binding mediated by CD8 (Fig. 3, C and
D). Therefore, the impact of CD8 on ligand binding was more
pronounced with receptors containing the -CPM (wild type and TM
control) compared with the receptor lacking this motif (-CPM mutant)
(Fig. 3). The importance of the -CPM is illustrated when comparing
the CD8-mediated ligand binding increase observed on the -CPM mutant
and the TM control TCRs. These two receptors differ only by the absence
or presence of the -CPM (Fig. 1A).
The ligand binding experiments were repeated on
CD8+ hybridomas expressing the chimeric T1 TCRs
in the presence of a CD8 blocking mAb, H35-17. Ligand binding
observed in the presence of this Ab was similar to the results obtained
on CD8- hybridomas (data not shown). Because a
mAb directed against the CD8 chain completely blocks the increase in
ligand binding this enhancement can be attributed to CD8
heterodimers rather than CD8 homodimers.
The fact that the absence of the -CPM precludes proper CD8
participation in the binding of pMHC ligands suggests that the -CPM
is an important structural feature orchestrating TCR/CD8
cooperation.
IL-2 production of hybridomas expressing wild-type or chimeric T1 TCRs
We examined the ability of -CPM mutant receptors to transduce a
signal by measuring the IL-2 produced in response to peptide ligands.
To determine whether the functional deficit observed with hybridomas
expressing -CPM-deficient TCRs was dependent on the potency of the
peptide tested, we performed IL-2 assays using three ligands with
varying potencies for the T1 TCR. Fig. 4A shows the IL-2 responses of
CD8- and CD8+ hybridomas
expressing the various receptors when stimulated with ligands of high
(Ag11.3; Fig. 4A, a–c), medium
(P255S; Fig. 4A, d–f) or low (P255A;
Fig. 4A, g–i) affinities. Stimulation
of CD8- hybridomas (Fig. 4A, open
symbols) generally resulted in lower IL-2 production than the
stimulation of CD8+ hybridomas (Fig. 4A, filled symbols). When comparing IL-2 production of the
various hybridomas stimulated by the high-affinity Ag11.3 ligand, CD8
significantly increased the IL-2 responses of hybridomas expressing the
wild-type, -CPM mutant, or TM control receptor (Fig. 4A,
a–c). A similar CD8-mediated increase in IL-2
production was observed when the various hybridomas were stimulated
with the medium-affinity P255S peptide variant (Fig. 4A,
d–f). Interestingly, a clear difference in the
CD8 participation was observed when comparing the responses to the
low-affinity P255A ligand. Hybridomas expressing the wild-type T1 TCR
(Fig. 4Ag) or the TM control TCR (Fig. 4Ai)
showed a significant CD8-mediated increase in IL-2 production when
stimulated with the weak P255A peptide. In contrast, -CPM mutant
hybridomas failed to produce any detectable IL-2, even when CD8 was
present (Fig. 4Ah).
To quantify the CD8-mediated differences between the wild-type, -CPM
mutant, and TM control receptors, we reanalyzed the IL-2 data obtained
with CD8+ hybridomas to determine the sensitivity
of each receptor for each Ag. We calculated the relative sensitivity of
each TCR by determining the peptide concentration required for
half-maximal IL-2 production for each hybridoma line and comparing it
to the P255S concentration required for half-maximal IL-2 production
from the CD8+ hybridoma expressing wild-type
receptor; this was defined as 1.0 (see Materials and Methods
for calculation).
The results are shown in Fig. 4B, where the three ligands
have been ordered according to their potency. Although the wild-type,
-CPM mutant, and TM control receptors showed the highest sensitivity
for the high-affinity Ag11.3 peptide, the -CPM mutant TCR displayed
a 30-fold reduced sensitivity for this ligand. Similar observations
were made with the intermediate-affinity P255S peptide. Again, the
-CPM mutant receptor was 10- to 30-fold less sensitive than the
wild-type or TM control receptors. However, in response to the
low-affinity P255A peptide, the -CPM mutant receptor was strikingly
unresponsive. Compared with the wild-type receptor, the -CPM mutant
TCR was at least 10,000-fold less responsive to this low-affinity
ligand. Therefore, we conclude that the -CPM is of particular
importance in mediating TCR/CD8 cooperation in functional responses to
weak ligands.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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thymocytes
expressing self-MHC-restricted TCRs survive and differentiate (positive
selection), while thymocytes, whose receptors are self-MHC reactive,
undergo apoptosis (negative selection) (43, 44, 45, 46, 47). Although
thymocytes can discriminate between different antigenic ligands, which
induce positive or negative selection, the mechanisms by which the TCR
couples ligand binding to distinct cellular responses are still
unclear.
We have previously identified a highly conserved motif in the constant
region of the TCR chain, -CPM, which is required for the positive
selection of thymocytes (24). Thymocytes expressing
-CPM mutant TCRs have specific signaling defects in responses to
positive-selecting ligands, while their responses to negative-selecting
ligands are unaffected (25). While it seems that the
-CPM plays an important role in generating positive selection
signals, how the -CPM functions is still unclear.
To further characterize the defects observed with -CPM mutant
receptors, we used a hybridoma cell line that expressed a wild-type or
-CPM mutant form of the T1 TCR, in either the presence or the
absence of the CD8 coreceptor. Because the -CPM mutant and wild-type
TCRs differ in their TM and cytoplasmic domains as well, we included
the TM control TCR, which provides a direct control for the role of the
-CPM (Fig. 1).
To test whether -CPM-deficient TCRs bind ligand defectively, we
performed photoaffinity labeling studies (29) with the
different TCR mutants expressed on hybridoma cell lines. Relative
affinities were determined for the strong Ag11.3 ligand at 0°C and at
37°C, and for the weak P255A ligand at 0°C. Two major observations
were made. First, there was no obvious (TCR-intrinsic) ligand binding
defect observed with -CPM mutant receptors when assessed on
CD8- hybridomas (Fig. 3, B–D, open bars). Therefore, the -CPM mutation
does not decrease ligand binding per se. However, a significant
difference in ligand binding was observed with
CD8+ hybridomas when comparing wild-type, TM, and
-CPM mutant TCRs (Fig. 3, B–D, filled bars).
Our results clearly show that the -CPM mutant TCR has a significant
defect in engaging CD8 for ligand binding. Photoaffinity labeling
performed with the weak P255A ligand variant showed a similar picture.
The inability of CD8 to participate in ligand binding was due to the
absence of the -CPM, because ligand binding to the TM control TCR
was comparable to that observed on the wild-type TCR (Fig. 3, B and D, filled bars). Taken together, these
results imply that the -CPM plays an important role in orchestrating
the cooperation between the TCR and CD8, which is required to enhance
the binding of both high- and low-affinity ligands.
The functional consequences of defective TCR/CD8 cooperation were also
studied using IL-2 assays. Interestingly, CD8 enhanced IL-2 production
from -CPM mutant hybridomas when stimulated with ligands of high
(Ag11.3) or medium (P255S) potency. However, no IL-2 production could
be detected when hybridomas expressing the -CPM mutant receptor were
stimulated with the weak P255A ligand, not even in the presence of CD8
(Fig. 4Ah). Because CD8- hybridomas
expressing the wild-type and TM control receptors showed a substantial
IL-2 response to P255A peptide, loading limitations on the APC do not
account for the failure of the -CPM mutant hybridomas to
respond.
When the IL-2 responses of CD8+ hybridomas were
analyzed for their peptide dose dependency, i.e., receptor sensitivity,
similar observations were made. As shown in Fig. 4B, -CPM
mutant receptors were 10- to 30-fold less sensitive than wild-type and
TM control receptors in response to peptides of high (Ag11.3) or medium
(P255S) potencies. Strikingly, the differences mediated by the -CPM
were much more pronounced in the case of the weak P255A peptide.
Comparing CD8+ hybridomas, the -CPM mutant
receptor is 3,000- to 10,000-fold less sensitive in the response to
P255A than the wild-type and TM control receptors. Furthermore, the
wild-type receptor is only 10-fold less sensitive to the low-affinity
ligand P255A compared with the medium-affinity ligand P255S. In
contrast, the -CPM mutant receptor is 10,000-fold less sensitive to
the low-affinity P255A ligand compared with the medium-affinity P255S
ligand. This underscores the particular importance of the
-CPM-mediated TCR/CD8 cooperation in response to weak ligands.
The fact that the functional defect of the -CPM mutation is so
pronounced with low-affinity ligands is surprising, because the binding
of both strong (Ag11.3) and weak (P255A) ligands is similarly affected
by the absence of the -CPM (Fig. 3). Previous studies measuring the
kinetics of the Ag11.3, the P255S, and the P255A binding to the
wild-type T1 TCR showed a clear correlation between their off-rates and
their functional potencies (42). Therefore, the high
affinity (and slow off-rate) of the Ag11.3 ligand for the T1 TCR might
compensate for the poor TCR/CD8 cooperation observed with the -CPM
mutant receptor. In this context, high-affinity pMHC ligands tend to be
more CD8 independent. In contrast, the reduced TCR/CD8 cooperativity
observed with the -CPM mutant receptors has a catastrophic effect on
weak ligands, which are more dependent on CD8 assistance to initiate
intracellular signaling cascades. These observations are reminiscent of
the phenotype displayed by thymocytes expressing -CPM mutant TCRs.
These mutant thymocytes cannot undergo positive selection and are
specifically unresponsive to low-affinity ligands
(25).
Although the precise mechanism responsible for mediating TCR/CD8
cooperation is not known, we propose a model in which the CD8
molecules are recruited to the TCR complex by the -CPM.
Support for such a model comes from crystallographic studies of many
murine and human TCR-pMHC complexes, which show a conserved orientation
of the pMHC over the TCR, placing the MHC coreceptor binding site over
the -chain side of the TCR (34, 48, 49, 50, 51, 52, 53). Furthermore,
certain V domains show a preference for selection into the CD4 or
CD8 subset of mature T cells (54). This could be explained
by a preference of certain V domains to physically interact with CD4
or CD8. Such an arrangement would likely recruit the membrane
proximal domain of the coreceptor to the -chain constant region, an
event that requires an intact -CPM.
Thymocytes from CD8 knockout mice were specifically blocked in
undergoing positive selection (9, 15, 18, 55), which
emphasizes the critical role played by the coreceptor in responding to
weak ligands. While studies by Bosselut et al. (21) have
clearly shown that the extracellular, TM, and cytoplasmic domains of
CD8 contribute to its ability to support positive selection, little
is known about the structural elements of the TCR, which mediate the
engagement between the receptor and the coreceptor. The studies
presented in this work suggest that the -CPM is an important
structural element mediating TCR/CD8 cooperation.
Whether the association of the CD8 coreceptor to the TCR is mediated by
a direct interaction of CD8 with the -CPM has not yet been ruled
out. Because proper association of CD3 to the TCR chain also
requires the -CPM (24, 25), CD3 may associate with
the coreceptor as well. Consistent with this idea is the observation
that thymocytes from CD3-deficient mice cannot undergo positive
selection (27, 28).
These studies provide evidence that the -CPM plays an
important role in mediating TCR/CD8 cooperativity. Although not
considered in these studies, a similar role might be played by the
-CPM in case of cooperation with the CD4 coreceptor, because the
-CPM mutation blocks the positive selection of a class II
MHC-restricted TCR as well (24). The precise mechanism by
which the cooperation between the TCR and its coreceptor allows a
"reading" of ligand affinity and to what extent this controls the
decisions taken during positive and negative selection require
additional work.
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Acknowledgments |
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, and Ramona Leibnitz for
some of the cDNA constructs. We are grateful to T. Hayden, H. Kohler,
and M. Dessing for flow cytometric support. We also thank J. Gatfield,
E. Meier, and G. Werlen for valuable discussion and J. Gatfield, B.
Hausmann, and G. Werlen for reviewing the manuscript. |
Footnotes |
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2 Abbreviations used in this paper: Cyto, cytosolic; -CPM, -chain connecting peptide motif; TM, transmembrane; CP, connecting peptide; PbCS, Plasmodium berghei circumsporozoite.
Received for publication May 2, 2002. Accepted for publication July 9, 2002.
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) and
CD8+ (
) hybridomas expressing the wild-type T1 TCR are
shown in a, d, and g.
Shown in b, e, and h are
IL-2 responses of CD8- (
) and CD8+ (•)
hybridomas expressing the 
) or presence (
) of the CD8
coreceptor molecules. B, Receptor sensitivities of
CD8+ hybridomas to peptide ligands of varying potency.
Relative receptor sensitivities of CD8+ hybridomas
expressing the various T1 TCRs were calculated from the peptide
concentration required for half-maximal IL-2 synthesis in each
hybridoma line and normalized to the receptor sensitivity calculated
from CD8+ hybridomas expressing the wild-type receptor
responding to P255S (see Materials and Methods for
calculation of receptor sensitivity). The numbers indicate the
different T1 TCRs tested: 1, wild type; 2, 
