By
From the Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland
Using H-2Kd-restricted photoprobe-specific cytotoxic T lymphocyte (CTL) clones, which permit assessment of T cell receptor (TCR)-ligand interactions by TCR photoaffinity labeling, we observed that the efficiency of antigen recognition by CTL was critically dependent on the half-life of TCR-ligand complexes. We show here that antigen recognition by CTL is essentially determined by the frequency of serial TCR engagement, except for very rapid dissociations, which resulted in aberrant TCR signaling and antagonism. Thus agonists that were efficiently recognized exhibited rapid TCR-ligand complex dissociation, and hence a high frequency of serial TCR engagement, whereas the opposite was true for weak agonists. Surprisingly, these differences were largely accounted for by the coreceptor CD8. While it was known that CD8 substantially decreases TCR-ligand complex dissociation, we observed in this study that this effect varied considerably among ligand variants, indicating that epitope modifications can alter the CD8 contribution to TCR-ligand binding, and hence the efficiency of antigen recognition by CTL.
Modifications of antigenic peptides can affect CTL responses in a diverse manner, such as provoking either
antagonism, weak or strong responses or selective activation of Fas (CD95/APO-1) dependent cytotoxicity (1).
As to the mechanism, it has been shown that TCR antagonists provoke hypophosphorylation of CD3 The observation that blocking of CD8, or in the case of
T helper cells CD4, can convert weak peptide agonists into
antagonists (7, 13, 14), indicated that the way T cells perceive antigen is determined in part by the coreceptor. Since
coreceptors are associated with the cytoplasmic tyrosine kinase p56lck, which plays a key role in T cell activation, it
has been proposed that the particular pattern of TCR signaling seen using partial agonists and antagonists is the result of inefficient recruitment of coreceptor to TCR (7). On
the other hand, it has been shown that CD8, by coordinate
binding of MHC molecules that interact with TCR, significantly strengthens the avidity of TCR-ligand binding by
decreasing the dissociation of TCR-ligand complexes (15,
16). The intimate participation of CD8 in TCR-ligand interactions raises the question whether CD8 may play a role
in aberrant CTL function by modulating the dynamics of
TCR-ligand interactions.
To assess TCR-ligand binding and its dependence on
CD8, we used H-2Kd-restricted CTL clones that are specific for a photoreactive peptide derivative and thus permit
measurement of TCR-ligand interactions by TCR photoaffinity labeling (4, 15, 17). To this end the Plasmodium
berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI) was modified by replacing PbCS S-252 with iodo-4-azidosalicylic acid (IASA) and conjugating PbCS K-259
with 4-azidobenzoic acid (ABA). Selective photoactivation
of the IASA group permitted covalent attachment of the
conjugate to Kd and photoactivation of the ABA group to
TCR. By testing 12 peptide derivative variants on 7 CTL
clones, we have previously identified 12 cases for which the
efficiency of antigen recognition (cytotoxicity) and TCR-ligand binding (TCR photoaffinity labeling) diverged by five-fold or more (4). As these divergences often did not
correlate with the avidity of TCR-ligand binding, we examine here whether they are related to the dissociation
rates of TCR-ligand complexes and how these depend on
the coreceptor CD8.
Chromium Release Assay.
The generation of the IASA-YIPSAEK(ABA)I-specific CTL clones S4, S14, S17, and T1 and their
culture conditions have been described previously (4, 17). Antigenic activities of peptide derivatives were determined in a 51Cr-release assay as described (4, 17). In brief, 51Cr-labeled P815 cells
(2 × 103 per well) were incubated in DMEM supplemented with
FCS (5%) and Hepes (10 mM) with 10-fold dilutions of peptide
derivative ranging from 10 Interferon Kd Photoaffinity Labeling.
Synthesis and photoaffinity-labeling
procedures were performed essentially as described (4, 17, 19). In
brief, for photoaffinity labeling of cell-associate Kd molecules,
P815 mastocytoma cells were incubated with the respective peptide derivatives for 60 min at 37°C, followed by UV irradiation at
TCR Photoaffinity Labeling.
TCR photoaffinity labeling was
performed as described (4, 15, 17, 19), except that the samples
were incubated at 26°C for 30-60 min before UV irradiation at
312 ± 40 nm. For TCR-ligand complex dissociation experiments CTL (6 × 106-2 × 107 cells/ml) were preincubated in medium with 125I-labeled Kd-peptide derivatives complexes (1-6 × 107 cpm/ml for 90-150 min at 0-4°C or for 30-60 min in medium containing SF1-1.1.1 Fab TCR-ligand Binding.
TCR-ligand binding was determined
by a direct binding assay as previously described (4), except that
the incubations of CTL (1.2 × 106 cells/200 µl) with 125I-labeled
Kd-peptide derivative complexes (3-10 × 106 cpm/incubation)
were performed for 30-60 min at 26°C. Specific TCR-ligand
binding was calculated by subtracting from the total cell-associated cpm, the cpm measured in presence of anti-Kd mAb 20-8-4S
(10 µg/ml). The specific binding (cpm) for the wild-type (wt)
ligand for a given clone was referred to as 1 and the binding of
the variant ligands expressed relative to this value. Due to high
background the difference between TCR-ligand binding in the presence and absence of SF1-1.1.1 Fab To assess the significance of TCR-ligand complex dissociation for CTL function, we selected two cases, in which
antigen recognition was more efficient than TCR-ligand
binding (strong agonists) (peptide derivative variant P255A
on T1 CTL and S256A on S17 CTL) and two examples
where recognition was less efficient than binding (weak agonists) (P255S on S17 CTL and E258A on S14 CTL) (Fig.
1 a). Since perforin dependent cytotoxicity, as mainly assessed in the used chromium release assay, is a rapid CTL
response and takes place at very low peptide concentrations
(20, 21), we also assessed the interferon-
Assessment of the kinetics of TCR-ligand complex dissociation showed that for both strong agonists the dissociation was significantly faster than for the wild-type ligand
(Fig. 2, a and c). In contrast, for the two weak agonists dissociation was markedly slower, even though in one case
(E258A on S14 CTL) TCR-ligand binding was weak (Fig.
2, b and c). For sake of increased accuracy, these kinetics were assessed at 26°C. With S17 CTL kinetics were also
measured at 37°C (Fig. 2 d), showing that on living CTL
TCR-ligand complex dissociation increases considerably
with temperature, as has been reported for T1 CTL (15).
These findings are consistent with the concept of serial TCR
engagement (21, 23, 24), namely that CTL activation depends on the frequency of serial TCR engagement, which
is determined by the rate of TCR-ligand complex dissociation.
To test this hypothesis, we repeated these experiments in
the presence of Fab Exceptions to this rule were the cases where blocking of
CD8 participation in TCR-ligand binding dramatically impaired antigen recognition. As it has been shown that blocking of CD4 can convert agonists into antagonists (7, 13), we
examined whether, in the presence of SF1-1.1.1 Fab
It is interesting to note that the half-life of TCR-ligand
complexes varied considerably among different CTL clones
(Fig. 2, a-c), which may explain why certain CTL clones
are more prone to TCR antagonism than others (4). For
example with S17 CTL, which exhibited remarkably slow
dissociation kinetics, TCR antagonism was never observed,
even when testing large panels of epitope variants, whereas
the opposite was true for S14 CTL and, to a lesser extent,
T1 CTL, especially in the presence of SF1-1.1.1 Fab A striking finding of our study is that the contribution of
CD8 to TCR-ligand binding varied not only among CTL
clones, but also among epitope variants on given clones (Fig.
4 a). While the former differences can be explained in part
by variations in CD8 and TCR expression (Fig. 4 a), the
latter indicate that epitope modifications can alter the avidity
of CD8 participation in TCR-ligand binding. This explains,
at least in part, why blocking of CD8 accelerated TCR-ligand complex dissociation in a diverse manner, and why
one cannot reliably predict from the avidity of TCR-ligand
binding the dissociation rates and hence the functional phenotype of epitope variants (Figs. 1 and 2 and reference 4). Moreover, several ligand variants displayed different ratios
of TCR
The present study shows that the half-life of TCR-ligand complexes is of critical importance for CTL activation and is determined to a considerable degree by the
coreceptor CD8. The finding that epitope modifications
can alter the participation of CD8 in TCR-ligand binding,
and hence TCR-ligand complex dissociation, reveals a new
principle by which CD8 can affect CTL function. However CD8 plays other roles in CTL function as well, such as
mediating CTL-target cell adhesion and participation in
CTL activation by CD8-dependent signaling (29, 30), which
must also be taken into account for a comprehensive understanding of the role of CD8 in normal and aberrant
CTL function.
chains, which
fail to recruit and activate the tyrosine kinase ZAP-70, thus
impairing the ZAP-70-NFAT pathway of T cell activation (1, 5). Two main hypotheses have been proposed to explain these phenomena. First, ligand modification may result in accelerated dissociation of TCR-ligand complexes
and thus perturb the cascade of biochemical events that is
initiated by TCR-ligation (8). Alternatively, ligand
modification may confer, via conformational changes in
TCR, qualitative changes in signaling by TCR-associated molecules (11, 12).
6-10
13 M. Cloned CTL (6 × 103
per well) were added and after 4 h of incubation at 37°C the 51Cr
content of supernatants was determined. The specific lysis was calculated as 100 × (experimental
spontaneous release)/(total
spontaneous release). The relative antigenic activities were calculated by dividing the concentrations of IASA-YIPSAEK(ABA)I
required for half-maximal lysis by that required for the variant
peptide derivatives. For sake of comparison the relative antigenic
activities of the compounds were normalized by dividing by the
relative Kd competitor activity, which expresses the ability of a
peptide derivative to bind to Kd (4, 17). In experiments where
Fab
fragments of the anti-Kd
3 mAb SF1-1.1.1 were used, and
in the antagonist assays, the peptide derivatives were covalently
attached to P815 cell-associated Kd (see below).
Assay.
The CTL were incubated with sensitized
target cells under the same conditions as for the cytotoxic assay,
except that peptide derivative concentrations ranged from 10
6-
10
11 M. After 24 h incubation the IFN-
content in supernatants was assessed by ELISA as previously described (18).
350 nm. Preparation of 125I-labeled soluble covalent Kd-peptide
derivative complexes was performed as described (4). The stock
solutions had concentrations of 1-5 × 108 cpm/ml and specific
radioactivities of ~2,000 Ci/mMol.
(20 µg/ml). Aliquots (100-700 µl)
were added to 10-ml volumes of 26°C medium containing 0.7%
FCS and anti-Kd
1 mAb 20-8-4S (10 µg/ml), which prevents
TCR-ligand binding (15), and incubated at 26°C for the indicated periods. After UV irradiation for 20 s with a 500 W UV B
lamp (Dr. Hönle Inc., Munich, Germany, equipped with an infrared and an UV 296-nm filter), TCR were immunoprecipitated
and analyzed by SDS-PAGE. TCR photoaffinity labeling was quantified by phosphor-imaging and normalized relative to the wild-type as described (4). Mean values and standard deviations were
calculated from three to six independent experiments.
was assessed by TCR
photoaffinity labeling. Each experiment was performed in triplicates and repeated at least twice.
(IFN-
) response,
which requires higher peptide concentrations and sustained
TCR signaling for extended periods of time (21). In spite
of these differences both CTL responses were remarkably
similar, except that on S14 CTL for E258A the IFN-
production was more efficient than cytotoxicity (Fig. 1 b). Occasional divergences between cytolytic and IFN-
CTL responses among peptide variants have also been observed in other systems (24).
Fig. 1.
Antigen recognition, IFN- production and TCR-ligand
binding for IASA-YIPSAEK(ABA)I (wt) and variants on cloned CTL.
The normalized relative antigenic activities (cytotoxicity) and TCR-ligand binding at 26°C are shown as open and full bars, respectively (a),
and the IFN-
production as open bars (b). All values were normalized relative to IASA-YIPSAEK(ABA)I. The cases for which the CTL response was
5-fold lower than TCR-ligand binding are shown in red
and those for which it was
5-fold higher in green. TCR antagonists are
shown in purple. The variants tested include IASA-YIASAEK(ABA)I (P255A), IASA-YIPSAAK(ABA)I (E258A), IASA-YISSAEK(ABA)I
(P255S), IASA-YIPAAEK(ABA)I (S256A), IASA-YILSAEK(ABA)I
(P255L), and the CTL clones T1, S14, and S17. Some of the experiments
were performed in the presence of SF1-1.1.1 Fab
. Each experiment was
performed in triplicates and the mean values and standard deviations were calculated from at least two experiments.
[View Larger Versions of these Images (37 + 31K GIF file)]
Fig. 2.
TCR-ligand complex dissociation kinetics on cloned T1,
S17 and S14 CTL. Aliquots of CTL, preincubated in the absence or presence of SF1.-1.1.1 Fab with soluble covalent complexes of Kd and
IASA-YIPSAEK(ABA)I or variants, were diluted into aliquots of DMEM
containing anti-Kd
1 mAb 20-8-4S and UV irradiated after the indicated
periods of incubation. All kinetic experiments were performed at 26°C, except the one shown in d, which was assessed at 37°C. The TCR photoaffinity labeling observed at time 0 was defined as 100%.
1/2 designates
the time required for 50% dissociation.
[View Larger Version of this Image (38K GIF file)]
fragments of the anti-Kd
3 mAb SF1-1.1.1, which prevents participation of CD8 in TCR-ligand binding, but not CD8 dependent adhesion (15). Remarkably, in the presence of this reagent the efficiency of antigen
recognition (cytotoxicity) was often unchanged or even increased (Fig. 1 a). This was also true for IFN-
production;
indeed, in two instances blocking of CD8 substantially increased this response (wt and P255S on S17 CTL) (Fig. 1 b).
However in other cases SF1-1.1.1 Fab
dramatically reduced the cytotoxic or IFN-
response (P255A on T1, wt
and E258A on S14 CTL) (Fig. 1). With the exception of the latter cases, blocking of CD8 reduced TCR-ligand binding more than it decreased the efficiency of antigen recognition, as a result the efficiency of antigen recognition was
increased relative to TCR-ligand binding; i.e., agonists or
weak agonists were converted into strong agonists (Fig. 1).
TCR-ligand complex dissociation was always considerably
faster in the presence of SF1-1.1.1 Fab
than in its absence
(Fig. 2). This is consistent with reports showing that CD8
stabilizes TCR-ligand binding by decreasing dissociation of
TCR-ligand complexes (15, 16). Our finding that acceleration of TCR-ligand complex dissociation increased the relative efficiency of antigen recognition supports the concept
that CTL activation depends on the frequency of serial
TCR engagement. It has been reported that CD8 expression on CTL can be downmodulated by antigen (25, 26),
which is likely to have similar effects as the CD8 blocking
experiments described here.
, the
variants P255A and E258A antagonized the cytotoxic responses of T1 and S14 CTL, respectively. As shown in Fig.
3, this was indeed the case. In the presence of this reagent the recognition of IASA-YIPSAEK(ABA)I by T1 (Fig. 3 a)
and S14 CTL (Fig. 3 b) was significantly reduced on target
cells that expressed covalent Kd-P255A or Kd-S256A complexes, compared to target cells that expressed irrelevant Kd-P255H complexes (4). Consistent with these findings
we observed in both cases predominant pp21
chain phosphorylation in the presence, but not in the absence of SF1-1.1.1 Fab
(data not shown). This form of
chain phosphorylation has been shown to be preferentially or exclusively induced by TCR antagonists (1, 5). Remarkably, in both cases exceedingly fast TCR-ligand complex dissociations were observed (Fig. 2, e and f). The only other
comparably fast dissociation was recorded for variant P255L
on S14 CTL, which is an antagonist for this clone (Fig. 2 b
and reference 4). These observations are in agreement with
the kinetic proofreading concept (8, 9) and a study by Lyons et al. (10) demonstrating that TCR-ligand complex
dissociation is more rapid for antagonists than for agonists.
Taken collectively our findings indicate that acceleration of
TCR-ligand complex dissociation increases the efficiency
of antigen recognition by CTL up to a critical threshold, beyond which TCR engagement results in aberrant TCR
signaling and TCR antagonism.
Fig. 3.
Blocking of CD8 converts agonists P255A and S256A into
antagonists for T1 and S14 CTL, respectively. T1 (a) or S14 CTL (b) were incubated in the presence of IASA-YIPSAEK(ABA)I 109 M in (a),
10
7 M in (b) and SF1-1.1.1 Fab
(20 µg/ml) with 51Cr-labeled P815
cells, expressing Kd molecules crosslinked with variant P255A, 10
9 M (a)
or E258A, 10
8 M (b) or P255H, 10
9 M in a and 10
8 M in b, a derivative not recognized by either TCR (4). The mean values and standard deviations were calculated from triplicate values of a representative experiment.
[View Larger Version of this Image (20K GIF file)]
(Figs.
1 and 2, reference 4 and unpublished data).
versus
chain photoaffinity labeling (Fig. 4 b).
Similar findings were obtained at 0-4°C (unpublished data),
i.e., in the absence of metabolically active cellular processes,
suggesting that epitope modification can induce either conformational changes in TCR or slightly alter the orientation
of TCR-ligand binding, as a result of which CD8 may participate more or less avidly in TCR-ligand binding. This
view is consistent with the observation that anti-TCR mAb
and their Fab
can substantially affect T cell responses (11,
12), and suggests that the conformational changes induced by
such reagents, can affect T cell function by interfering with
TCR-coreceptor cooperation. It is also interesting to note that TCR-ligand binding apparently can induce structural
changes in the
3 domain of MHC class I molecules,
which may alter their interaction with CD8 (27, 28).
Fig. 4.
Epitope modifications alter the contribution of CD8 to
TCR-ligand binding. The degree of CD8 participation in TCR-ligand
binding is expressed as the ratio of TCR-ligand binding in the absence, divided by the binding in the presence of SF1-1.1.1 Fab (a). The surface
expression of TCR and CD8 was assessed by flow cytometry after staining with antibodies H57-597 and H35-17, respectively. The values indicate mean fluorescence intensities for a representative experiment. (b) Different ligand variants exhibit different ratios of TCR-
versus -
chain
TCR photoaffinity labeling on T1 and S17 CTL. An autoradiogram of
SDS-PAGE (10%, reducing conditions) of a representative experiment is
shown.
[View Larger Versions of these Images (29 + 30K GIF file)]
Address correspondence to Immanuel F. Luescher, Ludwig Institute, Lausanne Branch, Ch. des Boveresses 155, 1066 Epalinges, Switzerland. Phone: 41 21 692 5988; Fax: 41 21 653 4474; E-mail: iluesche{at}eliot.unil.ch
Received for publication 7 July 1997 and in revised form 10 September 1997.
Dr. Denis Hudrisier was supported by an "Association Française pour la Recherche Thérapeutique" fellowship.We thank C. Horvath and P. Bassanini for excellent technical assistance, Dr. C. Servis for peptide and conjugate synthesis, Dr. P. Romero for helpful discussions and Anna Zoppi for preparing the manuscript.
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