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*
Ludwig Institute for Cancer Research, Lausanne Branch, and
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production)
correlated with the rate of TCR-ligand complex dissociation, but not
the avidity of TCR-ligand binding. While peptide antagonists exhibited
very rapid TCR-ligand complex dissociation, slightly slower
dissociation was observed for strong agonists. Conversely and
surprisingly, weak agonists typically displayed slower dissociation
than the wild-type agonists. Acceleration of TCR-ligand complex
dissociation by blocking CD8 participation in TCR-ligand binding
increased the efficiency of Ag recognition in cases where dissociation
was slow. In addition, permanent TCR engagement by TCR-ligand
photocross-linking completely abolished sustained intracellular calcium
mobilization, which is required for T cell activation. These results
indicate that the functional CTL response depends on the frequency of
serial TCR engagement, which, in turn, is determined by the rate of
TCR-ligand complex dissociation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Madrenas et al. argued
that premature TCR-ligand complex dissociation will limit the
phosphorylation of CD3/ and CD3-associated ZAP-70 by
p56lck (11). Alternatively, according to the
second concept, epitope modifications provoke conformational changes in
TCR that affect the association of TCR with CD3/ and thus
qualitatively alter TCR signaling (12, 13).
In support of the kinetic proofreading concept, Lyons et al. showed
that antagonist peptides exhibit faster TCR-ligand complex dissociation
than agonists (10). In addition, it has been demonstrated that
antagonists elicit only limited phosphorylation of CD3/, with a
preponderance of pp21-phosphorylated -chain that is unable to bind
ZAP-70, a PTK that plays a crucial role in the induction of the NF-AT
pathway of T cell activation (14, 15, 16). Together these data convincingly
explain the observation that peptide antagonists not only fail to
elicit a functional T cell response, but can inhibit recognition of an
agonist.
However, there exist several observations that seem difficult to explain by the kinetic proofreading concept. For example, it has been reported that cytolytic CTL responses can be antagonized by very low concentrations of altered viral peptides (17, 18). Also, this concept fails to explain how certain altered peptide ligands can induce some, but not other, T cell effector functions. Moreover, we have previously observed that peptide variants that were efficiently recognized by cloned CTL (strong agonists), exhibited faster TCR-ligand complex dissociation than those that were inefficiently recognized (weak agonists) (5). This suggests that when TCR ligation results in integral TCR signaling, the magnitude of the functional CTL response is dependent on the frequency of serial TCR engagement. The serial TCR engagement model of T cell activation proposes that during T cell-APC encounter, ligands on APC are serially engaged by TCR of T cells, which permits sustained TCR signaling, even when the ligand density is low (19, 20, 21). For our study we used CTL clones that permit assessment of TCR-ligand interaction by TCR photoaffinity labeling. These clones were H-2Kd restricted and specific for a derivative of the PbCS peptide252–260 (SYIPSAEKI) containing photoreactive iodo-4-azidosalicylic acid (IASA) in place of PbCS S-252 and 4-azidobenzoic acid (ABA) on PbCS K-259 (4, 5, 22). Selective photoactivation of the IASA group permitted cross-linking to Kd, and photoactivation of the ABA group permitted cross-linking to TCR.
In the present study we examined a larger panel of epitope variants and
CTL clones to verify this hypothesis. In addition, we addressed the
question of how blocking of CD8 affects Ag recognition and TCR-ligand
complex dissociation. CD8 plays various roles in CTL function such as
1) by co-ordinate binding of TCR-bound MHC molecules, CD8 strengthens
TCR-ligand binding, primarily by decreasing TCR-ligand complex
dissociation (23, 24); 2) following TCR/CD3 ligation, CD8 acquires an
increased affinity for MHC class I molecules and can act as an adhesion
molecule (25, 26); and 3) by virtue of the associated
p56lck, CD8 is involved in the phosphorylation
of CD3/ and ZAP-70 (14, 15). Since anti-CD8 mAb can
differentially affect these CD8 functions and hence have diverse
effects on functional CTL responses (23), we used in the present study
Fab' fragments of the anti-Kd
3 mAb SF1-1.1.1, a
reagent that selectively blocks CD8 participation in TCR-ligand
binding, without affecting CD8-dependent adhesion and the CD8-mediated
signaling it involves (25, 26).
We report that the efficiency of Ag recognition (cytotoxicity and
IFN- production) is directly related to the rates of TCR-ligand
complex dissociation, and that these, in turn, are determined largely
by CD8.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Amino acids and other chemicals for peptide and conjugate synthesis were obtained from Bachem (Bubendorf, Switzerland), Sigma (Buchs, Switzerland), and Neosystems (Strasbourg, France). Synthesis of peptides and conjugates, in radioactive and nonradioactive forms, was performed as previously described (4, 5, 22). In brief, all peptides were synthesized on an ABI 431 solid phase peptide synthesizer (Applied Biosystems Instruments, Foster City, CA), using F-moc for transient N-terminal protection. The PbCS peptide derivatives were synthesized using F-moc-Tyr(H2PO3)-OH as second residue, F-moc-Lys(ABA)-OH in position 8, and ASA-ONSu for N-capping. Following reverse phase HPLC purification on a C18 column (1 x 25 cm, 5-µm particle size, Marcherey & Nagel, Oensingen, Switzerland), the conjugates were iodinated with 125I or nonradioactive iodine in the presence of chloramine-T, followed by dephosphorylation with alkaline phosphatase. All peptide derivatives were characterized by mass spectrometry on an LDI 7000 mass spectrometer (Linear Scientific, Reno, CA) and UV spectrometry on an ABI 1000S diode array spectrometer. After lyophilization, nonradioactive peptides and peptide derivatives were reconstituted in PBS at 1 mM. The radioactive conjugates were immediately used for Kd photoaffinity labeling; their specific radioactivity was approximately 2000 Ci/mmol.
Cytolytic assays
Cloned CTL were obtained from BALB/c x C57 BL/6 F1 mice immunized with IASA-YIPSAEK(ABA)I as previously described (22). They were stimulated weekly with irradiated BALB/c splenocytes and irradiated peptide derivative-pulsed P815 cells in the presence of IL-2 (EL4 supernatant). 51Cr-labeled P815 cells were used as targets in a chromium release assay as previously described (4, 5, 22). In brief, sensitized target cells (5 x 103/well) were incubated in microtiter plates for 1 h at 37°C in medium containing 10-fold dilutions of peptides, followed by UV irradiation (see below). Cloned CTL (1.5 x 104/well) were added, and after 4-h incubation at 37°C, the 51Cr content of the supernatants was determined. In some experiments target cells were preincubated with SF1-1.1.1 Fab' (20 µg/ml) for 15 min at 37°C. The specific lysis was calculated as 100 x [(experimental - spontaneous release)/(total - spontaneous release)]. The relative antigenic activities were calculated by dividing the concentration of IASA-YIPSAEK(ABA)I required for half-maximal lysis, by that required for the variant peptide derivatives. The relative Kd competitor activity, expressing the Kd binding of the different peptide derivatives (4, 22), was used to normalize the relative antigenic activities by dividing them by the corresponding relative Kd competitor activities. In all experiments used for calculating relative antigenic activities, the slopes of the plots for specific lysis vs peptide concentration were parallel, as shown, for example, in Ref. 4.
IFN- production
P815 cells (5 x 103/well) were sensitized
with the respective peptide derivatives, as described in the previous
section, and incubated with cloned CTL (1.5 x
104/well) for 24 h at 37°C. The IFN- content of
the supernatants was determined by ELISA, using for absorption
anti-IFN- mAb R4–6A2 and for detection biotinylated
anti-IFN- mAb AN18. To detect the biotinylated Ab, horseradish
peroxidase-conjugated streptavidin (Amersham, Arlington Heights, IL)
was used, followed by incubation with o-phenylenediamine
hydrochloride (Sigma). The color was measured at 490 nm using an ELISA
reader (MR7000, Dynatech, Chantilly, VA). The relative antigenic
activities were calculated and normalized as described in the previous
section. For calculation of relative antigenic activities, only
experiments in which the slopes of the titrated functions were parallel
were used.
Kd and TCR photoaffinity labeling
All photoaffinity labeling procedures were performed as
previously described (4, 5, 22). In brief, for Kd
photoaffinity labeling, P815 cells resuspended in DMEM supplemented
with FCS (0.5%) and HEPES (10 mM) were incubated with peptide
derivative at 37°C for 2 to 3 h, followed by UV irradiation at
350 nm with a 3000-watt SupUVA Sun UV irradiator (Mutzhas, Munich,
Germany). After washing (three times) the cells were incubated at
37°C for 1 h with 51Cr and used in a cytolytic
assays as previously described (5, 22). Alternatively,
125I-labeled peptide derivatives were incubated with
soluble Kd (50 µg) in PBS (200 µl) and
ß2m (2.5 µg/ml; Sigma) for 1 to 2 h at room
temperature, followed by UV irradiation at 350 nm. The samples were
diluted with PBS (300 µl) containing PbCS peptide252–260
(10 µM) and after 12 h of incubation at 20 to 25°C were subjected
to gel filtration fast protein liquid chromatography. The purified
soluble Kd-peptide derivative complexes had concentrations
of 0.7 to 2.5 x 109 cpm/ml and were used within 1 mo.
For TCR photoaffinity labeling, cloned CTL were resuspended in DMEM
supplemented with FCS (1%) and HEPES (10 mM; 6–12 x
106 cells/ml) and incubated with 125I-labeled
Kd-peptide derivative complexes (0.6–4 x
107 cpm/ml) at 26°C for 1 to 1.5 h. After UV
irradiation for 20 s with a 500-watt UV B irradiator (Dr.
Hönle, Inc., Munich, Germany), equipped with a shutter as well as
an infrared and UV 296-nm filter, the cells were detergent lysed and
TCR immunoprecipitated with anti-TCR Cß mAb H57-597. The
immunoprecipitates were analyzed by SDS-PAGE (10%, reducing
conditions), and the covalent TCR-ligand complexes were quantified with
a SF PhosphorImager and the ImageQuant software (Molecular Dynamics,
Sunnyvale, CA). SDs were calculated using Student’s t test
from at least three different experiments. TCR photoaffinity labeling
was normalized by dividing the labeling intensity of the variant ligand
by that of the wild-type ligand.
Kinetics of TCR-ligand interactions
Kinetics of TCR-ligand complex dissociation were determined by
TCR photoaffinity labeling as previously described (5, 23). In brief,
cloned CTL (6–20 x 106 cells/ml) were
preincubated with soluble
Kd-125IASA-YIPSAEK(ABA)I or variants (1–6
x 107 cpm/ml) in the presence or the absence of SF1-1.1.1
Fab' (20 µg/ml) for 2 to 3 h at 0 to 4°C. At time zero
aliquots were diluted into 10-ml aliquots of DMEM containing
anti-Kd1 mAb 20-8-4S (10 µg/ml). After the
indicated periods of incubation at 26°C, the samples were UV B
irradiated, and TCR photoaffinity labeling was assessed as described in
the previous section. Mean values and SDs were calculated from three to
six independent experiments. The kinetics of intercellular TCR-ligand
binding were determined similarly as described previously (27).
Briefly, aliquots of P815 cells (5 x 106), previously
Kd photoaffinity labeled with
125IASA-YIPSAEK(ABA)I, were mixed at 0 to 4°C with S4 CTL
at an E:T cell ratio of 1:3, centrifuged for 1 min at 1500 x
g, and incubated at 37°C for the indicated periods. After
UV B irradiation, TCR photoaffinity labeling was assessed as described
above. Mean values and SDs were calculated from two to six
experiments.
CD3/-chain phosphorylation
For -chain phosphorylation, P815 (3 x
106 cells/incubation), previously Kd
photoaffinity labeled with the indicated peptide derivatives, were
mixed in the presence or the absence of SF1-1.1.1 Fab' (20 µg/ml)
with cloned CTL (1 x 107 cells/incubation) in DMEM,
supplemented with 5% FCS, centrifuged at 1500 x g for
1 min, and incubated for 8 to 10 min at 37°C. After centrifugation
(20 s at 14,000 x g), cells were lysed in 0.5
ml of cold lysis buffer (1% Triton X-100, 50 mM Tris (pH 7.5), 150 mM
NaCl, 10 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin, and 10 mM
Na3VO4). Detergent-insoluble materials were
removed by centrifugation (15 min at 14,000 x
g) and membrane filtration and subjected to
immunoprecipitation using anti--chain mAb H146.
Immunoprecipitates were washed (three times) in lysis buffer, boiled
for 3 min in reducing SDS-PAGE sample buffer, resolved on 15%
SDS-polyacrylamide gels, transferred on polyvinylidene difluoride
membranes (New England Nuclear, Boston, MA), and immunoblotted with
anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid,
NY). The blots were developed by the enhanced chemiluminescence
technique (Amersham). Each experiment was performed two to five times.
To take into account the variations among experiments, the films of all
experiments were evaluated by densitometry on a Hirschmann Elscript
densitometer (Hirschmann Gerätebau, Unterhachingen, Germany). For
each experiment and each case, the pp23/pp21 ratio was calculated from
pp23 and pp21 densitometer readings and used to calculate the mean and
SD. Alternatively, the pp23 and pp21 densitometer readings were
combined, and the ratio between -/+ SF1–1.1.1 Fab' values was
determined.
Calcium mobilization
To assess intracellular calcium ([Ca2+]i) mobilization, S4 CTL were incubated with indo-1/AM (Sigma; 2 µM, 1 x 106 cells/ml) at 37°C for 45 min. After washing (three times), CTL were resuspended at 1 x 106 cells/ml and used within 2 h. P815 cells, either untreated or previously Kd photoaffinity labeled with IASA-YIPSAEK(ABA)I, were mixed with S4 CTL at an E:T cell ratio of 1:3 in 5-ml polypropylene tubes (Falcon, Oxnard, CA), sedimented by centrifugation (1 min at 1500 x g), and incubated in a water bath at 37°C for 1 min. The calcium-dependent fluorescence of indo-1 in T cells was assessed by flow cytometry on a FACStar cytofluorometer (Becton Dickinson, Mountain View, CA) by gating on a forward light scattering corresponding to conjugates. The total time of recording was 15 min. In blocking experiments, intracellular calcium was measured for 3 min before UV irradiation at 312 ± 40 nm with a 90-watt UV lamp (BioBlock, Illkirch, France) and then measured for the remaining time. The results presented are representatives of eight experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To compare the efficiency of Ag recognition with TCR-ligand
binding, we tested seven variants of the PbCS peptide derivative
IASA-YIPSAEK(ABA)I on the CTL clones S4, S14, S17, and T1 (Fig. 1
). Three of these variants contained
alanine in place of PbCS P255, S256, and E258, respectively, and four
others contained K, L, N, or S in place of PbCS P255. The antigenic
activities were assessed in a chromium release assay, and TCR-ligand
binding was determined by TCR photoaffinity labeling at 26°C; both
were normalized relative to values for the wild-type peptide derivative
(wt). In 10 cases the normalized antigenic activity and TCR
photoaffinity labeling deviated by fivefold or more. Four of these
cases were weak agonists, i.e., Ag recognition was less efficient than
TCR-ligand binding, and four were strong agonists, i.e., recognition
was more efficient than TCR-ligand binding. While weak agonists were
observed on all four CTL clones, strong agonists were limited to S17
and T1 CTL. In only one case was TCR-ligand binding observed in the
absence of detectable cytotoxicity (P255L on S14 CTL). We have shown
previously that this variant is an antagonist for S14 CTL (4). It is
noteworthy that in some instances the efficiency of Ag recognition
differed depending on whether the peptide derivative was cross-linked
to Kd (Fig. 1) or not (4, 5) (e.g., variant E258A on
S4 CTL or variants P255L and P255N on S17 CTL).
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, it emerges that the efficiency of Ag recognition did not
correlate with TCR-ligand binding. We therefore examined whether it was
related to the rates of TCR-ligand complex dissociation. As shown in
Figure 2, for strong agonists, TCR-ligand
complex dissociation was more rapid than for the wild-type peptide
derivative (e.g., variants S256A, P255N, and P255L on S17 CTL).
Conversely, for all the weak agonists, TCR-ligand complex dissociation
was slower than for the wt. An exception to this rule was variant
P255L, for which TCR-ligand complex dissociation on T1 was remarkably
slow, but Ag recognition was only slightly less efficient than
TCR-ligand binding (Figs. 1 and 2). By contrast, on S14 CTL this
variant was an antagonist and exhibited very rapid TCR-ligand complex
dissociation, with a half-life of only approximately 15 s. It is
noteworthy that all dissociation kinetics in this study were performed
at 26°C. This reduced temperature was used to increase the accuracy
of measurement, as the dissociation increases significantly with the
temperature (5, 23).
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response, which requires higher peptide
concentrations and sustained TCR signaling for extended periods of time
(29). As shown in Figure 3, IFN-
production showed essentially the same patterns as the cytotoxic
response (Fig. 1). In only a few instances were deviations of up to
threefold observed (e.g., the IFN- production was higher than
cytotoxicity on S17 CTL for variant P255S, whereas the opposite was
true on S14 for variant S256A). Occasional differences between these
two CTL responses have been observed in other systems (30). Thus,
despite the different activation requirements, the two CTL responses
were generally similar.
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Taken collectively these results suggest that the magnitude of the
functional CTL response was determined by the rates of TCR-ligand
complex dissociation. To verify this conclusion we repeated these
experiments in the presence of SF1-1.1.1 Fab', which, by blocking
participation of CD8 in TCR-ligand binding, accelerates TCR-ligand
complex dissociation (22, 23, 27). Blocking of CD8 in several
instances, especially on S17 and T1 CTL, either had no significant
effect on Ag recognition or even increased it (e.g., wt, P255A, and
P255S on S17 or P255L on T1 CTL; Figs. 1 and 3). Since blocking of CD8
always considerably decreased TCR-ligand binding, the efficiency of Ag
recognition was significantly increased relative to that of TCR-ligand
binding. Thus, agonist or weak agonists were converted to strong
agonists (e.g., wt, P255A, S256A, and P255S on S17; P255L and S256A on
T1; and E258A on S4 CTL).
In contrast, there were nine cases, mainly on S14 and S4 CTL, where
blocking of CD8 dramatically impaired or abolished Ag recognition. For
two of these cases (P255A on T1 and S256A on S4) we have previously
shown that blocking of CD8 resulted in antagonism (5). Essentially the
same findings were obtained for the IFN- response; actually in some
instances blocking of CD8 increased cytokine production more than
cytotoxicity (e.g., wt and P255S on S17 CTL; Figs. 1 and 3). Also, in
all cases where blocking of CD8 abolished cytotoxicity, IFN-
production was undetectable as well, except for variant P255A, which
elicited a weak IFN- response.
In the presence of SF1-1.1.1 Fab', TCR-ligand complex dissociation was
considerably faster than in its absence (Table I). For all cases where blocking of CD8
abolished detectable Ag recognition, TCR-ligand complex dissociation
was very rapid, with half-lives in the range of 10 to 23 s. The
only comparably rapid dissociation was observed on S14 CTL for variant
P255L, which is an antagonist for this clone (Fig. 2). In contrast,
when TCR-ligand complex dissociation was slower than this
(t1/2 = >23 s), Ag recognition was
typically most efficient (e.g., S17 CTL clone, on which dissociations
were slower than on the other clones). Together these findings support
the conclusion that the efficiency of Ag recognition increases with the
rate of TCR-ligand complex dissociation up to a critical value, beyond
which TCR antagonism prevails.
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While blocking of CD8 contribution to TCR-ligand binding by
SF1-1.1.1 Fab' always reduced TCR-ligand binding, the degree of
reduction varied among CTL clones as well as among epitope variants on
given clones. As shown in Figure 4, the
ratio of TCR photoaffinity labeling in the absence divided by the
labeling in the presence of SF1-1.1.1 Fab' for the wild-type ligand was
highest for S17 CTL (13.6-fold), lowest for T1 CTL (5.6-fold),
and intermediate for the S4 and S14 CTL clones (11.7- and 8.6-fold,
respectively). While these differences are explained at least in part
by clone-specific differences in CD8 and TCR expression (5), the
differences observed on given clones among peptide derivative variants
indicate that epitope modifications can change the CD8 contribution to
TCR ligand binding. For example, on S17 CTL, blocking of CD8 TCR-ligand
binding was decreased by 25-fold in variant P255L, but only about
6-fold in variants P255A and P255S.
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-chain phosphorylation, Ag recognition,
and TCR-ligand complex dissociation
Among the earliest TCR signaling events are PTK-mediated
phosphorylations of CD3/ (14, 15). We therefore examined the
phosphorylation of -chain, namely the pp21 and pp23 phospho forms,
that are induced by TCR engagement. While the pp21 form is
predominantly elicited by peptide antagonists, the pp23 form is induced
by agonists and can bind ZAP-70 (6, 7, 14). As shown in Figure 5, the intensity and ratio of pp21 vs
pp23 -chain phosphorylation varied considerably among the different
cases.
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-chain phosphorylation. In the
presence of SF1-1.1.1 Fab' this phosphorylation increased, which is
consistent with the finding that blocking of CD8 increased the Ag
recognition (Figs. 1 and 3). This was also observed for the peptide
derivatives variants P255A, P255S, and S256A (Fig. 5 and data not
shown). Moreover, variant P255S elicited a weaker and variant S256A
elicited a more extensive -chain phosphorylation than the wt, which
is in accordance with the finding that the former derivative was a weak
and the latter a strong agonist on S17 CTL (Figs. 1 and 3).
On T1 CTL, the wt peptide derivative and variants P255A and S256A
induced similar -chain phosphorylation in terms of both overall
intensity and pp21 to pp23 ratio (Fig. 5). By contrast, the weak
agonist P255S elicited less efficient -chain phosphorylation, mainly
of the pp21 form. In the presence of SF1-1.1.1 Fab' -chain
phosphorylation increased slightly in variant S256A and remained
unchanged for the wt, but decreased significantly, mainly the pp23
form, in variants P255A and P255S. The latter -chain phosphorylation
pattern is typical for peptide antagonists, which is consistent with
the finding that upon blocking of CD8, the variants P255A and P255S
were antagonists for T1 CTL (5) (data not shown).
On S4 CTL, the -chain phosphorylation induced by the wt ligand
decreased slightly upon blocking of CD8, especially the pp23 form (Fig. 5). For variant S256A this decrease was more pronounced, which is in
accordance with the finding that blocking of CD8 abolished recognition
of this agonist (Figs. 1 and 3). This was also true for variant P255K.
On S14 CTL extensive -chain phosphorylation, especially of the pp23
form, was observed only in the wt peptide derivative and variants P255A
and S256A, compounds that were efficiently recognized (Figs. 1, 3, and 5). For the weak agonist E258A, little -chain phosphorylation,
mainly of the pp21 form, was observed. Upon blocking of CD8, -chain
phosphorylation, namely of the pp23 form, was significantly reduced in
all cases, except for variant P255A. The remaining weak -chain
phosphorylation, consisting mainly of the pp21 form, is reminiscent of
peptide antagonist, which is consistent with the lack of detectable Ag
recognition in these instances (Figs. 1 and 3).
It is noteworthy that on S17 CTL the pp23/pp21 ratio for the wt was
lower than that on the other clones. This is likely to be accounted for
by slower kinetics of pp23 accumulation on S17 CTL due to remarkably
slow TCR-ligand complex dissociation on this clone (Fig. 2).
Permanent TCR engagement abrogates TCR signaling
Based on the observation that weak agonist typically exhibited
slow TCR-ligand complex dissociation and elicited inefficient -chain
phosphorylation, we next examined what effect permanent TCR engagement
has on CTL activation. In our system this can be accomplished by
photocross-linking of intercellular TCR-ligand complexes. To this end,
indo-1-labeled S4 CTL were incubated with P815 cells expressing
covalent Kd-IASA-YIPSAEK(ABA)I complexes and
[Ca2+]i was monitored in the presence or the
absence of TCR-ligand photocross-linking. S4 CTL exhibited significant
calcium-dependent fluorescence upon incubation with P815 cells that
were sensitized with 0.3 or 30 nM of IASA-YIPSAEK(ABA)I (Fig. 6, B and D).
This calcium mobilization was Ag specific, since it was not observed
upon incubation with untreated P815 cells (Fig. 6A).
Importantly, following UV irradiation,
[Ca2+]i levels rapidly dropped to background
levels regardless of whether the target cells expressed a high or a low
ligand density (Fig. 6, C and E). This
effect was not due to UV irradiation damage of the CTL, since S4 CTL
that were UV irradiated before incubation with sensitized targets
exhibited the same calcium mobilization as untreated CTL (Fig. 6F). On the other hand, UV irradiation of sensitized
P815 cells before incubation with S4 CTL abolished significant calcium
mobilization (G). This is consistent with the finding that the ABA
group of IASA-YIPSAEK(ABA)I constitutes an essential part of the
epitope recognized by S4 and related CTL (22, 27) and demonstrates that
photolysis of this group destroys nominal ligand for S4 TCR.
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A, the TCR photoaffinity
labeling reached a maximum after approximately 4 min. After longer
incubations, TCR-ligand binding decreased gradually by about 20% after
20 min. This decrease was prevented by TCR photocross-linking after 4
min of incubation. These results indicate that in the previous
experiment (Fig. 6) the cells that were subjected to TCR
photocross-linking had an average TCR engagement that was equal to or
higher than that in the experiment without TCR-ligand
cross-linking.
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1 mAb 20-8-4S, which blocks
specific TCR-ligand binding (27), was subtracted. The amount of
cross-linked TCR was determined in the same way, except that the
incubations were followed by UV irradiation and incubation with 20-8-4S
mAb at 37°C. By dividing cross-linked by bound ligand, a value of
0.45 was obtained. Thus, for S4 TCR approximately 45% of the bound
ligand became cross-linked upon UV irradiation, indicating that
intercellular TCR-ligand photocross-linking can diminish TCR engagement
by up to 2.2-fold.
In the experiment shown in Figure 6, the same calcium
mobilization in S4 CTL was observed when the target cells were
sensitized with 0.3 or 30 nM of IASA-YIPSAEK(ABA)I. To assess TCR
engagement under these conditions, we used again TCR photoaffinity
labeling with P815 cells expressing radiolabeled
Kd-IASA-YIPSAEK(ABA)I complexes. As shown in Figure 7C, TCR photoaffinity labeling increased with the ligand
density on the target cells. If one defines the intercellular TCR
photoaffinity labeling observed with 30 nM
125IASA-YIPSAEK(ABA)I as 100%, the labeling observed with
target cells pulsed with 0.3 nM was approximately 11%. Thus, by
increasing the peptide concentration by 100-fold, TCR engagement
increased by 9-fold. Together these results indicate that the drop in
[Ca2+]i shown in Figure 6, C and
E, is not accounted for by inefficient TCR-ligand
cross-linking.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production), TCR-ligand binding, and TCR-ligand complex dissociation
kinetics of seven peptide derivative variants on four CTL clones
indicated that the magnitude of the functional CTL response is related
to the rate of TCR-ligand complex dissociation and not the
avidity of TCR-ligand binding (
Figs. 1–3). This relation was
bimodal in nature. While the efficiency of Ag recognition was maximal
when TCR-ligand complex dissociation was rapid, it decreased when
dissociation either further increased and, surprisingly, also when it
decreased. The former observation is in accordance with reports showing
that peptide antagonists exhibit faster TCR-ligand complex dissociation
than agonists (10). For some of these cases we have previously shown
that peptide derivative variants can inhibit the recognition of
wild-type peptide derivative in situations where CD8 was blocked or not
(4, 5). Moreover, in all cases we have examined in which Ag recognition
was undetectable or extremely inefficient, faint -chain
phosphorylation, mainly of the pp21 form, was observed (Fig. 5). This
pattern of -chain phosphorylation is a hallmark of peptide
antagonists (6, 7, 11, 16). These findings are in accordance with the
kinetic proofreading concept, according to which too short TCR
engagement results in incomplete TCR signaling and no or very limited
activation of T cell effector functions (9, 10, 31). It is interesting
to note that for all the cases we have examined, antagonism was
observed when the half-lives of the TCR-ligand complexes were in the
range of 9 to 23 s. Since this was true for different CTL clones
and ligand variants as well as in situations in which CD8 was blocked
or not, it appears that there exists a critical threshold below which
TCR engagement results in aberrant TCR signaling and antagonism.
Moreover, our results revealed that different CTL clones have different
tendencies for TCR antagonism. When testing a larger panel of peptide
derivative variants, antagonists were observed only for S14 CTL (4).
Similarly, acceleration of TCR-ligand complex dissociation by blocking
of CD8 participation in TCR-ligand binding on S14 CTL converted
agonists into antagonists in all but one case (Figs. 1 and 3).
Conversely, on the S17 CTL clone, antagonism was never observed, and
blocking of CD8 often increased the efficiency of Ag recognition (Figs. 1 and 3). From kinetic experiments we know that TCR-ligand complex
dissociation on S14 CTL typically was considerably faster than that on
S17 CTL (Fig. 2). It thus appears that the clone-specific
susceptibilities for antagonism are related to clone-specific
differences in TCR-ligand complex dissociation rates.
A main finding of the present study is that peptide derivative variants
that were recognized less efficiently than expected from their
TCR-ligand binding (weak agonists) exhibited slower TCR-ligand complex
dissociation than those that were recognized efficiently (strong
agonists). Consistent with this, we observed that weak agonists induced
less efficient -chain phosphorylation and typically less of the pp23
form than strong agonists (Figs. 1, 3, and 5). The finding that brief
TCR engagement resulted in more efficient TCR signaling than prolonged
engagement is not consistent with the kinetic proofreading concept
(8, 9, 10). Instead, this observation is in agreement with the concept of
serial TCR engagement, according to which TCR sequentially engage
ligand on APC (19, 20, 21). Indeed, one expects that when TCR-ligand
complex dissociation is rapid, the rate of serial TCR engagement will
be higher than when dissociation is slow. Our data are consistent with
the view that in the absence of antagonism, the magnitude of the
functional T cell response is directly related to the frequency of
serial TCR engagement.
This conclusion is supported by the finding that blocking of CD8 can
increase the efficiency of Ag recognition (
Figs. 1–3). While blocking
of coreceptor participation in TCR-ligand binding always accelerated
TCR-ligand complex dissociation, it had different effects on Ag
recognition. If dissociation became too rapid, blocking of CD8 can
convert agonists and weak agonists into antagonists. This is consistent
with studies showing that blocking of CD4 can convert agonists and weak
agonists into antagonists on Th cells (11, 32). In the study by
Madrenas et al. blocking of CD4 has been shown to result in the same
pattern of CD3/ phosphorylation as elicited by peptide antagonist,
i.e., a decrease in phosphorylation and a preponderance of pp21
phospho- (11). While most of our data are in accordance with this,
others suggest more complex relations in some cases. For example, for
variants P255A and P255S on T1 CTL, blocking of CD8 dramatically
reduced Ag recognition, but only moderately reduced the pp23/pp21 ratio
and the overall intensity of phospho- (Figs. 1 and 5). Also, on S17
CTL, in cases where blocking of CD8 augmented the efficiency of Ag
recognition, the overall -chain phosphorylation was increased rather
than the pp23/pp21 ratio. These patterns suggest that CD8 may influence
TCR signaling in a quantitative as well as qualitative manner (see
below). On the other hand, if TCR-ligand complex dissociation remained
above a critical limit, blocking of CD8 enhanced the functional CTL
response, either absolutely or relative to TCR-ligand binding (
Figs. 1–3). Since in our experiments, artifacts that anti-CD8 mAb may
cause are ruled out, this observation argues in favor of the concept of
serial TCR engagement.
Another observation that supports this conclusion is that blocking of
serial TCR engagement by photocross-linking of TCR-ligand complexes
rapidly abolished sustained intracellular calcium mobilization in S4
CTL, an obligatory early event of T cell activation (14, 31) (Fig. 6).
Consistent with this, we observed that irreversible TCR engagement also
abrogated Ag-specific IFN- production by S4 CTL and provoked barely
detectable -chain phosphorylation (unpublished observations).
Together, these findings strongly suggest that serial or sequential TCR
engagement (our data do not distinguish whether single ligands serially
engage many TCR or whether single TCR sequentially engage different
ligands) is a requirement for TCR signaling and that its frequency
determines the magnitude of CTL activation.
It has been recently shown that incubation of TCR and ligand at concentrations close to the equilibrium constant leads to the formation of TCR-ligand aggregates, suggesting that TCR aggregation may be critical for T cell activation, as has been observed for other receptor systems (33). Our finding that permanent TCR engagement abrogates TCR signaling does not rule out this possibility, but stipulates that TCR aggregation would be a dynamic event, taking place in the course of serial TCR engagement.
In terms of TCR signaling it is not clear how permanent TCR engagement
abolishes CTL activation. In view of reports showing that
hyperphosphorylation of ZAP-70 results in binding of the phosphatase
SHP-1 to ZAP-70, which then dephosphorylates ZAP-70 (34, 35), it is
tempting to speculate that permanent TCR ligation activates a
phosphatase-mediated extinction of TCR signaling. One would indeed
expect that upon permanent TCR ligation, CD8-associated
p56lck remains in the vicinity of CD3/ and
CD3/-associated ZAP-70 and hence hyperphosphorylates these molecules
(11). Since weak agonists typically exhibit slow TCR-ligand complex
dissociation, they may partially activate the same inhibitory
mechanism. This would explain the inefficient CD3/ phosphorylation
observed for weak agonist (Fig. 5).
A surprising finding was that blocking of CD8 reduced the avidity of
TCR-ligand binding to different degrees (Fig. 4). While such variations
among different CTL clones are explained at least in part by
clone-specific differences in CD8 and TCR expression (5), those
observed among epitope variants on given clones strongly suggest that
epitope modifications can alter the CD8 contribution to TCR-ligand
binding. Since similar changes were also observed at 0 to 4°C, i.e.,
in the absence of metabolically active cellular processes (our
unpublished observations), it appears that epitope modifications may
induce either conformational changes in TCR or ligand or slightly alter
the orientation in which they interact, as a result of which CD8 can
more or less avidly participate in TCR-ligand binding. This view is
consistent with the observation that epitope modifications can provoke
changes in the ratio of TCR - vs ß-chain photoaffinity
labeling (5).
The finding that epitope modifications can alter the participation of
CD8 in TCR-ligand binding is likely to have implications for Ag
recognition by CTL. For example, it will influence the rates of
TCR-ligand complex dissociation. Since these, in turn, determine the
efficiency of Ag recognition (see above), this implies that epitope
modifications can affect functional CTL responses by altering the
avidity of CD8 contribution to TCR-ligand binding. This differential
involvement of CD8 in TCR-ligand binding probably also explains at
least in part why the functional phenotype of epitope variants cannot
be predicted from the avidity of TCR-ligand binding (Figs. 1 and 3).
Moreover, since CD8 brings p56lck to CD3/, it
is conceivable that topologic changes in the docking of CD8 to
TCR-ligand complex alters the relative extents by which
p56lck phosphorylates the different ITAM of
CD3/. Since ITAM are not identical in terms of initiating downstream
signaling cascades (36, 37, 38), this may explain how epitope modifications
can result in selective activation of CTL functions, such as activation
of Fas, but not perforin-dependent cytotoxicity or differentially
activate IFN- production and cytotoxicity (3, 30, 38) (Figs. 1 and 3). It is interesting to note that blocking of CD8 generally had more
dramatic effects on Ag recognition and -chain phosphorylation on S14
and S4 CTL than on S17 and T1 CTL (Figs. 1 and 5 and our unpublished
data). Interestingly, the latter CTL clones are CD8 independent (i.e.,
their cytolytic responses are not affected or are little affected by
anti-CD8 mAb), whereas the former are CD8 dependent (5). It thus is
conceivable that for CD8-independent clones CD8-associated
p56lck is less critical for cell activation than
it is for CD8-dependent clones.
Our preferred interpretation of the results presented in this study is
schematically shown in Figure 8. The main
points we would like to make are 1) that serial TCR engagement is a
prerequisite for CTL activation; 2) that the magnitude of the
functional CTL response is dependent on the frequency of serial TCR
engagement; and 3) that its frequency is essentially determined by the
rate of TCR-ligand complex dissociation. Although this scheme explains
most of our data, it is not meant to be a unifying concept for T cell
activation; rather, it suggests that aberrant T cell function is
probably best explained by a combination of several principles and that
the coreceptor plays an important role.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. I. F. Luescher, Ludwig Institute for Cancer Research, Ch. des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address:
3 Abbreviations used in this paper: PTK, protein tyrosine kinase; IASA, iodo-4-azido-salicyclic acid; ABA, 4-azidobenzoic acid; [Ca2+]i, intracellular calcium; wt, wild type; ITAM, immunoreceptor tyrosine-based activation motif.
Received for publication December 26, 1997. Accepted for publication March 9, 1998.
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stands for a change in, i.e., a change in
TCR-ligand complex dissociation.