The Efficiency of Antigen Recognition by CD8⁺ CTL Clones Is Determined by the Frequency of Serial TCR Engagement

Peptide and peptide derivative synthesis

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 × 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 K^d photoaffinity labeling; their specific radioactivity was approximately 2000 Ci/mmol.

Cytolytic assays

Cloned CTL were obtained from BALB/c × 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 × 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 × 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 × [(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 K^d competitor activity, expressing the K^d binding of the different peptide derivatives (4, 22), was used to normalize the relative antigenic activities by dividing them by the corresponding relative K^d 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-γ production

P815 cells (5 × 10³/well) were sensitized with the respective peptide derivatives, as described in the previous section, and incubated with cloned CTL (1.5 × 10⁴/well) for 24 h at 37°C. The IFN-γ content of the supernatants was determined by ELISA, using for absorption anti-IFN-γ mAb R4–6A2 and for detection biotinylated anti-IFN-γ 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.

K^d and TCR photoaffinity labeling

All photoaffinity labeling procedures were performed as previously described (4, 5, 22). In brief, for K^d 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 K^d (50 μg) in PBS (200 μl) and β₂m (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 peptide_252–260 (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 K^d-peptide derivative complexes had concentrations of 0.7 to 2.5 × 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 × 10⁶ cells/ml) and incubated with ¹²⁵I-labeled K^d-peptide derivative complexes (0.6–4 × 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 Cβ 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 × 10⁶ cells/ml) were preincubated with soluble K^d-¹²⁵IASA-YIPSAEK(ABA)I or variants (1–6 × 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-K^dα1 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 × 10⁶), previously K^d 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 × 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/ζ-chain phosphorylation

For ζ-chain phosphorylation, P815 (3 × 10⁶ cells/incubation), previously K^d 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 × 10⁷ cells/incubation) in DMEM, supplemented with 5% FCS, centrifuged at 1500 × g for 1 min, and incubated for 8 to 10 min at 37°C. After centrifugation (20 s at 14,000 × 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 × g) and membrane filtration and subjected to immunoprecipitation using anti-ζ-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 × 10⁶ cells/ml) at 37°C for 45 min. After washing (three times), CTL were resuspended at 1 × 10⁶ cells/ml and used within 2 h. P815 cells, either untreated or previously K^d 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 × 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.

Strong agonists exhibit fast and weak agonists slow TCR-ligand complex dissociation

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 K^d (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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FIGURE 1.

Cytotoxicity and TCR photoaffinity labeling (26°C) on S17, T1, S4, and S14 CTL in the absence or the presence of SF1-1.1.1 Fab′ (20 μg/ml). The antigenic activities (open bars) and TCR photoaffinity labeling (full bars) were both normalized relative to the wt peptide derivative IASA-YIPSAEK(ABA)I. For strong agonists, shown in vertically striped bars, Ag recognition was fivefold or more higher than TCR photoaffinity labeling, and for weak agonists, shown in gray-shaded bars, the opposite was true. Shown in diagonally striped bars are cases where TCR photoaffinity labeling, but not Ag recognition, was detectable (antagonists). The mean values and SDs were calculated from at least two independent experiments.

From Fig. 1⇑, 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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FIGURE 2.

TCR-ligand complex dissociation. Aliquots of cloned CTL, preincubated with soluble K^d-peptide derivative complexes, were diluted in medium containing anti-K^dα1 mAb 20-8-4S and UV irradiated after the indicated periods of incubation at 26°C. The TCR were immunoprecipitated from detergent lysates, analyzed by SDS-PAGE (10%, reducing) and quantified by phosphorimaging. TCR photoaffinity labeling at time zero was referred to as 100%. The kinetics for strong agonists are shown in bold, those for weak agonists in dotted lines, and those for antagonists in broken lines. The half-life expresses the time required for 50% dissociation. The mean values and SDs were calculated for three to five independent experiments.

Since cytolytic CTL responses are rapid and can occur at very low peptide concentrations (28), we assessed for some of the cases the corresponding IFN-γ response, which requires higher peptide concentrations and sustained TCR signaling for extended periods of time (29). As shown in Figure 3⇓, IFN-γ 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-γ 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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FIGURE 3.

Comparison between cytotoxicity and IFN-γ production. Relative antigenic activities (open bars) and IFN-γ production (full bars), both normalized relative to IASA-YIPSAEK(ABA)I, are shown. The values for the cytotoxicity were taken from Figure 1⇑, and the IFN-γ values were calculated from two to four independent experiments, as described for Figure 1⇑.

Blocking of CD8 increased or abolished functional CTL response

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-γ 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-γ production was undetectable as well, except for variant P255A, which elicited a weak IFN-γ 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 (t_1/2 = >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.

Epitope modifications can alter CD8 participation in TCR-ligand binding

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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FIGURE 4.

Epitope modifications affect CD8 participation in TCR-ligand binding. The normalized TCR photoaffinity values in the absence were divided by those in the presence of SF1-1.1.1 Fab′. The values were taken from Figure 1⇑.

Correlation between CD3/ζ-chain phosphorylation, Ag recognition, and TCR-ligand complex dissociation

Among the earliest TCR signaling events are PTK-mediated phosphorylations of CD3/ζ (14, 15). We therefore examined the phosphorylation of ζ-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 ζ-chain phosphorylation varied considerably among the different cases.

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FIGURE 5.

ζ-Chain phosphorylation on S17, T1, S4, and S14 CTL. Cloned CTL were incubated in the absence or the presence of SF1-1.1.1 Fab′ (20 μg/ml) with P815 that were K^d photoaffinity labeled with the indicated peptide derivative using a 1-μM concentration. After detergent lysis, immunoprecipitated TCR/CD3/ζ were subjected to SDS-PAGE and Western blotting using for detection anti-phosphor-tyrosine mAb. The gels show one individual experiment, and the mean values and SDs indicated represent the averages of two to five different experiments. pp21/pp23 indicates the ratio of pp23 to pp21 phospho-ζ, and ζ −/ζ + indicates the ratio of the total phospho-ζ (pp21 and pp23) in the absence to the total phospho-ζ in the presence of SF1-1.1.1 Fab′.

Incubation of S17 CTL with P815 cells expressing covalent K^d-IASA-YIPSAEK(ABA)I complexes resulted in the formation of pp21 and less pp23 ζ-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 ζ-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 ζ-chain phosphorylation in terms of both overall intensity and pp21 to pp23 ratio (Fig. 5⇑). By contrast, the weak agonist P255S elicited less efficient ζ-chain phosphorylation, mainly of the pp21 form. In the presence of SF1-1.1.1 Fab′ ζ-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 ζ-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 ζ-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 ζ-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 ζ-chain phosphorylation, mainly of the pp21 form, was observed. Upon blocking of CD8, ζ-chain phosphorylation, namely of the pp23 form, was significantly reduced in all cases, except for variant P255A. The remaining weak ζ-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 ζ-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 K^d-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. 6⇓A). 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. 6⇓F). 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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FIGURE 6.

Permanent TCR engagement abolishes calcium mobilization on S4 CTL. Cloned S4 CTL, previously labeled with indo-1, were incubated with P815 cells that were either untreated (A) or K^d photoaffinity labeled with IASA-YIPSAEK(ABA)I using 0.3-nM (B and C) or 30-nM (D, E, G, and H) concentrations at an E:T cell ratio of 1:3. After 3 min of incubation at 37°C, the cultures were UV irradiated (C and E) or not (A–B and D). Alternatively, as controls, either the S4 CTL (F) or the target cells (G) were UV irradiated before incubation. Calcium-dependent fluorescence of indo-1 was assessed by flow cytometry over a period of 15 min. A representative experiment of four is shown.

To determine whether abrogation of calcium mobilization by TCR cross-linking was due to inefficient TCR-ligand photocross-linking, we assessed the kinetics of intercellular TCR-ligand binding. To this end, P815 expressing radiolabeled K^d-IASA-YIPSAEK(ABA)I complexes was incubated with cloned S4 CTL and UV irradiated after different periods of incubation. As shown in Figure 7⇓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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FIGURE 7.

Effect of TCR-ligand photocross-linking on TCR-ligand engagement. A, The kinetics of intercellular S4 TCR-ligand binding were assessed by TCR photoaffinity labeling. C, P815 cells P815 cells previously K^d photoaffinity labeled with ¹²⁵IASA-YIPSAEK(ABA)I using a 1-nM concentration or as specified were incubated for the indicated periods of time at 37°C with cloned S4 CTL at an E:T cell ratio of 1:3. After UV irradiation, the immunoprecipitated TCR were analyzed by SDS-PAGE and phosphorimaging. One hundred percent corresponds to the maximal TCR photoaffinity labeling. B, Alternatively S4 CTL were incubated with soluble monomer K^d-¹²⁵IASA-YIPSAEK(ABA)I complexes and UV irradiated (cross-linked) or not (bound) in the absence or the presence of anti-K^dα1 mAb 20–8–4S.

Next, we determined the efficiency of TCR photocross-linking, i.e., the fraction of engaged TCR that became cross-linked upon UV irradiation. The amount of TCR-bound ligand was measured by incubating S4 CTL with radiolabeled covalent ligand and by separating cell-associated and free ligand by spinning the cells through an oil gradient. From this value the nonspecific binding, i.e., cell-associated counts per minute in presence of anti-K^dα1 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 K^d-IASA-YIPSAEK(ABA)I complexes. As shown in Figure 7⇑C, 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.