The immunodominant T cell determinant of type II collagen (CII) recognized by DBA/1 mice (I-Aq) is CII 260–267. The aims of this study were to determine the role of the amino acid residues within CII 245–270 in T cell signal transduction. To that end, we utilized I-Aq-restricted, CII-specific T cell hybridomas and examined tyrosine phosphorylation of TCR-ζ following stimulation with either wild-type CII 245–270 or a panel of analogue peptides. A variety of patterns occurred, ranging from increased phosphorylation of TCR-ζ to either partial or a complete abrogation of phosphorylation. Critical substitutions also completely abrogated the phosphorylation of ZAP70, a downstream molecule in TCR-ζ signaling. Evaluation of the supernatants of the T cell hybridomas for cytokine production in response to the peptides revealed a close correlation between the induction of phosphorylation of TCR-ζ and the amount of cytokine induced. Selected analogue peptides were tested as tolerogens in neonatal mice. Analogues that did not induce the phosphorylation of ζ chain, such as B3 (CII 251–270s263F→N), were completely unable to induce tolerance, while analogues that caused a partial phosphorylation, such as B6 (CII 251–270s267Q→T) and A3 (CII 245–270s269P→A), induced partial tolerance judged by intermediate degrees of suppression of arthritis. We conclude that discrete alterations in specific amino acid residues of antigenic peptides had profound effects on T cell signaling and that the signaling correlated with T cell cytokine secretion and T cell function in the induction of tolerance and suppression of arthritis.

Engagement of the TCR by an immunogenic peptide bound to class II MHC molecules is the critical event initiating T cell activation. Once the TCR is engaged effectively, cytoplasmic protein tyrosine kinases (PTK)3 are recruited (1, 2), causing phosphorylation of a variety of cellular substrates (3, 4, 5). Recent studies utilizing chimeric molecules and reconstituted receptors have demonstrated that tyrosine phosphorylation of TCR-ζ is a critical early biochemical event (3, 4, 5). Transmission of extracellular signals downstream of the TCR (4) occurs once TCR-ζ phosphorylation allows the recruitment of a critical PTK, ζ chain-associated protein (ZAP70) (6, 7, 8, 9, 10). Thus, phosphorylation of TCR-ζ and its association with ZAP70 are important early events in TCR signal transduction.

T cell responses to a series of analogue peptides containing site-directed substitutions are not identical to that obtained with wild-type peptide bound to the same I-A molecule (11). When T cells recognize an altered ligand, the signaling through the TCR appears to be dependent upon the specific sequence of the peptide bound by I-A. Sometimes partial signaling can result, so that the cytokine panel secreted is altered significantly or shut down altogether (11).

Previous studies have shown that DBA/1 (I-Aq) mice develop autoimmune arthritis when immunized with heterologous type II collagen (CII) and that CII 245–270 contains the immunodominant T cell determinant. Collagen-induced arthritis (CIA) can be prevented or down-regulated in an Ag-specific manner by prior injection of CII or CII 245–270 as a tolerogen. The core of the T cell determinant has been shown to be CII 260–267 (12). Therefore, it was of interest to investigate the exact signal(s) the T cell receives from the interaction of a panel of analogue peptides bound to the MHC. To this purpose, we utilized CII-responsive T cell hybridomas that were I-Aq restricted, and compared the phosphorylation of TCR-ζ in these cells following stimulation with CII 245–270 and its analogues upon presentation by I-Aq cells. Tyrosine phosphorylation of TCR-ζ was evident in the cells stimulated with wild-type CII 245–270. On the other hand, analogue peptides induced a variety of patterns ranging from increased phosphorylation of TCR-ζ to either partial or a complete abrogation of phosphorylation. Moreover, substitutions at certain critical residues completely abrogated the ability of the analogues to induce phosphorylation of ZAP70, a downstream molecule in TCR-ζ signaling. Consistent with the pattern of TCR-ζ phosphorylation, cytokine production in the supernatants of T cell hybridomas correlated with the ability of each analogue to induce TCR signaling. Partial signaling induced by the analogues with substitution within CII 260–270 correlated with the partial ability of these analogue peptides to induce tolerance and suppress arthritis in susceptible mice. We conclude that discrete alterations in specific amino acid residues of antigenic peptides have profound effects on T cell signaling, causing partial or incomplete signaling events through TCR. The signaling correlated with T cell cytokine secretion and may ultimately determine T cell function in the induction of tolerance and suppression of arthritis.

A monoclonal anti-phosphotyrosine Ab PY20 was purchased from Transduction Laboratory (Lexington, KY). Rabbit polyclonal Ab to ZAP70 was kindly provided by Dr. Joe Fargnoli, Bristol/Myers/Squibb (Princeton, NJ). Anti-TCR-ζ peptide polyclonal Ab was raised in rabbits using a short synthetic peptide (DTYDALHMQTLAPR) corresponding to amino acid residues 151 to 164 of the murine TCR-ζ chain sequence. A cysteine residue was added to the amino terminus of the peptide for coupling to maleimide-activated keyhole limpet hemocyanin. The procedures for immunization of rabbits were described elsewhere (13). The antisera were collected after the second Ag boost. In some experiments, an antiserum (K2) with the highest titer was further purified by affinity chromatography on a column of TCR-ζ 151–164 bound to Sepharose 4B. A specific rabbit antiserum to TCR-ζ was kindly provided by Dr. Jeffrey V. Ravetch, Laboratory of Biochemical Genetics, Memorial Sloan-Kettering Cancer Center (New York, NY) and was used as a positive control.

Native CII was solubilized from bovine articular cartilage by limited pepsin digestion and purified as described earlier (14).

The peptide representing α1(II)-CII 245–270 (15) and its analogues containing specific amino acid substitutions were chemically synthesized by a solid-phase procedure described previously (16) using an Applied Biosystems (model 430; Foster City, CA) peptide synthesizer.

Establishment and characterization of an APC line (M12Aq), α1(II)-CB11-reactive T cell hybridomas (qcII85.33, 4qcII40), and a CII 245–270-reactive T cell line (DBA/1αA2) have been described previously (17). To induce tyrosine phosphorylation of TCR-ζ in T cell hybridomas and the T cell line, the cells were treated as follows: M12Aq cells (6 × 106) were incubated with or without CII synthetic oligopeptides (10–500 μg/ml) in RPMI 1640/10% FBS at 37°C for 12 h. Then the cells were washed two times with PBS and resuspended in 0.5 ml of RPMI 1640/10% FBS medium and used as APC. The T cell hybridomas or T cells (1.2 × 107) were washed two times with PBS and resuspended in 0.5 ml of RPMI 1640/10% FBS in Eppendorf tubes. The washed APC were then added to the tubes containing T cells and spun at a low speed (500 × g) for 30 s, and the cell mixture was incubated at 37°C for 5 min. Following incubation, the cells were spun in a microcentrifuge (4000 × g) for 1 min, and the supernatants were discarded. Stimulation was terminated by adding 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.4, containing 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 50 mM NaF, 0.2 μM Na3VO4, 1 mM PMSF, 10 μg leupeptin per ml, and 10 μg aprotinin per ml). Insoluble materials were removed by centrifugation at 10,000 × g at 4°C for 15 min.

Cells were harvested, washed two times in RPMI 1640 medium free of methionine and cysteine (ICN, Costa Mesa, CA), resuspended in the same medium (1 × 107/ml) containing 5% dialyzed FBS, and incubated in 37°C for 15 min. After this, trans-35S-labeled methionine/cysteine (ICN) was added (0.1 mCi/ml) and the cells were cultured in 37°C for additional 3 h. Following labeling, the cells were collected by centrifugation, washed two times with cold PBS, and resuspended at 107 cells/ml in lysis buffer (20 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 10% glycerol, 50 mM NaF, 0.2 μM Na3VO4, 1 mM PMSF, 10 μg leupeptin per ml, and 10 μg aprotinin per ml). The detergent-soluble cell lysates were clarified by centrifugation at 10,000 × g at 4°C for 15 min.

For immunoprecipitation, the cell lysates were mixed with either antiserum or Ab and incubated on ice for 2 h. Then protein A-Sepharose BL-4 (Pharmacia, Piscataway, NJ) was added and the samples were rotated at 4°C for 30 min. Immunoprecipitates were washed extensively with lysis buffer before suspension in Laemmli’s sample buffer. For peptide competitive immunoprecipitation, the procedures were the same as for immunoprecipitation described above, except that 3 μg of anti-TCR-ζ Ab was incubated with a synthetic TCR-ζ 151–164 (50 μg peptide in 50 μl lysis buffer) on ice for 30 min before addition to the cell lysates.

Proteins were separated on a 12.5% SDS-PAGE gel and electrotransferred onto nitrocellulose membranes. After transfer, the membrane was dried at room temperature and washed twice in TBS-T buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Tween-20). The Western Blot analysis described previously was used with minor modification (18). Briefly, for detection of phosphorylation of TCR-ζ, the nitrocellulose membrane was blocked in TBS containing 5% BSA for 2 h, incubated with an anti-phosphotyrosine mAb PY20 (1 μg/ml; Transduction Laboratory) in TBS-T/5% BSA for 2 h, and washed four times with TBS-T. The membrane was then incubated with a sheep anti-mouse peroxidase-conjugated Ab (Amersham, Arlington Heights, IL) for 1 h and subjected to enhanced chemiluminescence detection (ECL Western blot kit; Amersham), according to the manufacturer’s protocol. For detection of TCR-ζ and ZAP70 proteins, the membranes were blocked in TBS containing 5% no-fat milk for 2 h and incubated with polyclonal rabbit Abs against TCR-ζ (affinity purified, 1 μg/ml) or ZAP70 (1/1000 dilution), respectively, followed by incubation with a sheep anti-rabbit peroxidase-conjugated Ab (Amersham).

T cell hybridomas were established by polyethylene glycol-induced fusion of lymph node cells with the TCR-α thymoma line, BW5147 (19). Briefly, lymph node cells were obtained from DBA/1 mice immunized with CII emulsified with CFA 10 days previously and cultured in vitro with α1(II) for 5 days, and in the presence of IL-2 for 3 days before fusion. Hybridoma cells reactive to CII and α1(II)-CB11 were cloned by limiting dilution to 0.3 cells/well. Ag presentation experiments were performed in 96-well microtiter plates in a total volume of 0.3 ml containing 4 × 105 syngeneic spleen cells, 105 T hybridoma cells, and peptide (10–500 μg/ml). Cell cultures were maintained at 37° in 5% humidified CO2 for 20 to 24 h, after which culture supernatant fluids were harvested and analyzed for IL-2. To determine IL-2 titers, 4000 HT-2 cells were added to diluted supernatant fluids, and after 16 to 20 h HT-2 cell viability was evaluated by visual inspection and by the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. IL-2 titers are reported as the reciprocal of the highest twofold serial dilution maintaining 90% viability of the HT-2 cells.

DBA/1 mice obtained from The Jackson Laboratory (Bar Harbor, ME) were maintained in groups of six in polycarbonate cages and fed standard rodent chow (Ralston Purina, St. Louis, MO) and water ad libitum. The environment was specific pathogen free, and sentinel mice were tested routinely for mouse hepatitis and Sendai viruses. Mice were immunized at 8 to 12 wk of age, as described previously (20).

For routine immunization, CII was dissolved in 0.01 N acetic acid and emulsified with an equal volume of CFA, as described previously (21). The resulting emulsion was injected intradermally into the base of the tail. Each mouse received a total volume of 0.05 ml containing 100 μg Mycobacterium tuberculosis and 100 μg Ag.

DBA/1 mice were tolerized neonatally within 48 h of birth by giving each mouse 100 μg of Ag emulsified with CFA i.p. Mice were immunized with CII at 8 wk of age and observed for the incidence of arthritis (15). The incidence and severity of arthritis were determined by examining and scoring each of the forepaws and hindpaws on a scale of 0 to 4, as described previously (15). There were two separate examiners, one of whom was unaware of the identity of the treatment groups. Each mouse was scored three times per week by visual examination beginning 3 wk postimmunization and continuing for 8 wk. The incidence of arthritis (number of animals with one or more arthritic limbs) was analyzed at each time point. Incidence and severity shown represent data taken 6 wk after immunization when the controls reached their peak.

To examine the role of signal transduction of the TCR-ζ chain in collagen-induced T cell responses, we generated Abs against murine TCR-ζ 151–164 in rabbits. Three antisera were obtained following immunization with the synthetic peptide, and the Abs were affinity purified through a column of TCR-ζ 151–164-Sepharose. To determine the specificity of the Abs, we labeled T cell hybridomas with [35S]methionine/cysteine, immunoprecipitated proteins from the cell lysates using different amounts of the Abs, and separated the immunoprecipitates on SDS-PAGE gels. As shown in Figure 1 A, a 16-kDa protein migrating with the predicted m.w. of murine TCR-ζ was detected by both affinity-purified Ab (1–3 μg) or antiserum (3 μl/sample). However, the same protein was not detected in the sample treated with preimmune serum. Moreover, this 16-kDa protein completely disappeared in the presence of an excess amount of the synthetic TCR-ζ 151–164 that was used to raise the Ab tested, suggesting that the Ab we produced specifically recognized the TCR-ζ chain protein.

FIGURE 1.

Characterization of an Ab against TCR-ζ chain. A, [35S]methionine/cysteine-labeled proteins were immunoprecipitated with 3 μl preimmune serum (lane 1), 3 μl anti-TCR-ζ antiserum (lane 6), or affinity-purified anti-TCR-ζ Ab at 1 μg (lane 2), 2 μg (lane 3), 3 μg (lane 4), or 3 μg affinity-purified anti-TCR-ζ Ab in the presence of 50 μg TCR-ζ chain peptide that was used to raise the Ab (lane 5). The immunoprecipitates were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane, and autoradiography is shown. B, Western Blot analysis using a specific anti-TCR-ζ Ab from Dr. Jeffrey V. Ravetch, Memorial Sloan Kettering Cancer Center. The same membrane from above was immunoblotted with the specific Ab to TCR-ζ chain. C, Anti-TCR-ζ antiserum recognized phosphorylated forms of TCR-ζ chain. A collagen-specific T cell line (DBAαA2) was treated with collagen peptide CII 245–270 (+) or untreated (−). The TCR-ζ chain protein was immunoprecipitated and transferred onto nitrocellulose membrane. The membrane was first blotted with anti-phosphotyrosine mAb (αpTyr, left panel), then stripped and reprobed with anti-TCR-ζ antiserum obtained from Dr. Ravetch (αTCR-ζ, right panel). The positions of the phosphorylated TCR-ζ and unphosphorylated TCR-ζ are shown by arrows to the right of the figure. The 16-, 18-, and 21-kDa bands were not detected in immunoprecipitates from a B cell lymphoma line (data not shown).

FIGURE 1.

Characterization of an Ab against TCR-ζ chain. A, [35S]methionine/cysteine-labeled proteins were immunoprecipitated with 3 μl preimmune serum (lane 1), 3 μl anti-TCR-ζ antiserum (lane 6), or affinity-purified anti-TCR-ζ Ab at 1 μg (lane 2), 2 μg (lane 3), 3 μg (lane 4), or 3 μg affinity-purified anti-TCR-ζ Ab in the presence of 50 μg TCR-ζ chain peptide that was used to raise the Ab (lane 5). The immunoprecipitates were separated by 12% SDS-PAGE and transferred to nitrocellulose membrane, and autoradiography is shown. B, Western Blot analysis using a specific anti-TCR-ζ Ab from Dr. Jeffrey V. Ravetch, Memorial Sloan Kettering Cancer Center. The same membrane from above was immunoblotted with the specific Ab to TCR-ζ chain. C, Anti-TCR-ζ antiserum recognized phosphorylated forms of TCR-ζ chain. A collagen-specific T cell line (DBAαA2) was treated with collagen peptide CII 245–270 (+) or untreated (−). The TCR-ζ chain protein was immunoprecipitated and transferred onto nitrocellulose membrane. The membrane was first blotted with anti-phosphotyrosine mAb (αpTyr, left panel), then stripped and reprobed with anti-TCR-ζ antiserum obtained from Dr. Ravetch (αTCR-ζ, right panel). The positions of the phosphorylated TCR-ζ and unphosphorylated TCR-ζ are shown by arrows to the right of the figure. The 16-, 18-, and 21-kDa bands were not detected in immunoprecipitates from a B cell lymphoma line (data not shown).

Close modal

To further confirm the specificity of the Ab, we performed Western blot analysis using an Ab specific to TCR-ζ (a gift from Dr. Ravetch). Both our purified Abs and antiserum clearly precipitated a 16-kDa protein that was recognized specifically by the anti-TCR-ζ Ab. No 16-kDa protein was observed using preimmune serum or in the presence of TCR-ζ peptide (Fig. 1 B). A faint band slightly greater than 16 kDa probably represents a background component, as it was not able to compete with the TCR-ζ peptide and the same band was also observed in the preimmune serum control.

To determine whether the Ab we generated was able to precipitate the tyrosine-phosphorylated TCR-ζ chain, we induced tyrosine phosphorylation of TCR-ζ in a T cell line (DBA/1αA2) by culturing the T cells with the peptide CII 245–270. TCR-ζ chain protein was immunoprecipitated from the cell lysates with anti-TCR-ζ antiserum and subjected to Western blot analysis using anti-phosphotyrosine mAb (PY20). A total of 18 kDa of protein detected in the CII 245–270-treated sample was phosphorylated significantly compared with that of the unstimulated sample. Moreover, a 21-kDa band was detected only in the CII 245–270-stimulated, but not in the unstimulated sample (Fig. 1,C). Kinetic study shows that both the 18-kDa and the 21-kb bands are detectable as early as 2 min, and phosphorylation persists for at least 20 min after stimulation (data not shown). In each experiment, the membranes were stripped and reprobed with anti-TCR-ζ Ab, and an equal amount of 16-kDa protein was detected. Although 18-kDa phosphoproteins were not immunoblotted by the Ab (Fig. 1 C, right panel), our preliminary experiments have shown that increasing the salt concentrations in the lysis buffers and the wash buffer has no significant effect on the detection of phosphorylation of TCR-ζ chain by the Ab, suggesting that phosphorylated TCR-ζ was immunoprecipitated directly by the Ab, but not coimmunoprecipitated with unphosphorylated TCR-ζ chain (data not shown).

Our previous studies have identified CII 260–267 as the core of the immunodominant T cell determinant contained within CII recognized by I-Aq-restricted T cells. Nevertheless, it remained unclear how each residue affected signaling through the TCR. To determine the requirement of these individual residues for tyrosine phosphorylation of the TCR, we synthesized a panel of analogue peptides in which type I collagen residues replaced their type II collagen residues. The conserved glycine residues were replaced with alanine (Fig. 2,A). The ability of these analogues to induce tyrosine phosphorylation of TCR-ζ chain in T hybridomas was analyzed by culturing the hybridomas with each peptide, then followed by anti-phosphotyrosine immunoblotting. Substitution of individual residues at 260 (peptide B4), 261 (peptide A6), 263 (peptide B3), 264 (peptide B2), and 266 (peptide B1) completely abrogated the ability of the peptides to induce phosphorylation of TCR-ζ (Fig. 2 B). Replacement of the conserved glycine at residue 262 with alanine (peptide G39) partially affected its ability to induce phosphorylation of TCR-ζ, while substitution of the conserved glycine 265 (peptide B42) did not differ from the wild-type peptide. Another substitution at position 267 (peptide B6) induced minimal phosphorylation of TCR-ζ. Taken together, these data indicate that residues 260, 261, 263, 264, and 266 are all absolutely critical for induction of TCR-ζ phosphorylation in response to CII 260–267, and that residues 262 and 267 have an intermediate level of importance.

FIGURE 2.

The effect of substitution in CII 260–267 on phosphorylation of TCR-ζ chain. A, Sequences of analogue peptides with individual substitutions in CII 260–267 are shown. Sequence for A9 is also shown as a negative control. B indicates hydroxyproline. B, Phosphorylation of TCR-ζ chain in response to analogue peptides (50 μg/ml) shown in A. The TCR-ζ chain proteins were immunoprecipitated from the cells stimulated with the analogue peptides. The membrane was first blotted with anti-phosphotyrosine Ab (top panel) and then reprobed with TCR-ζ chain Ab (bottom panel). C, IL-2 production in response to wild-type peptide (A2) or analogue peptides in T hybridomas. The cells were cultured in the presence of A2 or analogue peptides as well as APCs for 12 h. The production of IL-2 in the supernatants from the cell cultures was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and is displayed as U/ml.

FIGURE 2.

The effect of substitution in CII 260–267 on phosphorylation of TCR-ζ chain. A, Sequences of analogue peptides with individual substitutions in CII 260–267 are shown. Sequence for A9 is also shown as a negative control. B indicates hydroxyproline. B, Phosphorylation of TCR-ζ chain in response to analogue peptides (50 μg/ml) shown in A. The TCR-ζ chain proteins were immunoprecipitated from the cells stimulated with the analogue peptides. The membrane was first blotted with anti-phosphotyrosine Ab (top panel) and then reprobed with TCR-ζ chain Ab (bottom panel). C, IL-2 production in response to wild-type peptide (A2) or analogue peptides in T hybridomas. The cells were cultured in the presence of A2 or analogue peptides as well as APCs for 12 h. The production of IL-2 in the supernatants from the cell cultures was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and is displayed as U/ml.

Close modal

To examine whether the alteration of phosphorylation of TCR-ζ by the analogue peptides was linked to the biologic function of T cells, we cultured the T hybridomas in the presence of the analogue peptides and measured IL-2 production in the cell supernatants. As shown in Figure 2 C, the peptides G39 and B42, which induced either partial or complete phosphorylation of TCR-ζ chain, were able to induce equal or even higher levels of IL-2 secretion as compared with that of wide-type peptide A2. In contrast, IL-2 production was abrogated completely by the five peptides that either failed to induce or induced very little TCR-ζ phosphorylation. A negligible level of IL-2 was produced in response to the other peptide, B4. These findings demonstrate a very close correlation between the degree of ζ chain phosphorylation and the production of lymphokine.

We further investigated the role of residues located outside the core T cell determinant, CII 260–267, in CII-induced signal transduction through TCR-ζ. Several CII analogue peptides containing one or more amino acid residue substitutions outside the CII 260–267 region were used for this purpose (Fig. 3,A). Consistent with results described in Figure 2, the peptides with substitutions within the CII 260–267 region abrogated the ability to induce phosphorylation of TCR-ζ. In addition, peptide A3, containing substitutions of residues 248, 250, and 269, showed a markedly reduced ability to induce phosphorylation of the TCR-ζ chain. In contrast, the other peptides containing substitutions outside the CII 260–267 region induced phosphorylation of TCR-ζ comparable with the level induced by wild-type CII 245–270 (Fig. 3 B). One surprising result was that obtained with peptide B5, which contained a substitution at position 258. This analogue peptide induced an increased amount of ζ chain phosphorylation compared with that of wild-type.

FIGURE 3.

The effect of substitution in CII 245–270 on phosphorylation of TCR-ζ chain in T hybridomas. A, Sequences of analogue peptides with substitutions in CII 245–270 are shown. B indicates hydroxyproline. B, Phosphorylation of TCR-ζ chain in response to analogue peptides (50 μg/ml) shown in A. The TCR-ζ chain proteins were immunoprecipitated from cells stimulated with analogue peptides. The membrane was first blotted with anti-phosphotyrosine Ab (top panel) and then reprobed with TCR-ζ chain Ab (bottom panel). C, IL-2 production in response to wide-type peptide or analogue peptides in T hybridomas. The cell culture and IL-2 production were determined as in Figure 2C.

FIGURE 3.

The effect of substitution in CII 245–270 on phosphorylation of TCR-ζ chain in T hybridomas. A, Sequences of analogue peptides with substitutions in CII 245–270 are shown. B indicates hydroxyproline. B, Phosphorylation of TCR-ζ chain in response to analogue peptides (50 μg/ml) shown in A. The TCR-ζ chain proteins were immunoprecipitated from cells stimulated with analogue peptides. The membrane was first blotted with anti-phosphotyrosine Ab (top panel) and then reprobed with TCR-ζ chain Ab (bottom panel). C, IL-2 production in response to wide-type peptide or analogue peptides in T hybridomas. The cell culture and IL-2 production were determined as in Figure 2C.

Close modal

The T hybridomas were cultured in the presence of the analogue peptides, and IL-2 production in the cell supernatants was measured. As shown in Figure 3 C, the peptides that induced complete phosphorylation of TCR-ζ chain were able to induce the secretion of equal levels of IL-2 as compared with that of wild-type CII 245–270 (peptide A2). In contrast, IL-2 production was abrogated completely by three of the peptides that failed to induce the TCR-ζ phosphorylation. A decrease in IL-2 by one dilution was noted in response to peptide A3, which gave a decrease in ζ chain phosphorylation. Peptide B5, which induced an increase in ζ phosphorylation, gave an IL-2 response that was similar to that of wild-type peptide. These findings again demonstrate a very close correlation between the degree of ζ chain phosphorylation and the production of cytokines by T cell hybridomas.

ZAP70 is a cytoplasmic tyrosine kinase and functions as a downstream molecule in TCR signaling by association with Ag recognition activation motif (ARAM) of the TCR-ζ chain via its two SH2 domains. Very likely, the T hybridomas stimulated with analogue peptides will have alterations in the phosphorylation of ZAP70 as well. To examine this, T cell hybridomas were stimulated with APCs previously pulsed with wild-type CII 245–270 (A2) or analogues A9 (which contain substitutions at 260, 261, and 263) and A10 (which contains substitutions at 266, 267, and 269). ZAP70 was immunoprecipitated using anti-ZAP70 Ab, and immunoprecipitates were probed with an anti-phosphotyrosine Ab. A 70-kDa ZAP70 protein was detectable, which was phosphorylated in the immunoprecipitates from the cells stimulated with A2. However, no ZAP70 phosphorylation was observed in the cells treated with analogue A9 or A10 (Fig. 4,A), even though an equal amount of ZAP70 protein was precipitated (Fig. 4,B). As shown in Figure 4,A, an 18-kDa phosphoprotein that was detected in all samples probably represents a background phosphorylation component of TCR-ζ. The phosphorylation of this 18-kDa protein was much stronger in the A2-treated sample. Besides the 18-kDa protein, a large amount of a 21-kDa protein with the same size as the phosphorylated form of TCR-ζ was also observed in the A2-treated sample. To confirm that TCR-ζ was indeed coimmunoprecipitated with ZAP70, we reprobed the same membrane with anti-TCR-ζ Ab. A 16-kDa protein, which represented unphosphorylated form of TCR-ζ, was equally detected (Fig. 4 C). These data suggested that although ZAP70 was associated consistently with the TCR-ζ chain, only phosphorylated TCR-ζ resulted in the activation of ZAP70. Conversely, failure to induce phosphorylation of TCR-ζ by A9 and A10 may account for the inactivation of ZAP70 in the TCR signal-transduction pathway.

FIGURE 4.

ZAP70 phosphorylation and association with TCR-ζ chain in T hybridomas in response to CII peptides. ZAP70 was immunoprecipitated from T hybridomas that were untreated or exposed to CII peptide wild-type peptide CII 245–270 (A2), A9, and A10, and analyzed by anti-phosphotyrosine immunoblotting (A). The same membrane was stripped and reprobed with anti-ZAP70 Ab (B) and anti-TCR-ζ (C). Phosphorylated ZAP70 and TCR-ζ chain are shown by arrows to the right of the figure.

FIGURE 4.

ZAP70 phosphorylation and association with TCR-ζ chain in T hybridomas in response to CII peptides. ZAP70 was immunoprecipitated from T hybridomas that were untreated or exposed to CII peptide wild-type peptide CII 245–270 (A2), A9, and A10, and analyzed by anti-phosphotyrosine immunoblotting (A). The same membrane was stripped and reprobed with anti-ZAP70 Ab (B) and anti-TCR-ζ (C). Phosphorylated ZAP70 and TCR-ζ chain are shown by arrows to the right of the figure.

Close modal

To test the effects these analogues might have in vivo on the induction of tolerance, analogue peptides were administered i.p. to neonatal mice. Once the mice attained 8 wk of age, they were immunized with CII and observed for the incidence of arthritis. As shown in Table I, induction of tolerance with wild-type A2 reduces the incidence of arthritis from 89 to 20%, which is significant (p < 0.05). Similarly, the induction of tolerance using peptide analogues that resulted in TCR-ζ phosphorylation, specifically peptide B42 and B45, suppressed the incidence of arthritis to 30 and 20%, respectively (Table I). On the other hand, analogues that did not allow the development of any ζ chain phosphorylation, such as B3, were completely unable to induce tolerance. The incidence of arthritis following tolerization with B3 was no different from that induced with the control OVA (88 and 89%, respectively). The induction of tolerance with analogues that induced partial phosphorylation, namely B6 and A3, induced intermediate degrees of suppression of arthritis with incidences of 56 and 50%, respectively. Peptide B5, which induced an increased degree of phosphorylation of TCR-ζ, was quite effective as a tolerogen, giving a final incidence of arthritis of 13%, which is a slightly lower incidence than that observed using wild-type peptide. Taken together, these data indicate that the degree of phosphorylation of the TCR-ζ induced following stimulation of T cells with analogue peptides correlated strongly with T cell function in vivo, as shown by the ability of these peptides to induce tolerance and suppress arthritis.

Table I.

Identification of residues critical for the induction of tolerance in collagen-induced arthritis utilizing CII peptides with amino acid

substitutionsa

PeptidePeptides Used to Induce ToleranceIncidence of Arthritis
250 255 260 265270
A2 P T G P L G P K G Q T G E L G I A G F K G E Q G P K 4/20 (20%)** 
B5 -------- B------------ 1/8 (13%)** 
B3 ------------ N------- 7/8 (88%) 
B42 -------------- A----- 3/10 (30%)** 
B6 ---------------- T--- 12/21 (57%)b 
B45 ----------------- A-- 2/10 (20%)** 
A3 ------------------------ A - 8/16 (50%)b 
OVA  16/18 (89%) 
PeptidePeptides Used to Induce ToleranceIncidence of Arthritis
250 255 260 265270
A2 P T G P L G P K G Q T G E L G I A G F K G E Q G P K 4/20 (20%)** 
B5 -------- B------------ 1/8 (13%)** 
B3 ------------ N------- 7/8 (88%) 
B42 -------------- A----- 3/10 (30%)** 
B6 ---------------- T--- 12/21 (57%)b 
B45 ----------------- A-- 2/10 (20%)** 
A3 ------------------------ A - 8/16 (50%)b 
OVA  16/18 (89%) 
a

Groups of DBA/1 mice were administered 100 μg of each peptide i.p. emulsified with IFA within 24 h of birth. Mice were then immunized with CII when they were 8 wk of age and observed for the incidence of arthritis. Results shown indicate the incidence of arthritis at 6 wk postimmunization. B indicates hydroxyproline. A dash (-) indicates identify of the residue with the wild-type sequence.

b

p ≤ 0.03 using Fisher’s exact test; ** p ≤ 0.003 using Fisher’s exact test.

The immunodominant T cell determinant of type II collagen, CII 260–267, recognized by sensitized T cells from DBA/1 mice (I-Aq), is also the most effective determinant for inducing tolerance and suppressing the arthritis of CIA. To evaluate cell signaling through the TCR in response to this determinant, we compared the phosphorylation of TCR-ζ in CII-specific hybridoma T cells following stimulation with either CII 245–270 or a panel of analogues presented by I-Aq cells. Tyrosine phosphorylation of TCR-ζ was evident in the cells stimulated with wild-type CII 245–270. On the other hand, analogue peptides induced a variety of patterns ranging from increased phosphorylation of TCR-ζ to either partial or a complete abrogation of this phosphorylation.

The TCR consists of ligand-specific α/β chain heterodimers and four invariant chains, CD3γδε (21, 22) and TCR-ζ (23, 24). All are required for effective membrane expression. T cell activation, however, requires engagement of TCR by an antigenic peptide bound to class II MHC molecule, together with costimulatory signals provided by APCs. These processes initiate multiple intracellular biochemical reactions that lead to clonal expansion and a variety of effector functions (25). One of the earliest biochemical events following T cell engagement by antigenic peptides is the activation of multiple cytoplasmic PTK. A ligand-specific engagement results in phosphorylation of a variety of cellular substrates (3, 4, 5). Although TCR has no intrinsic protein kinase activity, recent studies utilizing chimeric molecules and reconstituted receptors have demonstrated that CD3ε chain and TCR-ζ are responsible for TCR signal transduction (1, 2). These signal-transduction molecules contain a common 17-amino-acid-sequence motif comprising two critical tyrosine and leucine residues (YXXL), which are referred to as ARAM (26, 27) or tyrosine activation motif (TAM) (28).

Increasing evidence has accumulated showing that the phosphorylation of TCR-ζ correlates with the activation of T cells. Several studies have demonstrated recently that the cytoplasmic domain of TCR-ζ itself is sufficient to induce T cell activation, and expression of chimeric molecules containing portions of the cytoplasmic domain of TCR-ζ can effectively deliver signals (2, 29, 30). Furthermore, deficiency in TCR-ζ eliminated T cell functions (31), whereas expression of the TCR-ζ in T cell hybridoma can restore the cell reactivity (32). Failure of signal transduction through the TCR could block the signal required for cytokine release in T cells. In the present study, we demonstrated that the cytokine secretion of CII-specific hybridomas correlated well with the TCR-ζ phosphorylation. Hybridomas that failed to induce phosphorylation of TCR-ζ also failed to produce IL-2 in response to CII analogues, supportive that optimal signals provided by immunogenic peptides result in both cytokine production and TCR-ζ phosphorylation. Using T hybridomas stimulated with analogue peptides, we have identified six residues of CII 260–267 (260, 261, 263, 264, 266, and 267) in which substitution disrupts the production of IL-2. In parallel experiments, stimulation of the hybrids with the analogues completely disrupts phosphorylation of the TCR-ζ chain (analogue B6 is the exception in that it induces no IL-2 secretion, but does induce limited phosphorylation of TCR-ζ). Analogue peptides that induce TCR-ζ phosphorylation comparable with that induced by wild-type peptide in CII-specific hybridomas also cause secretion of IL-2 comparable with that induced by wild-type. On the other hand, certain interesting analogues induce an incomplete phosphorylation of the TCR-ζ. In these cases, the cytokine production ranges from undetectable to only a one-tube dilution below wild-type level.

Tyrosine-phosphorylated ARAMs of TCR-ζ, following TCR engagement, function as a binding site for SH2-containing PTKs. This leads to the recruitment of a critical PTK, the ζ chain-associated protein (ZAP70), via ZAP70’s tandem SH2 domains (6, 7, 8, 9, 10). Involvement of ZAP70 results in phosphorylation of downstream substrates, allowing transmission of extracellular signals beyond the TCR (4). A reduction in signaling through the TCR may interfere with the recruitment of ZAP70 to the membranes of T cells, which is the means whereby signals from the TCR can be delivered downstream. We demonstrate that substitutions at six of the critical residues completely abrogated the ability of the analogues to induce phosphorylation of ZAP70, a downstream molecule in TCR-ζ signaling. Thus, we confirm the association of phosphorylated TCR-ζ with ZAP70 as a critical event in TCR signal transduction.

Three residues, specifically CII 262, 267, and 269, have been identified in which analogues appear to induce a weak or partial phosphorylation of the TCR-ζ chain. It has been suggested that partial agonists of the TCR alter the immune response to the immunogen by inducing differential signaling through the TCR, and this differential signaling can be detected by changes in the biochemical status of the T cell, especially the TCR-CD3 complex and its associated proteins. Traditionally, T cell activation and the resulting effector functions have been thought to be binary events, being either on or off. However, recent studies have indicated that various T cell effector functions can be uncoupled from each other. Thus, the TCR is similar to other receptor systems that have been pharmacologically studied, in which alteration of the ligand presented to the T cell has identified receptor agonists, partial agonists, and antagonists. In our studies, we demonstrate that T hybridomas stimulated with CII analogue peptide substitutions at positions 262, 267, and 269 decreased the phosphorylation of the TCR-ζ chain. Using a CII-specific T cell hybridoma, these correlated precisely with a decrease in production of IL-2 compared with that produced in response to wild-type peptide. We were unable to detect any significant cytokine production in the absence of phosphorylation of the TCR-ζ chain. Two of the analogues capable of inducing partial phosphorylation, specifically substitutions at 263 and 267, were felt to be TCR contact residues rather than anchor residues for binding of the peptide to the I-Aq molecule (33). The identification of incomplete phosphorylation with substitutions at residue 269 and increased phosphorylation at residue 258 was unexpected, as both are positioned outside the core of the T cell determinant previously identified. Their exact function in T cell activation is unclear, although other investigators have identified residues outside the core that play a role in either stabilization of the tertiary structure of the peptide, or possibly interaction with the CD4+ accessory molecule (34).

Collagen-induced arthritis is an experimental autoimmune animal model characterized by inflammatory polyarthritis as well as humoral and cellular mediated autoimmune responses to CII, which are similar to features observed in human rheumatoid arthritis (35, 36, 37). T cell responses to CII play an important role in the induction and development of CIA (38) as well as in the induction of tolerance and subsequent suppression of autoimmune arthritis. Mechanistically, the induction of tolerance to CII appears to change the response of CII-specific T cells by altering their cytokine production. Spleen cells obtained from mice tolerized with CII and stimulated by CII in vitro had significantly increased levels of IL-4 and IL-10 (34, 39), and reduction in the levels of the complement-fixing IgG2 Abs specific for CII in comparison with controls (34). Thus, it appears likely that tolerance to CII induces T cells to secrete Th2-type cytokine profiles that modulate inflammation and markedly decrease incidence and severity of autoimmune arthritis.

To test the functional significance of the varying degrees of phosphorylation of the TCR-ζ, analogue peptides containing individual substitutions within CII 245–270 were administered to neonatal mice before challenging immunization to determine their ability to induce tolerance and suppression of arthritis. As expected from the hybridoma data, the analogue containing a substitution at positions 263, which disrupts TCR-ζ chain phosphorylation, was incapable of inducing tolerance and suppression of arthritis. Analogue peptides containing substitutions that appear to induce a weak or partial phosphorylation of the TCR-ζ chain, specifically CII 267 and 269, induced intermediate degrees of suppression of arthritis compared with that induced with wild-type peptide. Therefore, we observe that TCR-ζ phosphorylation correlated very well with in vivo T cell function. Partial signaling induced by the analogues with substitution within CII 260–270 correlated with a partial ability of these analogue peptides to induce tolerance and suppress arthritis in susceptible mice. We conclude that discrete alterations in specific amino acid residues of antigenic peptides have profound affects on T cell signaling, causing partial or incomplete signaling events through TCR. The signaling correlated with T cell cytokine secretion and, ultimately, T cell function in inducing tolerance and suppression of arthritis.

We thank Dr. Joe Fargnoli, Bristol/Myers/Squibb (Princeton, NJ), for his generous gift of rabbit anti-ZAP70 antiserum, and Dr. Jeffrey V. Ravetch, Laboratory of Biochemical Genetics, Memorial Sloan-Kettering Cancer Center (New York, NY), for providing a specific rabbit antiserum to TCR-ζ.

1

This work was supported, in part, by USPHS Grant AR-39166, USPHS Grant AR-43589, and program-directed funds from Veterans Administration.

3

Abbreviations used in this paper: PTK, protein tyrosine kinase; ARAM, antigen recognition activation motif; CIA, collagen-induced arthritis; CII, type II collagen; TBS-T, Tris buffer saline-Tween.

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