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Study of the Mechanism of TCR Antagonism Using Dual-TCR-Expressing T Cells¹

La Jolla Institute for Allergy and Immunology, San Diego, CA 92121

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanism of action of TCR antagonists is incompletely understood. T cells expressing two distinct TCRs have been used to test competition for TCR occupancy as a potential mechanism. Previous studies with CD4 T cells showed that an antagonist for one TCR inhibited the response to the other TCR (cross-antagonism), whereas studies with CD8 cells failed to demonstrate cross-antagonism. To determine whether CD4 and CD8 cells were intrinsically different or whether the differences were the result of the use of different effector assays, we studied both CD4 and CD8 dual-TCR-expressing T cells. In the CD4 system, consistent with previous reports, cross-antagonism of proliferation was observed. In the CD8 system, cross-antagonism was observed using proliferation as readout but not when target cell cytolysis was used. These results suggest that different mechanisms may be involved in the inhibition of proliferation and inhibition of cytotoxic effector function, the latter only involving competition for TCR occupancy. Inhibition of proliferation appears to be more complex and other mechanisms such as sequestration of signaling molecules or negative signaling may be involved. The fact that 10- to 20-fold more antagonist was needed to achieve cross-antagonism compared with inhibition of the cognate TCR is consistent with the hypothesis that competition for TCR occupancy is also a major, albeit not sole, mechanism of antagonism of the proliferative responses of CD4 and CD8 cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The outcome of the interaction between the TCR and its MHC-peptide ligand varies significantly depending on the affinity and/or off-rate of the interaction. In the thymus, high avidity interactions lead to deletion, whereas lower avidity interactions result in positive selection (1, 2, 3, 4, 5). In the periphery, affinity of interaction with Ag may influence the type of effector cells that develop (e.g., Th1 vs Th2) (6, 7, 8) and the termination or induction of peripheral T cell tolerance (9, 10, 11). These changes in TCR affinity for an epitope are typically accomplished by a single conservative amino acid substitution at a TCR contact site. The initial reports that such altered peptide ligands (APL)³ could strongly affect the outcome of TCR engagement with peptide-MHC complexes demonstrated partial agonist activity (cytokine production in the absence of proliferation) (12) or antagonist activity whereby stimulation of T cells by agonist could be inhibited by the addition of certain APLs to cultures containing Ag-pulsed APCs and T cells (13, 14). The mechanism by which inhibition of T cell activation is accomplished by such antagonist APLs remains unresolved. Various mechanisms have been proposed including competition for engagement of the TCR by agonist peptide (13, 15, 16), consumption or sequestration of signaling molecules that are present in limiting quantities (17), and induction of negative regulatory signals (18, 19).

In recent years, T cells expressing two distinct TCRs have been used as a model system to specifically test the competition model of antagonism (15, 16, 18, 20). It is predicted that if competition for TCR occupancy were the sole mechanism by which antagonist peptides inhibit T cell activation induced by agonist stimulation, then stimulation by an agonist peptide specific for one TCR would not be inhibited by an antagonist peptide specific for the second TCR that had different Ag and antagonist specificity (i.e., cross-antagonism would not occur). In contrast, if either of the other two models, negative signaling or sequestration of signaling molecules, were operative, then cross-antagonism would be expected to occur. The results reported from laboratories that have used this system have been inconsistent. In two papers that used dual-TCR-containing CD4 T cells, cross-antagonism was demonstrated (18, 20); whereas in two reports that utilized dual-TCR-containing CD8 T cells, no cross-antagonism was detected (15, 16). Whether these discrepant results were due to intrinsic differences in the mechanisms of antagonism in CD4 and CD8 T cells or whether it was related to the different detection systems used in these studies, or other factors, is not clear. To address these issues, we have generated two types of dual-TCR-expressing T cells: a CD4 T cell line expressing the 5CC7 and DO11.10 TCRs which are specific for MCC/IE^k and OVA/IA^d, respectively (21, 22); and a CD8 T cell line expressing the P14 and OT-1 TCRs, specific for lymphocytic choriomeningitis virus (LCMV) glycoprotein/D^b and OVA/K^b, respectively (23, 24, 25). Using these lines, we have tested the ability of antagonist peptides to cross-antagonize a second unrelated TCR. The results obtained help to clarify the basis of some of the discrepancies in the literature and are discussed in light of the potential mechanisms involved in TCR antagonism.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice and cell lines

Homozygous P14 TCR-transgenic (rag2^-/-, B10), 5CC7 TCR-transgenic (rag2^-/-, B10.A), and DO11.10 TCR-transgenic (rag2^-/-, B10.D2) mice were purchased from Taconic (Germantown, NY) as were homozygous rag2^-/- (H2^b) mice. OT-1 (rag2^+/+, B6) mice were obtained from M. Croft (La Jolla Institute for Allergy and Immunology, San Diego, CA) and bred to rag2^-/- (H2^b) mice to generate OT-1 rag2^-/- mice. Homozygous P14-transgenic (rag2^-/-) and hemizygous OT-1-transgenic (rag2^-/-) mice were bred together to generate P14/OT-1 rag2^-/- F₁ mice transgenic for the two TCRs. The P14/OT-1 rag2^-/- mice were also bred back to homozygous P14 transgenic to generate P14^+/+/OT-1^+/- (rag2^-/-) mice. Homozygous 5CC7 rag2^-/- and DO11.10 rag2^-/--transgenic mice were bred together to generate 5CC7/DO11.10 rag2^-/- F₁ mice transgenic for the two TCRs. B10.A and B10.D2 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). LK35.2 cell, a B cell hybridoma that expresses I-A^d and I-E^k was provided by Dr. P. Marrack (Howard Hughes Medical Institute, Denver, CO) and LB27.4, a B cell hybridoma expressing D^b and K^b molecules (26), was purchased from American Type Culture Collection (Manassas, VA) as was the EL-4 T cell lymphoma.

Peptide synthesis

Peptides were synthesized on a Rainin Symphony synthesizer (Peptide Technologies, Washington, DC) as described (27) and purified by reverse phase HPLC. The identity of the peptides was substantiated by mass spectrometry. Purity was routinely 95% as measured by HPLC. The peptides used in this study were: OVA_HTL (OVA_323–339; ISQAVHAAHAEINEAGR), OVA_HTLH331K (ISQAVHAAKAEINEAGR), MCCp (moth cytochrome c_88–103; ANERADLIAYLKQATK), MCCpT102G (ANERADLIAYLKQAGK), GP33p (LCMV glycoprotein_33–41; KAVYNFATM), GP33p Y36S (KAVSNFATM), OVA_CTL (OVA_257–264; SIINFEKL), OVA_CTLK263P (SIINFEPL), HYp (male HY peptide; KCSRNRQYL), RPp (40-kDa bacterial ribosomal protein_89–96; GAYEFTTL), sperm whale myoglobin_106–118; FISEAIIHVLHSR), poly(A)-I-E^k (I-E^k-binding peptide; AAAAAAAIAYKEQWKK).

T cell lines

All T cell lines used in this study were generated from TCR-transgenic mice that were on a rag2-deficient background. T cell lines were generated by stimulating lymph node cells from the transgenic mice with irradiated spleen cells or mitomycin C-treated B hybridoma cells of the appropriate MHC haplotype together with Ag (peptide or protein). IL-2 (10 U/ml) was added to the cultures every 2–3 days in the form of mouse Con A supernatant. Cell lines were maintained by restimulation with Ag at 2-wk intervals. In the case of the 5CC7/DO11.10 T cell lines, the cell line was sorted for triple-positive cells (CD4⁺, V3⁺, and DO11.10 clonotype⁺) by FACS, and the sorted cells were maintained in culture by periodic stimulation with pigeon cytochrome c protein or OVA_HTL peptide.

Abs and cell surface staining

Cy-Chrome-labeled anti-CD4; APC-labeled anti-CD8; FITC-labeled anti-V 2, V11, and V8; and PE-labeled anti-V3 and V5 were purchased from BD PharMingen (San Diego, CA). FITC-labeled anti-DO11.10 clonotypic Ab was purchased from Caltag Laboratories (Burlingame, CA). For determining expression of 5CC7 and DO11.10 TCRs, cells were stained for CD4, V3 (5CC7), and the DO11.10 clonotype. To measure the expression of P14 and OT-1 TCRs, the cells were stained for CD8, V8 (P14), and V5 (OT-1). Surface expression of TCRs was analyzed on a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Proliferation assays

T cell proliferation assays were conducted as previously described (27). In brief, B hybridoma cells (LK35.2 or LB27.4) were used as APCs after treatment with mitomycin C. T cells (10⁵) were incubated with APCs (10⁵) in wells of flat-bottom 96-well plates containing various concentrations of the relevant peptides. After 48 h of culture, 1 µCi of [³H]thymidine was added. Cultures were harvested 16–18 h later, and [³H]thymidine incorporation into DNA was measured by liquid scintography. For antagonism assays, the APCs were prepulsed with suboptimal concentrations of agonist peptide for 2 h at 37°C in RPMI containing 10% FCS. Unbound peptides were removed by washing, and the peptide-loaded APCs (10⁵) were added to flat-bottom 96-well plates containing a wide concentration range of antagonist peptides or control peptides. The cultures were incubated at 37°C for 1 h after which time T cells (10⁵) were added, and the cultures were treated as described above to determine [³H]thymidine incorporation.

⁵¹Cr release assay

EL-4 target cells were prelabeled with sodium [⁵¹Cr]chromate and washed, and 5 x 10³ cells were transferred to wells of a round-bottom 96-well plate containing peptides at various concentrations. The plates were incubated for 2 h at 37°C before addition of T cells (10⁵) to achieve an E:T ratio of 20:1. The plates were then centrifuged briefly and incubated at 37°C for 4–5 h, at which time ⁵¹Cr release into the culture supernatant was measured in a Wallac MicroBeta Trilux gamma counter (Perkin-Elmer, Wellesley, MA). Spontaneous ⁵¹Cr release was determined in cultures of EL-4 cells without effector cells, and total ⁵¹Cr release was determined by adding 10% Triton X-100 instead of effector cells. ⁵¹Cr release was calculated as: [(sample - spontaneous release)/(maximum release - spontaneous release)] x 100. For antagonism assays, the ⁵¹Cr-labeled EL-4 cells were prepulsed with suboptimal doses of agonist peptide for 2 h at 37°C. The unbound peptides were removed by washing and the agonist-pulsed EL-4 cells (5 x 10³) were then transferred to wells of a round-bottom 96-well plate containing various quantities of antagonist or control peptides and incubated for 1 h before addition of T cells. The remainder of the assay was performed as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of dual-TCR-expressing T cell lines

Homozygous 5CC7 rag2^-/- and DO11.10 rag2^-/- mice were bred with one another to generate F₁ mice (5CC7/D011.10) transgenic for both TCRs. CD4⁺ T cell lines were generated by stimulating lymph node cells of the 5CC7/DO11.10 mice with OVA_HTL or pigeon cytochrome c protein. Because FACS analysis showed that a considerable number of cells lacked reactivity with the DO.11.10 anticlonotypic Ab, the cell line was sorted for CD4⁺, V3⁺, and DO11.10⁺ cells. Fig. 1A shows the TCR expression levels of the sorted cell line. All CD4⁺ T cells of the 5CC7/DO11.10 cell line expressed both receptors. Compared with cell lines made from the single TCR-transgenic parental mice, the DO11.10 receptor on the dual-TCR cell line was expressed at relatively high levels (within 2-fold of the DO11.10 single-TCR cell line), whereas the 5CC7 receptor on the dual-TCR line was expressed at 10–15% of the level of the single-TCR 5CC7 line (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. TCR expression of CD4+ and CD8+ restricted dual-TCR T cell lines. A, T cell line derived from 5CC7/DO11.10 TCR-transgenic mouse (see Materials and Methods) was stained for CD4, V3 (5CC7), and DO11.10 clonotypic determinants. The TCR expression of CD4+ cells is shown. Almost all cells expressed both 5CC7 and DO11.10 TCRs. B, a T cell line derived from dual-P14/OT-I TCR-transgenic mouse was stained for CD8, V8, and V5. TCR expression of CD8+ cells is shown; 96% of CD8+ cells expressed both V8(P14)- and V5(OT-1)-containing TCRs.

Specificity of agonist and antagonist peptides for 5CC7 and DO11.10 TCRs

Preliminary to conducting experiments on the dual-TCR-expressing T cells, it was necessary to demonstrate the absence of any cross-reactivity of agonist and antagonist peptides for the second unrelated TCR. To do this, the 5CC7 and DO11.10 single-TCR T cells were stimulated with both cognate agonist peptides, (MCCp and OVA_HTL). As shown in Fig. 2, the 5CC7 line responded to MCCp but not to OVA_HTL, and the DO11.10 T cell line responded to OVA_HTL but not to MCCp. Next the specificity of the antagonist peptides, MCCpT102G (28) and OVA_HTLH331K (H. Grey, unpublished observations), were analyzed. Antagonism assays were performed using LK35.2 APCs that expressed both I-E^k and I-A^d. These cells were prepulsed with suboptimal amounts of agonist peptide followed by the addition of 100 µg of antagonist peptide and T cells. Proliferation of the T cells was determined after 3 days of culture. As shown in Table I, the DO11.10 response to OVA_HTL was inhibited by the OVA_HTLAPL, H331K, but not the MCC APL, T102G. Conversely, the 5CC7 response to MCC was inhibited by the MCC APL but not the OVA_HTLAPL. Thus, both agonist and antagonist peptides were specific for their respective TCRs and showed no cross-reactivity with the other TCR that was coexpressed on the dual-TCR cell line.

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Ag specificity of single TCR cell lines, 5CC7 and DO11.10 TCR. T cell lines expressing the 5CC7 TCR (A) or DO11.10 TCR (B) were cocultured with mitomycin C-treated LK35.2 cells as APCs in the presence of varying concentrations of OVAHTL () or MCCp (•) for 48 h. [3H]Thymidine (1 µCi) was added to each well, and the culture was harvested 16–18 h later and assayed for [3H]thymidine incorporation.

The same agonist and antagonist peptides used with the single-TCR lines were then used to analyze the ability of an antagonist for one TCR to inhibit the response of the dual-TCR T cell line that was stimulated by an agonist for the other receptor (cross-antagonism). As shown in Fig. 3A, the 5CC7/DO11.10 dual-TCR line responded to both OVA_HTL and MCCp. Similar to the single-TCR lines, the response to MCCp required 3- to 10-fold less peptide than that required to obtain a similar response to OVA_HTL. The results of antagonism assays, performed with suboptimal doses of agonist and a range of antagonist or control MHC-binding peptides are illustrated in Fig. 3, B and C. Fig. 3B shows the capacity of the different peptides to inhibit the proliferative response of the dual-TCR line when OVA_HTL was used as the agonist. The OVA_HTLAPL, H331K, inhibited the OVA_HTL response almost completely at 70 µM, with 40% inhibition requiring 1 µM. Although less efficient, definite inhibition of the response was also observed with the MCCp APL, T102G, with 40% inhibition being obtained at a concentration of 3 µM. In contrast, the two control MHC-binding peptides Myo_106–118 (I-A^d-binding peptide) and the control I-E^k-binding peptide showed no inhibitory capacity. Similarly, when the dual-TCR line was stimulated by a suboptimal concentration of MCCp, the proliferative response was inhibited by both APLs, MCCpT102G and OVA_HTLH331K. Again, the APL of the agonist, in this case MCCpT102G, was more effective, requiring 0.3 µM to achieve 40% inhibition, whereas 23 µM OVA_HTL H331K was required to achieve a similar level of inhibition. This experiment was performed four times. The summary of the four experiments is shown in Table II. In all four experiments, the OVA_HTL APL inhibited the OVA_HTL response and cross-inhibited the MCCp response. The MCC APL, although it effectively inhibited the MCCp response, achieved 40% inhibition of the OVA_HTL response in only two of the four experiments. In all instances, the APLs were considerably more effective at inhibiting the cognate response (e.g., OVA_HTL APL inhibition of OVA_HTL response) than the noncognate response (e.g., OVA_HTL APL inhibition of the MCCp response). The average noncognate-cognate ratio was 17:1 in the six instances in which the end point of 40% antagonism was achieved.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Ag and antagonist specificity of dual-5CC7/DO11.10 TCR T cell line. A, Proliferation of dual-5CC7/DO11.10 TCR T cells cultured with LK35.2 APC and various concentrations of OVAHTL () or MCCp (•). B and C, Antagonism assay. LK35.2 cells were prepulsed with suboptimal concentration of OVAHTL (0.06 µM) (B) or MCCp (0.02 µM) (C) for 2 h, washed, and incubated with varying concentrations of the antagonist APLs OVAHTLH331K () or MCC T102G (), or the I-Ad-binding control peptide MYOp (*) or I-Ek-binding control peptide poly(A)-I-Ek () for 1 h. T cells were then added to the wells, and their proliferative responses were measured as in Fig. 2. Cross-antagonism of OVAHTL-stimulated cells by MCCp APL and of MCCp-stimulated cells by OVAHTL APL H331K was observed.

As described above for the class II MHC-restricted 5CC7 and DO11.10 TCRs, the specificity of the class I MHC-restricted P14 and OT-1 TCRs for their respective agonists (GP33p and OVA_CTL) and antagonist APLs were determined. Both target cell cytolysis and proliferative responses were used as assay systems. The specificity of the agonist peptides OVA_CTL and GP33p and their respective antagonist APLs in a cytotoxicity assay is shown in Fig. 4. The P14 line killed EL-4 cells pulsed with nanomolar quantities of GP33p but no killing of OVA_CTL pulsed cells was observed, even at a concentration of 10 µM (Fig. 4A). Conversely, OT-1 cells killed EL-4 cells pulsed with picomolar amounts of OVA_CTL but did not kill EL-4 cells pulsed with GP33p (Fig. 4B). The antagonist APLs showed similar specificity in that the GP33p APL, Y36S, (29) inhibited the P14-induced killing of GP33p-coated target cells (Fig. 4C) but did not inhibit the killing of OVA_CTL-sensitized EL-4 cells by OT-1 (Fig. 4D) and the OVA_CTL APL, K263P, (1, 30) inhibited OT-1 killing of OVA_CTL-sensitized target cells but did not inhibit P14 killing of GP33p-sensitized targets. Control D^b- and K^b-binding peptides failed to inhibit either OT-1- or P14-mediated killing.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Ag and antagonist specificity of single TCR cell lines, P14 and OT-1 in cytotoxicity assay. EL-4 target cells were labeled with 51Cr and preincubated with the P14 agonist GP33p () or the OT-1 agonist OVACTL (•) for 1 h at 37°C before coculture with P14 T cells (A) or OT-1 T cells (B) at E:T 20:1. 51Cr release was measured after 4 h. C and D, Inhibition of cytotoxicity by antagonist peptides. Only GP33pY36S () inhibited killing of GP33p-sensitized EL-4 cells by P14 T cells (C), and only OVACTLK263P inhibited lysis of OVACTL-sensitized EL-4 cells by OT-1. D, GP33p Y36S (), OVACTLK263P (), control Db-binding peptide HYp (*), and control Kb-binding peptide RPp ().

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Absence of cross-antagonism of cytotoxicity in the dual-P14/OT-1 cell line. A, P14/OT-1 cells can kill EL-4 cells sensitized with GP33p () or OVACTL (•). B, Only GP33pY36S () can inhibit killing of GP33p (3 x 10-3 µM)-sensitized target cells. C, Only OVACTLK263P can inhibit killing of OVACTL (1 x 10-5 µM)-sensitized target cells by P14/OT-1 T cells. , GP33pY36S; , OVACTLK263P; *, control Db-binding peptide HYp; , control Kb-binding peptide RPp.

View larger version (36K): [in this window] [in a new window]   FIGURE 7. Summary of antagonism of CD8 dual-P14/OT-1 TCR T cells. A, Mean percentage inhibition from seven experiments that assayed the capacity of antagonist peptides to inhibit the specific net lysis of target cells by the dual-TCR cell line P14/OT-1. Only the cognate antagonists were capable of inhibition. Concentrations of agonists used in these experiments were: GP33p, 3 x 10-4-10-3 µM; OVACTL, 3 x 10-6-10-5 µM. B, Mean percentage inhibition of three experiments that assayed proliferative responses of the dual-P14/OT-1 cell line. Data were obtained from the highest dose (10 µM) of antagonist peptides. , GP33p APL; , OVACTL APL. The GP33 response demonstrated both cognate and noncognate (cross-) inhibition, whereas the OT-1 response was inhibited only by the cognate APL. Agonist concentrations used in proliferation assays were: GP33p, 0.3–3 µM; OVACTL, 3 x 10-5-10-4 µM.

View larger version (31K): [in this window] [in a new window]   FIGURE 6. Cross-antagonism of proliferation in the dual-P14/OT-1 T cell line. A and B, Specificity of antagonist peptides in their ability to inhibit proliferative response of single TCR-expressing cell lines. A, GP33p (3 x 10-2 µM)-induced proliferation of P14 is inhibited only by the GP33p APL. B, OVACTL (3 x 10-4µM)-induced proliferation of OT-1 is inhibited only by the OVACTL APL. C and D, Antagonism of proliferative response of P14/OT-1 dual-TCR cell line. C, Proliferative response of P14/OT-1 induced by GP33p (3 µM) is inhibited by both GP33p APL and OVACTL APL but not by control peptides. D, The proliferative response of P14/OT-1 induced by OVACTL (10-4µM) is inhibited only by the OVACTL APL but not by any of the other peptides. Symbols are the same as in Figs. 4 and 5.

It is not clear why cross-antagonism of the OVA_CTL response by the GP33p APL was not observed, but it is likely related to the relatively low expression of the P14 TCR in this cell line and the requirement for high concentrations of both agonist and antagonist to function with this receptor. A range of 0.2–10 µM concentration of the GP33p APL were required to inhibit the cognate response to GP33p. This high concentration requirement, together with the relative inefficiency of cross-antagonism, likely contributed to the failure to inhibit the noncognate response.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we investigated the mechanism of TCR antagonism by using cell lines that expressed two TCRs with different specificities and have evaluated whether engagement of one TCR by an antagonist peptide would result in inhibition of the activation of the T cell line when stimulated by an agonist specific for the other TCR. The major findings were: 1) when the readout in the class I-restricted dual-TCR system was target cell cytolysis, cross-antagonism was not detected; 2) when T cell proliferation was used as the readout for activation, both class I-restricted and class II-restricted dual-TCR-containing T cells could be cross-antagonized; 3) a quantitative assessment of the relative efficiency of inhibition by TCR antagonists when inhibiting an agonist specific for the same receptor as the antagonist (cognate inhibition) compared with its ability to cross-antagonize a second unrelated receptor (noncognate inhibition) indicated that inhibition of signaling via the noncognate receptor required 10- to 20-fold more antagonist than was required to inhibit the signaling of the cognate receptor.

These data help resolve some of the discrepancies previously reported using dual-TCR-expressing cell lines to explore the mechanisms of TCR antagonism. Robertson and Evavold (20) reported in a class II-restricted system that a HEL_48–62 APL was capable of inhibiting the response to MCC_88–103 in a dual-TCR line containing receptors specific for MCC_88–103 and HEL_48–62. Similarly, Dittel et al. (18) showed cross-inhibition using a T cell line with TCRs specific for myelin basic protein, Ac_1–11 and conalbumin_134–146. In contrast, in the two previous reports using dual-TCR-containing T cells restricted by class I MHC, cross-antagonism could not be demonstrated (15, 16). The study by Daniels et al. (15) used antagonism of target cell cytolysis as the readout, and the lack of cross-antagonism they observed agrees with our results using the same assay. The fact that we could readily detect cross-antagonism in the class I-restricted system when proliferation was used as the readout strongly suggests that there is no inherent difference in the mechanisms of antagonism between CD4 and CD8 cells, but rather, the readout of T cell activation determines whether mechanisms other than competition for TCR occupancy may also be involved. The second previous report that studied a class I MHC-restricted dual-TCR system was distinguished from the three other studies by the use of a T cell hybridoma rather than T cell lines (16). In this study, a TCR^- T cell hybridoma was transfected with the genes encoding two different TCRs along with the gene encoding CD8, thereby generating a CD8⁺ dual-TCR hybridoma. Using IL-2 production as the readout, no cross-antagonism was observed. Since IL-2 production by a hybridoma may have quite different signaling requirements than normal T cell lines, it is difficult to evaluate these results in the context of our results and those of the three other studies. That the assay system per se was not an issue was evidenced by our ability to demonstrate cross-antagonism of IL-2 production in the P14/OT-1 dual-TCR system (data not shown). Unfortunately, in the CD4 dual-TCR system, reciprocal experiments could not be performed, since this cell line was a poor producer of cytokines (IL-2, IFN-, IL-4) and had relatively low cytolytic activity.

Some of the possible mechanisms by which TCR antagonists inhibit agonist-induced T cell activation that have been previously considered are: 1) competition for TCR occupancy; 2) sequestration of downstream signaling molecules that are present in limiting quantities; 3) generation of a negative signaling pathway by the antagonist that suppresses the agonist-induced activation pathway. The argument against the classical model of competitive inhibition of agonist binding to the receptor has been the fact that there are more TCRs than there are agonist and antagonist-MHC complexes, thereby making it impossible to saturate the TCRs of a cell with antagonist peptide (14, 31). However, there are two considerations that mitigate this conclusion. First, only a portion of the TCRs of a cell localize to the immunological synapse and are thereby involved in the activation process (32). Second, and more importantly, there is considerable evidence that effective signaling via the TCR requires engagement of at least two agonist-MHC complexes by two TCRs creating a TCR oligomer signaling complex (33, 34, 35, 36, 37). Thus, inhibition of activation by a TCR antagonist could be accomplished by preventing only a fraction of the TCRs within the synapse from engaging an agonist peptide. Consistent with a competition model are studies that have used a panel of peptides that represent a hierarchy of activation capacity including strong agonists, weak agonists, partial agonists, and antagonists (29). At each level, peptides of a lower stimulatory ranking were capable of inhibiting the activation that characterized the higher ranking peptides. Such findings are more readily explained by a competition for TCR occupancy model than a model of negative signaling or sequestration, which would require a complex set of negative signals or sequestered molecules that acted at various stages in the signaling pathways leading to T cell activation. Furthermore, the finding of peptides with mixed antagonist and agonist characteristics depending on the concentration of peptide used is difficult to reconcile with a negative signaling model (30, 38, 39). Finally, the observation that effector cell cytolysis is not susceptible to cross-antagonism strongly supports a competitive inhibition model for TCR antagonism. We also interpret the finding that when cross-antagonism was observed it was only 5–10% as efficient as antagonism of the same TCR as support for a major, albeit not exclusive, involvement of competition for TCR occupancy in the mechanism of TCR antagonism.

However, the finding of cross-antagonism of T cell proliferation clearly indicates that mechanisms other than competition for TCR also play a role in TCR antagonism. Evidence in support of generation of a negative signal by the interaction of antagonist peptides with TCR has come from biochemical experiments that showed that preincubation of T cells with antagonist peptide followed by the removal of the T cells from antagonist-loaded MHC and their subsequent stimulation with agonist peptide resulted in inhibition of tyrosine phosphorylation, decreased Lck activity, and the association of SHP-1 phosphatase with the noncognate TCR that was not engaged by antagonist peptide in a dual-TCR T cell line (18). However, the relevancy of these biochemical findings to the biology of antagonism must be considered moot, since, when CD4 T cells are separately pulsed with antagonist-loaded APCs and with agonist-loaded APCs, no inhibition of a proliferative response was observed (18, 39).

There is little evidence for or against the model of sequestration of signaling molecules critical for activation. The model was initially proposed based on observations with mast cells and the finding that antagonist ligands inhibited the phosphorylation of FcRs engaged by agonist ligands whereas they stimulated phosphorylation of those receptors engaged by the antagonist (17). It was postulated that the antagonist ligand caused sequestration of the limited quantity of the receptor-associated protein tyrosine kinase, lyn, to the antagonist bound FcRs, leaving the agonist-associated receptors with insufficient kinase to initiate an effective signaling process. Whether a similar sequestration process might function in antagonism of TCR-mediated activation remains to be explored. More information on the signaling molecules that are engaged and/or activated after antagonist stimulation of T cells will be helpful in evaluating the sequestration model in the future. The finding that an early event of T cell activation, namely activation of a cytolytic pathway, cannot be cross-antagonized whereas later events such as cytokine production and proliferation can be cross-antagonized raises the possibility that cross-antagonism may operate on a late rather than early signaling event. This possibility is currently being investigated by studying the kinetics of cross-antagonism and determining which biochemical signaling pathways are sensitive to cross-antagonism.

	Acknowledgments

 
We thank Nancy Martorana for help in the preparation of the manuscript and Yan Fei Adams for excellent technical help.

	Footnotes

 
¹ This work was supported in part by National Institutes of Health Grant AI18634 (to H.M.G.). This is publication 532 from the La Jolla Institute for Allergy and Immunology.

² Address correspondence and reprint requests to Dr. Howard M. Grey, La Jolla Institute for Allergy and Immunology, 10355 Science Park Road, San Diego, CA 92121. E-mail address: hgrey{at}liai.org

³ Abbreviations used in this paper: APL, altered peptide ligands; LCMV, lymphocytic choriomeningitis virus.

Received for publication October 31, 2002. Accepted for publication February 28, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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