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The Journal of Immunology, 2008, 181, 1760 -1766
Copyright © 2008 by The American Association of Immunologists, Inc.
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TCR Antagonism by Peptide Requires High TCR Expression1

Daniel S. Jones, Peter Reichardt, Mandy L. Ford, Lindsay J. Edwards and Brian D. Evavold2

Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Current models of T cell activation focus on the kinetics of TCR-ligand interactions as the central parameter governing T cell responsiveness. However, these kinetic parameters do not adequately predict all T cell behavior, particularly the response to antagonist ligands. Recent studies have demonstrated that TCR number is a critical parameter influencing the responses of CD4+ T cells to weak agonist ligands, and receptor density represents an important means of regulating tissue responsiveness in other receptor ligand systems. To systematically address the impact of TCR expression on CD8+ T cell responses, mAbs to the TCR {alpha}-chain and T cells expressing two TCR species were used as two different methods to manipulate the number of available TCRs on P14 and OT-I transgenic T cells. Both methods of TCR reduction demonstrated that the efficacy of antagonist peptides was significantly reduced on T cells bearing low numbers of available receptors. In addition, the ability of weak agonists to induce proliferation was critically dependent on the availability of high numbers of TCRs. Therefore, in this report we show that TCR density is a major determinant of CD8+ T cell reactivity to weak agonist and antagonist ligands but not agonist ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Historically, the activation of T lymphocytes through the TCR has been considered remarkably Ag specific. However, more recent studies have identified variants of immunogenic peptides capable of dissociating the proliferative and effector functions of Ag-specific T cells (1, 2, 3). These observations have provided the rationale for a number of studies investigating the ability of peptides containing amino acid substitutions at TCR contact residues to mediate T cell activation. Such analog peptides, termed altered peptide ligands, can be categorized as agonists, weak agonists, or partial agonists according to their ability to stimulate various hierarchical T cell responses (3). In addition, antagonist ligands, which induce no measurable effector responses on their own, markedly inhibit T cell activation when presented concomitantly with agonist peptide (4).

Several reports have correlated peptide affinity for the TCR and/or MHC with ligand potency (5, 6, 7). As a result, current models have focused on the kinetics of TCR:ligand interactions as the pivotal factor in T cell activation, as well as the role of endogenous peptide:MHC (pMHC)3 in potentiating T cell activation (8, 9). However, the relative contribution of TCR density to T cell responsiveness has received little attention (10, 11, 12, 13, 14), although receptor density represents an important mechanism regulating the sensitivity and rapidity of responses in other receptor-ligand systems. Although T cells typically express tens of thousands TCRs, T cell responses to agonist pMHC complexes require remarkably few TCR molecules (15, 16). These results draw into question the role of these excess Ag receptors. Our work has demonstrated that the ability of CD4+ T cells to respond to weak ligands is critically dependent on high TCR expression (17, 18). Specifically, CD4+ T cells with as few as 1500 available TCRs were capable of responding to agonist peptides, whereas measurable responses to several weak agonists required dramatically more TCRs (~20,000) (17). These data provided evidence supporting a spare receptor theory of T cell activation. According to the spare receptor theory, agonist ligands achieve maximal responses by engaging only a fraction of available receptors while the excess receptors, termed "receptor reserve", are required for the induction of responses by less potent ligands (19). The excess receptors provide a means for generating responses to ligands encompassing a range of affinities and potencies. In this study, we investigated the impact of TCR density on the recognition of ligands of varying potency by CD8+ T cells. We demonstrate that the activation of CD8+ T cells by weak agonist, but not agonist peptides, requires the existence of a receptor reserve. One challenge to any model of T cell activation is to describe the action of antagonist pMHC complexes. As such, we investigated whether the spare receptor theory of T cell activation accurately describes the interactions of TCR molecules with antagonist ligands. Our results demonstrate that high TCR density is required for effective T cell antagonism, indicating that TCR-mediated responses function according to a spare receptor model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

OT-I transgenic mice (20) and P14 transgenic mice (21) were purchased from The Jackson Laboratory. OT-I (V{alpha}2Vβ5) and P14 (V{alpha}2Vβ8) TCRs were visualized with PE-conjugated mAbs to Vβ5 (BD Pharmingen) and Vβ8 (BD Pharmingen), respectively (21, 22). OT-I and P14 mice were bred to generate P14 x OT-I dual transgenic mice bearing T cells expressing both TCRs. All mice were maintained according to federal guidelines by the Emory University Department of Animal Resources (Atlanta, GA).

Cell culture and reagents

Cell culture medium consisted of RPMI medium 1640 (Mediatech) supplemented with 10% FBS (Mediatech), 2 mM L-glutamine, 0.01 M HEPES buffer, 100 µg/ml gentamicin (Mediatech), and 2 x 10–5 M 2-ME (Sigma-Aldrich). All peptides were synthesized using Fmoc chemistry on a Symphony/Multiplex Peptide synthesizer (Rainin), purified by HPLC (purity > 90%) and analyzed by mass spectrometry at the Emory University Department of Chemistry Core Facility (Atlanta, GA). The OT-I agonist OVA257–264 (SIINFEKL) and antagonist E1 (EIINFEKL), as well as the P14 agonist lymphocytic choriomeningitis virus (LCMV) gp33–41 (KAVYNFATM), antagonist 36S (KAVSNFATM), and weak agonist rat dopamine β-mono-oxygenase (rDBM) (KALYNYAPI) have all been previously described (23, 24, 25). The mAbs used included PerCP-conjugated anti-CD8{alpha} (BD Pharmingen), PE-conjugated anti-Vβ5 (clone MR9-4) (Pharmingen), PE-conjugated Vβ8 (clone F23.1) (BD Pharmingen), biotinylated and PE-conjugated anti-V{alpha}2 (clone B20.1) (BD Pharmingen), and PE-conjugated anti-IFN-{gamma} (clone XMG1.2) (BD Pharmingen). LCMV gp33–41:Db and OVA257–264:Kb monomers were assembled at the Emory University Tetramer Core Facility (Atlanta, GA) and multimerized with PE- or allophycocyanin-conjugated streptavidin (Molecular Probes).

T cell proliferation assays

Proliferation of Ag-specific T cells was assessed by [3H]thymidine incorporation (26). Briefly, transgenic splenocytes were incubated with varying concentrations of agonist peptides in HBSS for 2 h at 37°C. The cells were then washed to remove free peptide, and the splenocytes were cultured at 3 x 105 cells/well in a flat-bottom, 96-well plate. For antagonism assays, various concentrations of antagonist peptides were added to the appropriate wells. After 48 h, the cells were pulsed with 0.4 µCi of [3H]thymidine for 18–24 h, at which time the cells were harvested and the cpm were counted on a Matrix 96 direct β-counter (Packard Instruments).

Intracellular cytokine production

Transgenic splenocytes were incubated with various peptides in HBSS for 2 h at 37°C, washed, resuspended in complete medium, and cultured in a 24-well plate (3 x 106 cells/well) with 50 U/ml IL-2 (26). At day 4, the live cells were collected by centrifugation over a Ficoll gradient (Mediatech). EL-4 thymoma cells were incubated with various peptides in HBSS for 2 h at 37°C, washed, resuspended in cell culture medium, and transferred to flat-bottom, 96-well plates (2 x 105 cells/well). For antagonist assays, the EL-4 APCs were incubated with continuous antagonist peptides before the addition of T cells. T cells (2 x 105 cells/well) were added to each experimental well with 50U/ml IL-2 and incubated for 6 h at 37°C. The cells were then transferred to round-bottom, 96-well plates, fixed, and permeabilized (Caltag Laboratories). IFN-{gamma} production was detected intracellularly with PE-conjugated monoclonal rat anti-mouse IFN-{gamma} Abs (BD Pharmingen). In these experiments, CD8 was visualized with PerCP-conjugated monoclonal rat anti-mouse CD8{alpha} Abs (BD Pharmingen).

Determination of TCR level and tetramer staining

Quantitative analyses of TCR expression were achieved using Quantum R-PE microbeads with 500–50,000 molecules of equivalent soluble fluorochrome (MESF) (Bangs Laboratories) and PE-conjugated anti-V{alpha}2 (27). For tetramer staining, cells were incubated for 30 min on ice with 1 µg of tetramer per 1 x 106 cells. For Ab blockade of tetramer binding, cells were preincubated with indicated concentrations of Abs for 1 h at 37°C and then stained with tetramer as described. Flow cytometric data was collected on a BD FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Manipulation of TCR density and effective blockade of the pMHC binding site using mAbs to the TCR {alpha}-chain

As an initial step in our investigation of the relationship between TCR density and T cell responsiveness, the number of available TCRs in the presence of various concentrations of mAbs to the TCR {alpha}-chain was determined by quantitative flow cytometry (Fig. 1) (27). P14 or OT-I transgenic splenocytes were incubated with 2-fold dilutions of anti-V{alpha}2 (0.06–15 µg/ml) (Fig. 1A). The unoccupied TCRs were then visualized with R-PE-conjugated anti-V{alpha}2 mAbs and quantitated using Quantum R-PE microbeads. Quantitative analyses verified that the doses of Abs chosen resulted in a range of TCR densities (Fig. 1B). To determine whether Abs to the TCR {alpha}-chain successfully inhibited the TCR:pMHC interaction, P14 and OT-I T cells were incubated with gp33–41:Db and SIINFEKL/Kb tetramers, respectively, in the presence of anti-V{alpha}2 Abs. Importantly, anti-V{alpha}2 Abs inhibited tetramer staining in a dose-dependent manner, demonstrating that treatment of T cells with these Abs effectively masks the pMHC binding site of the TCR (>98% inhibition; Fig. 1C). In addition, the minimal expression of TCR required for tetramer staining of these two T cell lines was identified. These data indicated that OT-I requires fewer TCR (~1825), as compared with the P14 T cell, which required 4000–7000 TCR for detectable tetramer staining. Thus, inhibition of tetramer binding using anti-V{alpha}2 Abs demonstrated the effectiveness of Ab treatment for decreasing TCR density. These data also indicated differences in affinity of TCR for ligand, with the affinity of OT-I TCR for ligand being greater than that of P14 TCR.


Figure 1
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  		FIGURE 1. mAbs to V{alpha}2 provide a range of densities of available TCRs and effectively inhibit TCR:pMHC interaction. A, To establish the relationship between Ab concentration and available TCR density, the number of unbound TCRs on P14 or OT-I T cells at each corresponding Ab concentration was determined by quantitative flow cytometry. {alpha}-V{alpha}2, Anti-V{alpha}2. B, The concentrations of Abs used resulted in a range of TCR densities, representing ~5–100% of original levels. The data indicated that the anti-V{alpha}2 Abs were effective at masking both P14 ({blacksquare}) and OT-I ({circ}) TCRs. C, P14 and OT-I T cells that had been incubated with various concentrations of anti-V{alpha}2 mAbs were stained with gp33–41:Db and SIINFEKL:Kb MHC tetramers, respectively. Results demonstrated an inverse correlation between Ab concentration and tetramer binding, indicating that anti-V{alpha}2 mAbs effectively mask the pMHC binding site of the TCR.

 
TCR expression level is a critical parameter governing T cell responsiveness to suboptimal ligands

To assess whether agonist and weak agonist peptides exhibit differential requirements for TCR density, proliferation assays were conducted in the presence of various concentrations of mAbs to V{alpha}2 (Fig. 2). P14 T cells were stimulated with either the agonist peptide gp33–41 (Fig. 2A) or the weak agonist peptide rDBM (Fig. 2B) in the presence of the indicated concentrations of anti-V{alpha}2 Abs, resulting in a wide range of available TCR densities. Although the presence of high concentrations of anti-V{alpha}2 reduced the number of available TCRs by >95%, this reduction in TCR density had little effect on agonist-induced proliferation of P14 T cells (Fig. 2A). In contrast, proliferation induced by the weak agonist rDBM was dramatically inhibited at low densities of available TCRs (Fig. 2B). Of note, biotinylated and R-PE-conjugated Abs proved the most effective at inhibiting T cell responses and tetramer staining, respectively, indicating a role for steric hindrance in their ability to inhibit pMHC binding. We have previously demonstrated that higher concentrations of this Ab (50 µg/ml) completely abrogate the ability of both P14 and OT-I T cells to bind to agonist pMHC (28). However, the concentrations used in our experiments are sufficient to alter the functional response to weak ligands.


Figure 2
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  		FIGURE 2. mAbs to V{alpha}2 inhibited the function of P14 weak agonist and antagonist ligands but not agonist ligands. Naive P14 splenocytes were stimulated with the indicated concentrations of gp33–41 in the presence of anti-V{alpha}2 mAbs. A, mAb treatment has no effect on agonist stimulation of proliferation. B, The stimulatory capacity of the weak agonist, rDBM was decreased by the addition of anti-V{alpha}2 mAbs. C, The ability of the antagonist peptide (36S) to inhibit proliferation induced by stimulation with agonist (1 nM gp33–41) was decreased in the presence of increasing concentrations of anti-V{alpha}2 mAbs.

 
Next, we used anti-V{alpha}2 Abs to assess the role of TCR density in determining antagonist peptide efficacy. The impact of mAbs to V{alpha}2 on the ability of 36S to antagonize the ex vivo proliferation of P14 T cells following stimulation with gp33–41 is shown in Fig. 2C. Decreasing the number of TCRs available for productive interactions with pMHC ligands drastically reduced the ability of 36S to inhibit agonist-induced proliferation of P14 T cells. The addition of 4.7 µg/ml anti-V{alpha}2 resulted in the complete abrogation of TCR antagonism (Fig. 2C). Importantly, this observation was not unique to the P14 TCR, as similar results were obtained using OT-I (V{alpha}2Vβ5) T cells (Fig. 3, A and B). The presence of 15 µg/ml anti-V{alpha}2 resulted in the absolute loss of antagonist peptide function while maintaining the response of OT-I T cells to the agonist peptide OVA257–264 (Fig. 3, B and A, respectively). In conjunction with the results in Fig. 1, these data highlight the disparate requirements for TCR density exhibited by agonist and antagonist ligands. Specifically, the efficacy of antagonist ligands for both the P14 and OT-I TCRs was negatively impacted by a ~60% reduction in TCR density, whereas the agonist-induced responses remained intact with <5% of the TCRs remaining. Thus, reducing the number of available TCRs selectively inhibits the function of suboptimal ligands while leaving agonist-induced responses intact. These results also indicate that antagonist ligands make use of the receptor reserve to inhibit T cell activation.


Figure 3
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  		FIGURE 3. mAbs to V{alpha}2 inhibited the function of an OT-I antagonist but not that of the agonist ligand. A, Treatment with anti-V{alpha}2 mAbs had no effect on the agonist-induced stimulation of OT-I T cells as measured by the proliferation of naive OT-I splenocytes in response to the indicated concentrations of OVA257–264. B, The addition of anti-V{alpha}2 mAbs decreased the effectiveness of the antagonist E1 at inhibiting agonist-induced (1 nM OVA257–264) proliferation.

 
The correlation between antagonist peptide efficacy and TCR density is clearly illustrated in Fig. 4. Although the degree of maximal inhibition was dependent on the dose of the antagonist, the ability of both 36S and E1 to inhibit agonist-induced responses strongly correlated (r2 = 0.9–1.0) with the number of available TCRs at all doses (Fig. 4, A and B, respectively). Strikingly, a precipitous decline in antagonist efficacy was observed at TCR densities below ~5000 TCR/cell at all concentrations of antagonist peptide tested (Fig. 4, A and B).


Figure 4
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  		FIGURE 4. Antagonist peptide efficacy correlated with TCR density at all doses of antagonist ligand. The number of TCRs available at each dose of anti-V{alpha}2 mAb was plotted against the corresponding observed percentage inhibition of agonist-induced proliferation. Regression analysis was conducted to determine the degree of correlation between TCR density and antagonist efficacy (solid lines). Antagonism assays conducted in the presence of neutralizing Abs to V{alpha}2 revealed that the ability of both P14 (A) and OT-I (B) TCR antagonist to inhibit T cell activation correlated with TCR density (r2 = 0.9–1.0). In addition, at a given TCR density the inhibition of both P14 (A) and OT-I (B) T cell proliferation was more effective at higher doses of antagonist peptide.

 
Anti-V{alpha}2 Ab does not activate OT-I or P14 T cells

We verified that the clone of the anti-V{alpha}2 Ab used in these studies (B20.1) was nonstimulatory by treating P14 or OT-I T cells with up to 15 µg/ml Ab. This concentration of Ab did not result in up-regulation of CD25 or CD44 (Fig. 5A) proliferation (Fig. 5B) or cytokine production (data not shown). Thus, we conclude that any potential activating properties of this Ab would not be detectable in our readouts of proliferation and cytokine production.


Figure 5
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  		FIGURE 5. Anti-V{alpha}2 Ab does not activate OT-I cells. A, OT-I splenocytes were stimulated for 48 h with 5 µg/ml anti-V{alpha}2 Ab. Cells were stained for CD8, V{alpha}2, CD25, and CD44. Histograms were gated on CD8+ cells. The shaded histogram represents Ab-treated cells and the open histogram represents untreated cells. No significant difference in CD25 or CD44 up-regulation was detectable. B, Proliferation induced by the anti-V{alpha}2 Ab was assessed by culturing cells for 48 h with the indicated concentration of Ab. [3H]Thymidine was added and cells were cultured for an additional 18 h. Differences in proliferation were not statistically significant.

 
Differential expression of Vβ5 and Vβ8 on T cells isolated from P14 x OT-I dual transgenic mice results in limited responsiveness

Another means of reducing TCR expression is through the generation of T cells expressing two TCRs. This can be accomplished through the breeding of two TCR transgenic mice to yield mice bearing T cells that express both TCRs. As a result of the finite number of TCRs that can be expressed on the T cell surface, both TCRs cannot be present at parental levels. In this study, P14 TCR transgenic mice were bred to OT-I TCR transgenic mice to generate F1 progeny bearing dual receptor T cells (Fig. 6). Flow cytometric analyses indicated that while CD8+ T cells from P14 x OT-I F1 mice expressed nearly equivalent levels of Vβ5 as those isolated from parental OT-I mice, the Vβ8 TCR was expressed at ~15% of parental P14 levels (Fig. 6, A, and B, respectively). This data revealed that the OT-I and P14 receptors represented ~85% and 15% of the TCRs at the cell surface, respectively (Fig. 6). Tetramer staining of the OT-I TCR was similar to that of the single transgenic. However, tetramer staining of the P14 receptor was undetectable, which is to be expected based on Fig. 1, as 15% of the receptors would be ~3000 P14 TCR. Thus, the conserved expression of the OT-I TCR and the reduced expression of the P14 TCR are significant features of this model.


Figure 6
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  		FIGURE 6. P14 x OT-I T cells expressed lower Vβ8 relative to cells from parental single transgenics, but Vβ5 levels were maintained at near parental levels. A, Levels of Vβ5 expression on P14 x OT-I T cells (solid line) was similar to those on cells from OT-I mice (dotted line). P14 cells were included as a negative control (shaded). B, Vβ8 expression is decreased on dual receptor cells (solid line) relative to P14 single transgenic cells (dotted line). OT-I cells were included as a negative control (shaded). C, P14 (left panel) and dual TCR cells (right panel) exhibited similar proliferative responses to agonist stimulation ({square}), but responses to the weak agonist rDBM ({blacktriangleup}) were abrogated in the P14 x OT-I cells.

 
Despite this skewing of receptor expression, CD8+ T cells isolated from P14 x OT-I mice responded to both Ags in a dose-dependent manner as determined by [3H]thymidine incorporation and intracellular staining for IFN-{gamma} production (Fig. 7 and data not shown). Notably, although expression of the P14 TCR on P14 x OT-I T cells was reduced by ~85% relative to parental T cells, these cells remained remarkably sensitive to stimulation with the agonist gp33–41 as demonstrated by the similar dose-response curve exhibited by both single and dual receptor T cells (Fig. 6C, left panel). However, no proliferative response was observed when P14 x OT-I T cells were stimulated with the weak agonist, rDBM (Fig. 6C, right panel). Strikingly, these data indicate that while the response to agonist remains intact, the reduced P14 TCR expression of P14 x OT-I T cells renders these cells at least 10,000-fold less sensitive than parental P14 T cells to stimulation with the weak agonist.


Figure 7
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  		FIGURE 7. The OT-I antagonist (E1) but not the P14 antagonist (36S) potently inhibited the production of IFN-{gamma} by P14 x OT-I dual receptor T cells through either TCR. P14 x OT-I T cells were cultured for 4 days with gp33–41, restimulated with either 1 nM gp33–41 (A, left panel), or 1 nM OVA257–264 (B, left panel) for 6 h and stained for the presence of intracellular IFN-{gamma}. The addition of 10 µM E1 (A and B, center panels) but not 10 µM 36S (A and B, right panels) dramatically inhibited the responses of dual receptor T cells to either Ag. E1-mediated inhibition of both gp33–41 (C, left panel) and OVA257–264 (C, right panel) was dose dependent.

 
Cross-talk antagonism of IFN-{gamma} production by CD8+ P14 x OT-I dual receptor T cells

Dual TCR models have been used by a number of groups in an attempt to elucidate the mechanism of T cell antagonism. In such models, the presence of two independent TCR species minimizes or eliminates competition between agonist and antagonist pMHC complexes for available TCRs. Therefore, the ability of antagonist ligands for one TCR to inhibit the T cell responses induced via the second TCR, termed "cross-talk antagonism," would suggest that antagonists function either through the sequestration of intracellular molecules or the production of a negative signal. In these experiments, we examined whether antagonist peptides could inhibit IFN-{gamma} production through either conventional or cross-talk antagonism (Fig. 7). This assay allowed us to assess the inhibition of T cell activation on a per cell basis. Antagonists for the P14 and OT-I TCRs were tested for their ability to inhibit P14 x OT-I T cell responses induced by either the P14 agonist LCMV gp33–41 or the OT-I agonist OVA257–264 (Fig. 7). The OT-I antagonist E1 potently reduced the number of cells producing IFN-{gamma} in response to OVA257–264 (>85%; Fig. 7B, compare left and center panels; Fig. 7C, right panel). Furthermore, the antagonist for the OT-I TCR (E1) reduced the response of P14 x OT-I T cells to the P14 agonist (gp33–41) by >80% (Fig. A, compare left and center panels; Fig. 7C, left panel). However, the P14 antagonist 36S was unable to significantly inhibit the agonist-induced responses provoked through either the P14 TCR or the dominantly expressed OT-I TCR (Fig. 7C). Therefore, while the 36S antagonist for P14 potently inhibited both the proliferation (Figs. 2C and 4A) and the production of IFN-{gamma} (data not shown) by P14 single TCR-expressing T cells, minimal inhibition (~20%) of agonist-induced response in P14 x OT-I dual receptor T cells was observed (Fig. 7C) in concordance with the results from a previous study (29).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
According to the spare receptor theory, agonist ligands achieve maximal responses by engaging far fewer receptors than are required for the induction of responses by less potent ligands (19). Thus, receptor density represents an important mechanism for regulating cellular and, subsequently, tissue responsiveness. Our previous studies have shown that a spare receptor model accurately describes the responses of CD4+ T cells to weak agonist peptides typified by altered peptide ligands or autoimmune Ags (17, 30). In this study we extend our original observations and demonstrate that this model also describes the responses of CD8+ T cells to agonist and weak agonist as well as antagonist ligands. For both the P14 and OT-I systems, reducing the number of available TCRs drastically diminished both weak agonist and antagonist peptide potency while leaving responses to agonist ligands relatively unaffected (Figs. 2 and 3). It is possible that the antagonist peptides used in these experiments vary in potency. However, the data we have presented demonstrate a similar ability of E1 and 36S to antagonize their respective T cells (Figs. 2 and 3), allowing us to compare these two transgenic systems. These results suggest that high TCR density allows responsiveness to a wide variety of ligands. This ability to respond to suboptimal ligands has been shown to play a critical role in thymic selection (31, 32), and antagonist ligands have been shown to function as surrogate self-ligands in the promotion of the survival of naive CD8+ T cells (33). The finding that T cell responses to weak ligands are absolutely dependent on TCR density implies that the ability to perceive self-ligands and subsequently survive is also dependent on TCR density. This requirement may represent an important selective pressure in the maintenance of high TCR density. However, this ability to respond to a variety of ligands due to the existence of a receptor reserve may also contribute to T cell cross-reactivity (34).

The mechanism by which antagonist peptides inhibit T cell activation remains incompletely understood, although commonly proposed mechanisms include competition for available TCRs (4, 35, 36), the sequestration of intracellular signaling intermediates (37), and the production of a qualitatively unique negative signal by TCRs engaged by antagonist ligands (38, 39). Dual TCR models have been used by a number of groups in an attempt to elucidate the mechanism of T cell antagonism, as there is minimal competition for TCRs in such dual receptor systems (26, 35, 36, 39). Initial studies using CD8+ T cells failed to demonstrate cross-talk antagonism (29, 36), but another study and the data presented here report the opposite findings (29). In light of our current results, the establishment of TCR density as a critical parameter governing the efficacy of antagonist ligands provides new insights regarding the interpretation of these results from dual receptor models. In the initial studies in CD8+ dual TCR models where cross-talk antagonism was not observed, the T cells displayed similar albeit equally reduced TCR expression levels (36). Our current data and our previous CD4+ dual receptor model found substantial skewing of TCR expression such that one receptor was represented at near parental levels (26). Of interest, only antagonist ligands for the dominantly expressed receptor were capable of inhibiting T cell responses in trans (26).

The observation that the spare receptor theory accurately describes the action of TCR antagonists also provides correlative evidence for their ability to signal. Proponents of the competition model of TCR antagonism have suggested that, by virtue of their rapid dissociation rates, antagonist peptides interact with a large number of TCRs and thus merely prevent agonist complexes from triggering the number of TCRs required for activation (40). As such, the competition model predicts that as the number of available TCRs approaches the number required to support activation (~1500), antagonist pMHC complexes would need to sequester fewer TCRs to inhibit activation. Thus, whether considering either the total TCR population or the fraction of this population localized to the T cell:APC contact zone, a strict competition model predicts that antagonist peptides would be most effective at inhibiting the responses of Ag-specific T cells at low TCR density. Therefore, our observation that antagonist peptide efficacy is directly proportional to TCR density is inconsistent with the competition model of TCR antagonism. In contrast, if antagonist peptides function through the activation of intracellular signaling molecules, then the spare receptor theory predicts that sufficient TCR density is critical for the ability of antagonist ligands to produce a negative signal. The mediation of antagonism by a negative signal has been supported by the observation that the Src homology domain containing protein tyrosine phosphatase (SHP-1) is activated during T cell antagonism in CD4+ T cells and is required for effective inhibition of T cell activation by antagonist pMHC ligands (41, 42).

Our data from both the single and dual TCR transgenic systems highlight the significance of TCR number in governing the potency of weak agonist and antagonist peptides. It is important that our observations were consistent in both model systems, as this limits the potential alternative explanations for our findings. The initial findings were made using V{alpha}-specific Abs as a means to limit TCR access to pMHC Ag and were based on studies where mAbs have proven to be potent and effective competitors for binding at the cellular and purified protein levels (18, 43, 44). The high affinity interaction between TCR and Ab makes it unlikely that our data can be explained by the inability of the weak agonist and antagonist pMHC complexes to compete as effectively as agonist for TCR. The dual TCR system also eliminates this possibility. Similarly, the Ab treatment of the cells could alter the antagonists’ effects by altering signal transduction pathways, TCR dimerization, or some other stereological aspect of TCR:pMHC interaction. Again, these potentially confounding effects were mitigated by using the dual TCR system. The dual TCR system alone may allow for alternative interpretations in that the presence of two different TCRs could affect engagement or clustering in some manner. However, the consistent inhibition of responses to weak ligands that we have observed in these complementary approaches suggests that the reduction of TCR and the spare receptor model of T cell activation are the most likely explanations for the data presented here.

Peptide:MHC tetramer staining was included to demonstrate specificity of the anti-V{alpha}2 Ab in altering TCR density, but it also revealed several features related to Ag recognition by T cells. For example, fewer TCRs were needed to detect functional T cell responses (<800) as compared with tetramer staining (several thousand; Fig. 1 as compared with Figs. 2 and 3). This would suggest that although MHC tetramer staining is extremely valuable for assessing levels of response, it may also identify fewer Ag reactive cells than are actually present if the T cells are of sufficiently low affinity or express low numbers of Ag-reactive TCRs. We made use of T cells with multiple TCRs to confirm the minimal TCR density required for response to weak ligands, but dual receptor cells can avoid allelic exclusion in the thymus and are normally found in the periphery (45). Thus, T cells with low specific density could contribute to a functional T cell response and yet be undetectable by a specific peptide:MHC tetramer.

A recent study demonstrated that the proliferation, but not the cytolytic function, of CD8+ T cells is sensitive to cross-talk antagonism (29). In this study, we report that antagonist ligands for the OT-I TCR effectively inhibit IFN-{gamma} production induced by the P14 agonist in the P14 x OT-I dual TCR model. Although Yang et al. proposed that antagonist ligands may inhibit cytolysis through a different mechanism than proliferation, another possible explanation is that both proliferation and cytokine production require de novo mRNA and protein synthesis (46, 47) and, therefore, the antagonist ligand may have a greater opportunity to interrupt the signaling cascade. In contrast, cytolysis results from the release of preformed granules (48), potentially rendering the process more difficult to inhibit. Although the dependence of antagonist peptide efficacy on TCR density supports the ability of antagonist ligands to induce a negative signal, an answer to the question of whether distinct mechanisms are active in the inhibition of different effector functions will require further investigation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI056017. Back

2 Address correspondence and reprint requests to Dr. Brian D. Evavold, Department of Microbiology and Immunology, Emory University, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: evavold{at}microbio.emory.edu Back

3 Abbreviations used in this paper: pMHC, peptide:MHC interaction; LCMV, lymphocytic choriomeningitis virus; rDBM, rat dopamine β-mono-oxygenase. Back

Received for publication August 3, 2007. Accepted for publication May 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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