TCR antagonism by peptide requires high TCR expression

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.     

Cutting edge: dueling TCRs: peptide antagonism of CD4+ T cells with dual antigen specificities.

T cells expressing two different TCRs were generated by interbreeding 3A9 and AND CD4+ TCR transgenic mice specific for the hen egg lysozyme (HEL) peptide 48-62:I-Ak and moth cytochrome c (MCC) peptide 88-103:I-Ek peptide:MHC ligands, respectively. Peripheral T cells in the offspring express two TCR V beta-chains and respond to HEL and MCC. We observed minimal or no additive effects upon simultaneous suboptimal stimulation with both agonist peptides; however, an antagonist peptide for the 3A9 TCR was able to inhibit the response of the dual receptor T cells to MCC, the AND TCR agonist. This HEL antagonist peptide did not affect AND single transgenic T cells, indicating that the antagonism observed in the dual TCR cells is dependent on the presence of the HEL-specific 3A9 TCR. In contrast, anti-TCR Abs mediate receptor-specific antagonism. These results demonstrate that peptide antagonism exerts a dominant effect.     

Mechanisms for T cell receptor triggering

There is considerable controversy about the mechanism of T cell receptor (TCR) triggering, the process by which the TCR tranduces signals across the plasma membrane after binding to its ligand (an agonist peptide complexed with an MHC molecule). Three main types of mechanism have been proposed, which involve aggregation, conformational change and segregation. Here, we review recently published evidence for each type of mechanism and conclude that all three may be involved. This complexity may reflect the uniquely demanding nature of TCR-mediated antigen recognition, which requires the detection of a very weak 'signal' (very rare foreign peptide-MHC ligands) in the presence of considerable 'noise' (abundant self peptide-MHC molecules).     

Altered peptide ligand-mediated TCR antagonism can be modulated by a change in a single amino acid residue within the CDR3 beta of an MHC class I-restricted TCR

The Ag receptor of cytotoxic CD8+ T lymphocytes recognizes peptides of 8-10 aa bound to MHC class I molecules. This Ag recognition event leads to the activation of the CD8+ lymphocyte and subsequent lysis of the target cell. Altered peptide ligands are analogues derived from the original antigenic peptide that commonly carry amino acid substitutions at TCR contact residues. TCR engagement by these altered peptide ligands usually impairs normal T cell function. Some of these altered peptide ligands (antagonists) are able to specifically antagonize and inhibit T cell activation induced by the wild-type antigenic peptide. Despite significant advances made in understanding TCR antagonism, the molecular interactions between the TCR and the MHC/peptide complex responsible for the inhibitory activity of antagonist peptides remain elusive. To approach this question, we have identified altered peptide ligands derived from the vesicular stomatitis virus peptide (RGYVYQGL) that specifically antagonize an H-2Kb/vesicular stomatitis virus-specific TCR. Furthermore, by site-directed mutagenesis, we altered single amino acid residues of the complementarity-determining region 3 of the beta-chain of this TCR and tested the effect of these point mutations on Ag recognition and TCR antagonism. Here we show that a single amino acid change on the TCR CDR3 beta loop can modulate the TCR-antagonistic properties of an altered peptide ligand. Our results highlight the role of the TCR complementarity-determining region 3 loops for controlling the nature of the T cell response to TCR/altered peptide ligand interactions, including those leading to TCR antagonism.     

T cell antagonism is functionally uncoupled from the 21- and 23-kDa tyrosine-phosphorylated TCR zeta subunits

The functional effects of altered peptide ligands on T cells is proposed to involve differential intracellular signaling mediated by the 21- and 23-kDa tyrosine-phosphorylated derivatives of the TCR zeta subunit (p21 and p23). To understand the functional contribution of p21 and p23 to T cell development and T cell antagonism, we generated selected TCR zeta transgenic mice maintained on the P14 alphabeta TCR transgenic line such that p23 or both p21 and p23 were selectively eliminated. Importantly, one line (YF1,2) retains the constitutively tyrosine-phosphorylated p21 in the complete absence of inducible p23. We determined that T cell development was uncoupled from p21 and/or p23. Using a series of agonist, weak agonist, and antagonist peptides, we analyzed the role of each of the phosphorylated forms of TCR zeta on T cell activation and antagonism. In this study, we report that the proliferative responses of alphabeta P14 T cells to agonist peptides and the inhibition of proliferation resulting from antagonist peptide treatments was functionally uncoupled from p21 and/or p23. These results suggest that the mechanism of T cell antagonism is independent of the two phosphorylated TCR zeta derivatives.     

Differential CD3 zeta phosphorylation is not required for the induction of T cell antagonism by altered peptide ligands.

T cells recognize foreign Ags in the form of short peptides bound to MHC molecules. Ligation of the TCR:CD3 complex gives rise to the generation of two tyrosine-phosphorylated forms of the CD3 zeta-chain, pp21 and pp23. Replacement of residues in MHC-bound peptides that alter its recognition by the TCR can generate altered peptide ligands (APL) that antagonize T cell responses to the original agonist peptide, leading to altered T cell function and anergy. This biological process has been linked to differential CD3zeta phosphorylation and generation of only the pp21 phospho-species. Here, we show that T cells expressing CD3zeta mutants, which cannot be phosphorylated, exhibit a 5-fold reduction in IL-2 production and a 30-fold reduction in sensitivity following stimulation with an agonist peptide. However, these T cells are still strongly antagonized by APL. These data demonstrate that: 1) the threshold required for an APL to block a response is much lower than for an agonist peptide to induce a response, 2) CD3zeta is required for full agonist but not antagonist responses, and 3) differential CD3zeta phosphorylation is not a prerequisite for T cell antagonism.     

Surface-anchored monomeric agonist pMHCs alone trigger TCR with high sensitivity

At the interface between T cell and antigen-presenting cell (APC), peptide antigen presented by MHC (pMHC) binds to the T cell receptor (TCR) and initiates signaling. The mechanism of TCR signal initiation, or triggering, remains unclear. An interesting aspect of this puzzle is that although soluble agonist pMHCs cannot trigger TCR even at high concentrations, the same ligands trigger TCR very efficiently on the surface of APCs. Here, using lipid bilayers or plastic-based artificial APCs with defined components, we identify the critical APC-associated factors that confer agonist pMHCs with such potency. We found that CD4⁺ T cells are triggered by very low numbers of monomeric agonist pMHCs anchored on fluid lipid bilayers or fixed plastic surfaces, in the absence of any other APC surface molecules. Importantly, on bilayers, plastic surfaces, or real APCs, endogenous pMHCs did not enhance TCR triggering. TCR triggering, however, critically depended upon the adhesiveness of the surface and an intact T cell actin cytoskeleton. Based on these observations, we propose the receptor deformation model of TCR triggering to explain the remarkable sensitivity and specificity of TCR triggering.     

Alteration of cell surface sialylation regulates antigen-induced naive CD8+ T cell responses

The strength of interactions with APC instructs naive T cells to undergo programmed expansion and differentiation, which is largely determined by the peptide affinity and dose as well as the duration of TCR ligation. Although, most ligands mediating these interactions are terminally sialylated, the impact of the T cell sialylation status on Ag-dependent response remains poorly understood. In this study, by monitoring TCR transgenic CD8+ T cells, OT-I, we show that biochemical desialylation of naive OT-I T cells increases their sensitivity for agonist as well as partial agonist peptides. Desialylation enhances early activation and shortens the duration of TCR stimulation required for proliferation and differentiation, without increasing apoptosis. Moreover, desialylation of naive OT-I T cells augments their response to tumor-presented Ag. These results provide direct evidence for a regulatory role for sialylation in Ag-dependent CD8+ T cell responses and offer a new approach to sensitize or dampen Ag-specific CD8+ T cell responses.     

Partial T cell activation with an altered superantigenic ligand

T cells have the capacity to respond to ligands as full, weak, partial or null agonists, or indeed as antagonists. In the present paper, it is reported that staphylococcal enterotoxin B (SEB) mutated in a T cell receptor (TCR) contact site (SEBDelta61Y) behaves as an altered ligand for a T cell clone (AC20) that expresses the Vbeta17 TCR. The T cells were partially activated by SEBDelta61Y, as shown by TCR down-modulation and up-regulation of the IL-2 receptor. However, these cells did not secrete IL-2, IL-3, IL-4 or IFN-gamma, nor did they proliferate. Analysis of intracellular protein tyrosine phosphorylation after cellular activation provided further evidence that SEBDelta61Y could transduce a signal via the Vbeta17 TCR. The events following receptor ligation were clearly different when the T cells were stimulated with SEB or SEBDelta61Y, manifested as both quantitatively and qualitatively different patterns of phosphorylation of intracellular substrates. In contrast, only quantitative differences were apparent when a transfectant expressing the same alpha/beta TCR was stimulated with the different superantigens. Together, these results provide the first demonstration that altered TCR ligands are not restricted to peptides substituted at secondary TCR contact residues. Rather, an altered superantigenic ligand mutated in the TCR binding site can behave as a partial agonist.     

Induction of T cell anergy by low numbers of agonist ligands.

Engagement of TCR by its ligand, the MHC/peptide complex, causes T cell activation. T cells respond positively to stimulation with agonists, and are inhibited by antagonist MHC/peptide ligands. Failure to induce proper conformational changes in the TCR or fast TCR/MHC dissociation are the leading models proposed to explain anergy induction by antagonist ligands. In this study, we demonstrate that presentation of between 1 and 10 complexes of agonist/MHC II by unfixed APC induces T cell anergy that persists up to 7 days and has characteristics similar to anergy induced by antagonist ligand or TCR occupancy without costimulation. Furthermore, anergy-inducing doses of hemagglutinin 306-318 peptide led to the engagement of less than 1000 TCR/CD3 complexes. Thus, engagement of a subthreshold number of TCR by either a low density of agonist/MHC or a 2-3 orders of magnitude higher density of antagonist/MHC causes anergy. Moreover, we show that anergy induced by low agonist concentrations is inhibited in the presence of IL-2 or cyclosporin A, suggesting involvement of the calcineurin signaling pathway.     

In vivo modulation of T cell responses and protective immunity by TCR antagonism during infection

Infectious agents are known to express altered peptide ligands that antagonize T cells in vitro; however, direct evidence of TCR antagonism during infection is still lacking, and its importance in the context of infection remains to be established. In this study, we used a murine model of infection with recombinant Listeria monocytogenes and addressed three issues that are critical for assessing the role of TCR antagonism in the modulation of the immune response. First, we demonstrated that the antagonist peptide efficiently inhibited the ability of the agonist to prime naive TCR-transgenic T cells in vivo. Second, we showed clonal memory T cells were antagonized during recall responses, resulting in loss of protective immunity. Lastly, we observed that even in the context of a polyclonal response, TCR antagonism greatly inhibits the agonist-specific response, leading to altered hierarchy of immunodominance and reduced T cell memory and protective immunity. These results provide direct evidence of clonal TCR antagonism of naive and memory CD8 T cells during infection and demonstrate the effect of TCR antagonism on protective immunity. Thus, agonist/antagonist interactions may play an important role in determining the immunodominance and repertoire of T cell targets, and evaluation of immune responses and vaccine strategies may require examination of not only agonists but also antagonists and their interactions during an infection.     

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.     

Enhanced expression of lymphotactin by CD8+ T cells is selectively induced by enhancer agonist peptides of tumor-associated antigens

Human tumor-associated antigens (TAAs) are weak immunogens. One strategy for increasing the immunogenicity of TAAs is to generate altered peptide ligands. In the studies reported here, microarray technology has been used to compare gene expression profiles of a human T cell line that was derived from the peripheral blood of a cancer patient vaccinated with a carcinoembryonic antigen (CEA)-based vaccine. We compared the gene expression profiles of this CEA-specific CD8 T cell line when (a) stimulated with the native peptide used to derive this line vs. no peptide, and (b) stimulated with its TCR enhancer agonist epitope vs. no peptide. The results demonstrate that the effect on the T cell line, when stimulated with the agonist peptide, is not an enhanced quantitative expression of the same genes or gene sets induced by the native peptide, but is rather a nearly reciprocal upregulation of different gene sets. The gene for the chemokine lymphotactin, which was overexpressed only in T cells stimulated with the agonist peptide, stood out above all others. This finding was extended using other T cell lines, and another set of agonist and native peptides from another TAA. ELISPOT and ELISA assays for lymphotactin confirmed and extended these findings. These studies suggest a potential role for lymphotactin in the T-cell activation processes and subsequent anti-tumor events.     

Differential signalling by variant ligands of the T cell receptor and the kinetic model of T cell activation

The structural basis of T cell activation through the T cell receptor is still a major unresolved issue in T cell biology. The wealth of information on the generation and structure of T cell receptor ligands and the biochemistry of signal transduction from this receptor have been useful in the initial approach to explain how T cell activation occurs. More recently, the generation of variant T cell receptor ligands with partial agonist or antagonist properties, the determination of crystal structures for unengaged and engaged T cell receptors, and the kinetics of T cell receptor interactions with peptide:MHC molecule complexes have provided new insights on T cell receptor function. The common theme arising from these experiments is that the T cell receptor is a versatile signalling machine, with an inherent flexibility for ligand recognition that translates in different signalling patterns. Here, I will review the data on differential signalling from the T cell receptor upon recognition of partial agonist and antagonist ligands and how these data impact on a more general kinetic model of T cell receptor-mediated activation.     

Cutting edge: a test of the dominant negative signal model for TCR antagonism

The mechanism by which TCR antagonists interfere with T cell activation is unclear. One popular hypothesis is that incomplete early signaling events induced by these ligands dominantly inhibit the T cell's ability to respond to a copresented agonist ligand. Here we test this "dominant negative" signal hypothesis by studying T cells expressing two distinct MHC class I-restricted TCRs (2C and OT-I). Although responses through each TCR can be efficiently inhibited by their specific antagonists, we found no evidence for "cross-antagonism" in which an antagonist for receptor "A" blocks responses through receptor "B." Such inhibition would have been expected were the dominant negative signaling hypothesis correct, and alternative models for TCR antagonism are discussed.     

Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3 (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency of the SEC3 variants correlated with enhanced binding without any optimum in the binding range covered by native TCR ligands. Comparable studies using anti-TCR antibodies of known affinity confirmed these observations. By comparing the biological potency of the two sets of ligands, we found a significant correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength.     

T cell receptor recognition via cooperative conformational plasticity

Although T cell receptor cross-reactivity is a fundamental property of the immune system and is implicated in numerous autoimmune pathologies, the molecular mechanisms by which T cell receptors can recognize and respond to diverse ligands are incompletely understood. In the current study we examined the response of the human T cell lymphotropic virus-1 (HTLV-1) Tax-specific T cell receptor (TCR) A6 to a panel of structurally distinct haptens coupled to the Tax 11-19 peptide with a lysine substitution at position 5 (Tax5K, LLFG[K-hapten]PVYV). The A6 TCR could cross-reactively recognize one of these haptenated peptides, Tax-5K-4-(3-Indolyl)-butyric acid (IBA), presented by HLA-A*0201. The crystal structures of Tax5K-IBA/HLA-A2 free and in complex with A6 reveal that binding is mediated by a mechanism of cooperative conformational plasticity involving conformational changes on both sides of the protein-protein interface, including the TCR complementarity determining region (CDR) loops, Valpha/Vbeta domain orientation, and the hapten-modified peptide. Our findings illustrate the complex role that protein dynamics can play in TCR cross-reactivity and highlight that T cell receptor recognition of ligand can be achieved through diverse and complex molecular mechanisms that can occur simultaneously in the interface, not limited to molecular mimicry and CDR loop shifts.     

Modification of peptide interaction with MHC creates TCR partial agonists

We report the creation of TCR partial agonists by the novel approach of manipulating the interaction between immunogenic peptide and MHC. Amino acids at MHC anchor positions of the I-E(k)-restricted hemoglobin (64-76) and moth cytochrome c (88-103) peptides were exchanged with MHC anchor residues from the low affinity class II invariant chain peptide (CLIP), resulting in antigenic peptides with altered affinity for MHC class II. Several low affinity peptides were identified as TCR partial agonists, as defined by the ability to stimulate cytolytic function but not proliferation. For example, a peptide containing methionine substitutions at positions one and nine of the I-E(k) binding motif acted as a partial agonist for two hemoglobin-reactive T cell clones (PL.17 and 3.L2). The identical MHC anchor substitutions in moth cytochrome c (88-103) also created a partial agonist for a mCC-reactive T cell (A.E7). Thus, peptides containing MHC anchor modifications mediated similar T cell responses regardless of TCR fine specificity or antigen reactivity. This data contrasts with the unique specificity among individual clones demonstrated using traditional altered peptide ligands containing substitutions at TCR contact residues. In conclusion, we demonstrate that altering the MHC anchor residues of the immunogenic peptide can be a powerful method to create TCR partial agonists.     

Supra-agonist peptides enhance the reactivation of memory CTL responses

Single amino acid substitutions at TCR contacts may transform a natural peptide Ag in CTL ligands with partial agonist, antagonist, or null activity. We obtained peptide variants by changing nonanchor amino acid residues involved in MHC class I binding. These peptides were derived from a subdominant HLA-A2-presented, latent membrane protein 2-derived epitope expressed in EBV-infected cells and in EBV-associated tumors. We found that small structural changes produced ligands with vastly different activities. In particular, the variants that associated more stably to HLA-A2/molecules did not activate any CTL function, behaving as null ligands. Interestingly, T cell stimulations performed with the combination of null ligands and the natural epitope produced significantly higher specific CTL reactivation than reactivation of CTLs induced by the wild-type epitope alone. In addition, these particular variants activated memory CTL responses in the presence of concentrations of natural epitope that per se did not induce T cell responses. We show here that null ligands increased ZAP-70 tyrosine kinase activation induced by the natural epitope. Our results demonstrate for the first time that particular peptide variants, apparently behaving as null ligands, interact with the TCR, showing a supra-agonist activity. These variant peptides did not affect the effector T cell functions activated by the natural epitope. Supra-agonist peptides represent the counterpart of antagonists and may have important applications in the development of therapeutic peptides.     

Antagonism of antiviral and allogeneic activity of a human public CTL clonotype by a single altered peptide ligand: implications for allograft rejection

Alloreactive T lymphocytes are central mediators of graft-versus-host disease and allograft rejection. A public CTL clonotype with specificity for the alloantigens HLA-B*4402 and B*4405 is often expanded to large numbers in healthy HLA-B*0801(+) individuals, driven by cross-reactive stimulation with the common, persistent herpesvirus EBV. Since such alloreactive memory CTL expansions have the potential to influence transplantation outcome, altered peptide ligands (APLs) of the target HLA-B*0801-binding EBV peptide, FLRGRAYGL, were screened as specific antagonists for this immunodominant clonotype. One APL, FLRGRFYGL, exerted powerful antagonism of a prototypic T cell clone expressing this immunodominant TCR when costimulated with target cells presenting HLA-B*0801(FLRGRAYGL). Significantly, this APL also reduced the lysis of allogeneic target cells expressing HLA-B*4402 by up to 99%. The affinities of the agonist and antagonist complexes for the public TCR, measured using solution and solid-phase assays, were 8 and 138 muM, respectively. Surprisingly, the half-life of the agonist and antagonist complexes was similar, yet the association rate for the antagonist complex was significantly slower. These observations were further supported by structural studies that suggested a large conformational hurdle was required to ligate the immunodominant TCR to the HLA-B*0801 antagonist complex. By defining an antagonist APL against an immunodominant alloreactive TCR, these findings raise the prospect of exploiting such peptides to inhibit clinical alloreactivity, particularly against clonal T cell expansions that react with alloantigens.