Contribution of individual amino acids within MHC molecule or antigenic peptide to TCR ligand potency

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.     

Soluble, high-affinity dimers of T-cell receptors and class II major histocompatibility complexes: biochemical probes for analysis and modulation of immune responses

T cell receptors (TCR) and major histocompatibility complex (MHC) molecules are integral membrane proteins that have central roles in cell-mediated immune recognition. Therefore, soluble analogs of these molecules would be useful for analyzing and possibly modulating antigen-specific immune responses. However, due to the intrinsic low-affinity and inherent solubility problems, it has been difficult to produce soluble high-affinity analogs of TCR and class II MHC molecules. This report describes a general approach which solves this intrinsic low-affinity by constructing soluble divalent analogs using IgG as a molecular scaffold. The divalent nature of the complexes increases the avidity of the chimeric molecules for cognate ligands. The generality of this approach was studied by making soluble divalent analogs of two different classes of proteins, a TCR (2C TCR2Ig) and a class II MHC (MCCI-Ek2Ig) molecule. Direct flow cytometry assays demonstrate that the divalent 2C TCR2Ig chimera retained the specificity of the native 2C TCR, while displaying increased avidity for cognate peptide/MHC ligands, resulting in a high-affinity probe capable of detecting interactions that heretofore have only been detected using surface plasmon resonance. TCR2IgG was also used in immunofluorescence studies to show ER localization of intracellular peptide-MHC complexes after peptide feeding. MCCI-Ek2Ig chimeras were able to both stain and activate an MCC-specific T cell hybridoma. Construction and expression of these two diverse heterodimers demonstrate the generality of this approach. Furthermore, the increased avidity of these soluble divalent proteins makes these chimeric molecules potentially useful in clinical settings for probing and modulating in vivo cellular responses.     

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.     

The role of polymorphic amino acids of the MHC molecule in the selection of the T cell repertoire

Allelic variants of MHC molecules expressed on cells of the thymus affect the selection and the specificity of the T cell repertoire. The selection is based on either the direct recognition by the TCR of the MHC molecules, or the recognition of a complex determinant formed by self-peptides bound to MHC molecules. In an analysis of the T cell repertoire in bone marrow chimeras that express allelic forms of MHC class II molecules in the thymus epithelium, we find that amino acid substitutions that are predicted to affect peptide binding influence the selection of the T cell repertoire during thymic selection.     

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.     

Involvement of the same region of the T cell antigen receptor in thymic selection and foreign peptide recognition.

The Ag receptor (TCR) on T lymphocytes has been shown to be specific for foreign antigenic peptides bound to MHC-encoded molecules. During T cell differentiation in the thymus this same TCR mediates the recognition of MHC molecules in the absence of foreign Ag, a process termed positive selection. To analyze the structural relationship between MHC-restricted Ag recognition and positive selection, we characterized two different transgenic lines of mice bearing TCR specific for pigeon cytochrome c and the Ek class II MHC molecule. The two TCR expressed in these animals differed by only one amino acid in the V-J junction of the alpha-chain. In vitro, we find that this TCR difference alters Ag fine specificity. Analysis of transgenic animals demonstrates that this change in the putative third complementarity determining region of the TCR also alters the specificity of positive selection in the thymus. These results suggest that the diversity of a TCR region that can be shown to affect the specificity of foreign Ag recognition may be influenced by selection in the thymus. The findings presented here are discussed in relation to the possible role of self-peptides in positive selection.     

Amino acid substitutions in the putative MHC class II "dimer of dimers" interface inhibit CD4+ T cell activation

Activation of T lymphocytes is dependent on multiple ligand-receptor interactions. The possibility that TCR dimerization contributes to T cell triggering was raised by the crystallographic analysis of MHC class II molecules. The MHC class II molecules associated as double dimers, and in such a way that two TCR (and two CD4 molecules) could bind simultaneously. Several subsequent studies have lent support to this concept, although the role of TCR cross-linking in T cell activation remains unclear. Using DRA cDNAs modified to encode two different C-terminal tags, no evidence of constitutive double dimer formation was obtained following immunoprecipitation and Western blotting from cells transiently transfected with wild-type DRB and tagged DRA constructs, together with invariant chain and HLA-DM. To determine whether MHC class II molecules contribute actively to TCR-dependent dimerization and consequent T cell activation, panels of HLA-DR1beta and H2-E(k) cDNAs were generated with mutations in the sequences encoding the interface regions of the MHC class II double dimer. Stable DAP.3 transfectants expressing these cDNAs were generated and characterized biochemically and functionally. Substitutions in either interface region I or III did not affect T cell activation, whereas combinations of amino acid substitutions in both regions led to substantial inhibition of proliferation or IL-2 secretion by human and murine T cells. Because the amino acid-substituted molecules were serologically indistinguishable from wild type, bound antigenic peptide with equal efficiency, and induced Ag-dependent CD25 expression indicating TCR recognition, the reduced ability of the mutants to induce full T cell activation is most likely the result of impaired double dimer formation. These data suggest that MHC class II molecules, due to their structural properties, actively contribute to TCR cross-linking.     

Distinct footprints of TCR engagement with highly homologous ligands

T cell receptor engagement promotes proliferation, differentiation, survival, or death of T lymphocytes. The affinity/avidity of the TCR ligand and the maturational stage of the T cell are thought to be principal determinants of the outcome of TCR engagement. We demonstrate in this study that the same mouse TCR preferentially uses distinct residues of homologous peptides presented by the MHC molecules to promote specific cellular responses. The preference for distinct TCR contacts depends on neither the affinity/avidity of TCR engagement (except in the most extreme ranges), nor the maturity of engaged T cells. Thus, different portions of the TCR ligand appear capable of biasing T cells toward specific biological responses. These findings explain differences in functional versatility of TCR ligands, as well as anomalies in the relationship between affinity/avidity of the TCR for the peptide/MHC and cellular responses of T cells.     

Influenza epitope-specific CD8+ T cell avidity, but not cytokine polyfunctionality, can be determined by TCRβ clonotype

Cytokine polyfunctionality has recently emerged as a correlate of effective CTL immunity to viruses and tumors. Although the determinants of polyfunctionality remain unclear, there are published instances of a link between the production of multiple effector molecules and the peptide plus MHC class I molecule avidity of T cell populations. Influenza A virus infection of C57BL/6J mice induces CTL populations specific for multiple viral epitopes, each with varying proportions of monofunctional (IFN-γ(+) only) or polyfunctional (IFN-γ(+)TNF-α(+)IL-2(+)) CTLs. In this study, we probe the link between TCR avidity and polyfunctionality for two dominant influenza epitopes (D(b)NP(366) and D(b)PA(224)) by sequencing the TCR CDR3β regions of influenza-specific IFN-γ(+) versus IFN-γ(+)IL-2(+) cells, or total tetramer(+) versus high-avidity CTLs (as defined by the peptide plus MHC class I molecule-TCR dissociation rate). Preferential selection for particular clonotypes was evident for the high-avidity D(b)PA(224)-specific set but not for any of the other subsets examined. These data suggest that factors other than TCRβ sequence influence cytokine profiles and demonstrate no link between differential avidity and polyfunctionality.     

Cysteinylation of MHC class II ligands: peptide endocytosis and reduction within APC influences T cell recognition

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4(+) T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.     

Peptide binding inhibits aggregation of soluble MHC class II in solution

Affinity-purified major histocompatability complex (MHC) class II molecules are known to bind antigenic peptide in vitro. This peptide-bound MHC class II is known to undergo a change in structure upon stable binding of antigenic peptide. Previous results from our, and other laboratories, have suggested a relationship between MHC class II structure and peptide association that enables class II to enter into a stable conformation upon peptide binding. In this report we describe that stable binding of high-affinity antigenic peptide to MHC class II molecule results in transition of aggregated purified MHC class II proteins to a stable heterodimeric state. Such transition was demonstrated by using purified human HLA-DR2 class II molecule and high-affinity myelin basic protein (MBP) 83-102)Y83 peptide. Highly aggregated purified DR2 (high molecular weight; HMW) was first separated from heterodimer (low molecular weight: LMW) in the presence of 50-fold molar excess of MBP(83-102)Y83 peptide. We then show that the aggregated HMW preparation can be successfully converted into a stable dimer by further incubation with MBP(83-102)Y83 and changing various binding parameters such as pH, temperature, reducing agent, and peptide concentrations. Under optimized conditions, the highly aggregated inactive DR2 molecules can be completely loaded with the antigenic peptide. The transformed heterodimers with bound peptide prepared by this method are biologically active, as shown by their ability to induce the production of gamma-interferon by SS8T-transformed human T cells. These results suggest that in solution, MHC class II molecules may be aggregated in the absence of bound peptide. Such aggregated MHC class II molecules can be converted to stable and biologically active heterodimers in the presence of high-affinity antigenic peptide.     

Structural and functional consequences of altering a peptide MHC anchor residue

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.     

Modeling alternative binding registers of a minimal immunogenic peptide on two class II major histocompatibility complex (MHC II) molecules predicts polarized T-cell receptor (TCR) contact positions.

Several major histocompatibility complex class II (MHC II) complexes with known minimal immunogenic peptides have now been solved by X-ray crystallography. Specificity pockets within the MHC II binding groove provide distinct peptide contacts that influence peptide conformation and define the binding register within different allelic MHC II molecules. Altering peptide ligands with respect to the residues that contact the T-cell receptor (TCR) can drastically change the nature of the ensuing immune response. Here, we provide an example of how MHC II (I-A) molecules may indirectly effect TCR contacts with a peptide and drive functionally distinct immune responses. We modeled the same immunogenic 12-amino acid peptide into the binding grooves of two allelic MHC II molecules linked to distinct cytokine responses against the peptide. Surprisingly, the favored conformation of the peptide in each molecule was distinct with respect to the exposure of the N- or C-terminus of the peptide above the MHC II binding groove. T-cell clones derived from each allelic MHC II genotype were found to be allele-restricted with respect to the recognition of these N- vs. C-terminal residues on the bound peptide. Taken together, these data suggest that MHC II alleles may influence T-cell functions by restricting TCR access to specific residues of the I-A-bound peptide. Thus, these data are of significance to diseases that display genetic linkage to specific MHC II alleles, e.g. type 1 diabetes and rheumatoid arthritis.     

Molecular modeling of a T-cell receptor bound to a major histocompatibility complex molecule: implications for T-cell recognition.

The main functions of the T-cell receptor (TCR) involve its specific interaction with short and linear antigenic peptides bound to the major histocompatibility complex (MHC) molecules. In the absence of a 3D structure for TCR and for the TCR/peptide/MHC complex, several attempts to characterize the structural components of the TCR/peptide/MHC interaction have been made. However, this subject is still troublesome. In this paper a computer-based 3D model for a TCR/peptide/MHC complex (5C.C7/moth cytochrome c [MCC] peptide 93-103/I-Ek) was obtained. The complex surface shows a high complementarity between the 5C.C7 structure and the peptide/I-Ek molecule. The mapping of residues involved in the TCR/peptide/MHC interaction shows close agreement with mutational experiments (Jorgensen JL, Reay PA, Ehrich EW, Davis MM, 1992b, Annu Rev Immunol 10:835-873). Moreover, the results are consistent with a recent variability analysis of TCR sequences using three variability indexes (Almagro JC, Zenteno-Cuevas R, Vargas-Madrazo E, Lara-Ochoa F, 1995b, Int J Pept Protein Res 45:180-186). Accordingly, the 3D model of the 5C.C7/MCC peptide 93-103/I-Ek complex provides a framework to generate testable hypotheses about TCR recognition. Thus, starting from this model, the role played by each loop that forms the peptide/MHC binding site of the TCR is discussed.     

Mhc-guided processing: binding of large antigen fragments

Ever since the emergence of models for the processing and presentation of antigenic determinants by MHC class II molecules, the main view has been that proteins are unfolded, enzymatically cleaved into peptide lengths of about 12-25 amino acids and then loaded onto MHC class II molecules. There is, however, an alternative model stating that partially intact unfolding antigens are first bound by MHC class II molecules and then trimmed to fragments of a smaller size while remaining bound to the MHC class II molecule. In this analysis, we make the case that a considerable portion of the elutable peptide cargo belongs to this latter class.     

Functional evidence that conserved TCR CDR alpha 3 loop docking governs the cross-recognition of closely related peptide:class I complexes.

The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2K(b) and H-2K(bm8). Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2-4 and the neighboring MHC side chains. We found that CTL clones sharing TCR beta-chains exhibited disparate recognition patterns, whereas those with drastically different TCRbeta-chains but sharing identical TCRalpha CDR3 loops displayed identical functional specificity. This suggested that the CDRalpha3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:K(b) crystal structure. Importantly, these results indicate a remarkable conservation in CDRalpha3 positioning, and, therefore, in docking of diverse TCRalphabeta heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.     

A physiological ligand of positive selection is recognized as a weak agonist

Positive selection is a process that ensures that peripheral T cells express TCR that are self-MHC restricted. This process occurs in the thymus and requires both self-MHC and self-peptides. We have recently established a TCR transgenic (TCR(trans)(+)) mouse model using the C10.4 TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize a mitochondrial NADH dehydrogenase subunit 1-derived 9-mer peptide as the physiological ligand of positive selection. Here, we demonstrate that the NADH dehydrogenase subunit 1 self-peptide is seen by mature C10.4 TCR(trans)(+) T cells as a weak agonist and induces positive selection at a defined concentration range. We also found that the full-length cognate peptide, a strong agonist for mature C10.4 TCR(trans)(+) T cells, initiated positive selection, albeit at significantly lower concentrations. At increased peptide concentrations, and thus increased epitope densities, either peptide only induced the development of partially functional T cells. We conclude that successful positive selection only proceeded at a defined, yet fairly narrow window of avidity.     

Two MHC surface amino acid differences distinguish foreign peptide recognition from autoantigen specificity

KRN T cells can recognize two self MHC alleles with differing biological consequences. They respond to the foreign peptide RN(42--56) bound to I-A(k) or alternatively initiate autoimmune arthritis by interacting with a self Ag, GPI(282--294), on I-A(g7). Five surface amino acid differences between the two MHC molecules collectively alter which peptide side chains are recognized by the KRN TCR. In this study, it is shown that mutation of only two of these residues, alpha 65 and beta 78, in I-A(k) to their I-A(g7) counterparts is sufficient to allow recognition of the TCR contacts from GPI(282--294). To provide a detailed mechanism for the specificity change, the distinct contributions of each of these two mutations to the global effect on peptide specificity were analyzed. The alpha65 mutation is shown to broaden the spectrum of amino acids permissible at P8 of the peptide. In contrast, the beta 78 mutation alone blocks KRN TCR interaction with I-A(k) and requires the simultaneous presence of the alpha 65 mutation to preserve recognition. In the presence of the alpha 65 mutation, the beta 78 residue broadens peptide recognition at P3 and prevents recognition of the P8 L in RN(42--56), thus producing the observed specificity shift. These results localize the functionally relevant differences between the surfaces of two self-restricted MHC molecules to two residues that have counterbalanced positive and negative contributions to interaction with a single TCR. They highlight how subtle structural distinctions attributable to single amino acids can stand at the interface between foreign Ag responsiveness and pathogenic autoreactivity.     

An MHC class Ib-restricted TCR that cross-reacts with an MHC class Ia molecule

TCR transgenic 6C5 T cells recognize an insulin B chain epitope presented by the nonclassical class I MHC molecule, Qa-1(b). Positive selection of these T cells was shown previously to require Qa-1(b). Despite dedicated specificity for Qa-1(b), evidence presented in the current study indicates that 6C5 T cells can cross-recognize a classical class I molecule. Clonal deletion was observed unexpectedly in 6C5.H-2(bxq) mice, which do not express I-E MHC class II molecules and thus should not be subject to superantigen-mediated negative selection. 6C5 T cells were observed to respond in vivo and in vitro to spleen cells from allogeneic H-2(q) mice, and specificity was mapped to D(q). Evidence was obtained for direct recognition of D(q), rather than indirect presentation of a D(q)-derived peptide presented by Qa-1(b). Polyclonal CD8(+) T cells from class Ia-deficient K(b)D(b-/-) mice reacted in vitro to allogeneic spleen cells with an apparent frequency comparable to conventional class Ia-restricted T cells. Our results provide a clear example of a Qa-1-specific TCR that can cross-react with a class Ia molecule and evidence supporting the idea that this may be a common property of T cells selected by class Ib molecules.