T-cell Receptor Binding Site Research Articles

I-107 Long-range allosteric effects induced by Tax–HLA-A2 binding to an anti-HTLV-1 TCR: Implications for early T cell signaling

The mechanism whereby αβ T cell receptor (TCR) engagement by peptide–MHC (pMHC) is first communicated to the CD3 signaling apparatus of the TCR–CD3 complex, a process termed early T cell activation, is not well understood. To address the possibility that pMHC binding induces allosteric changes in TCR conformation and/or dynamics that are relayed to CD3, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and β chains of a human antiviral TCR (A6) that recognizes the Tax antigen from HTLV-1 bound to HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR V domains that propagate to 3 distinct sites in the C domains: (1) the Cβ FG loop that projects from the Vβ/Cβ interface, (2) a cluster of Cβ residues near the Cβ αA helix, a region involved in interactions with CD3, and (3) the Cα AB loop located at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. In addition, the pattern of long-range ligand-induced changes in TCR A6 seen by NMR is broadly similar to that predicted by MD simulations. We propose that the unique structure of the TCR β chain enables allosteric communication between the TCR binding sites for pMHC and CD3.

Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor

To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a “class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed “class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen–deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ∼50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.

Making Weak Antigens Strong: Cross-Linking Peptides to KLH with Maleimide

Haptens, which are small antigens such as peptides and drug compounds, are very weakly or nonimmunogenic by themselves and require the assistance of carrier proteins: complex molecules capable of eliciting a strong immune response in the host on injection. The haptens serve as epitopes for binding to the antibodies on the B-cell surface, and the carriers provide the MHC class II-T-cell receptor binding sites. Keyhole limpet hemocyanin (KLH) is one of the most widely used of such carrier proteins. KLH-hapten conjugates are commonly used in antibody generation in a variety of hosts such as mice, rats, and rabbits. Because KLH is harvested from mollusks, it is physiologically distant from mammalian species and less likely to produce antibodies that cross-react mammalian antigens. Maleimide activation of carrier proteins makes it possible to conjugate sulfhydryl-containing haptens, and hence this chemistry is widely used for conjugating KLH with haptens.

Computational Modeling of T Cell Receptor Complexes.

T-cell receptor (TCR) binding to peptide/MHC determines specificity and initiates signaling in antigen-specific cellular immune responses. Structures of TCR-pMHC complexes have provided enormous insight to cellular immune functions, permitted a rational understanding of processes such as pathogen escape, and led to the development of novel approaches for the design of vaccines and other therapeutics. As production, crystallization, and structure determination of TCR-pMHC complexes can be challenging, there is considerable interest in modeling new complexes. Here we describe a rapid approach to TCR-pMHC modeling that takes advantage of structural features conserved in known complexes, such as the restricted TCR binding site and the generally conserved diagonal docking mode. The approach relies on the powerful Rosetta suite and is implemented using the PyRosetta scripting environment. We show how the approach can recapitulate changes in TCR binding angles and other structural details, and highlight areas where careful evaluation of parameters is needed and alternative choices might be made. As TCRs are highly sensitive to subtle structural perturbations, there is room for improvement. Our method nonetheless generates high-quality models that can be foundational for structure-based hypotheses regarding TCR recognition.

Probing the Minimum Geometric Requirements for T-Cell Stimulation

The immune recognition process involves an elaborate arrangement of adhesion, costimulatory and signaling molecules organized into a stereotypic geometric structure known as the immunological synapse (IS). We have developed a versatile engineered platform to probe the minimum geometric requirements (in terms of spacing and stoichiometry) for T-cell stimulation. Arrays of metallic nanodots, ∼ 2-10 nm in size, to which a UCHT1 Fab antibody was bound, were created by nanolithography. These served as individual T-cell receptor (TCR) binding sites. The adhesion molecule ICAM-1 was either statically bound to a PEG-silane brush surrounding the nanodots, or it was allowed to move freely within the arrays in a supported lipid bilayer. T-cells were plated on arrays comprising individual TCR binding sites, small clusters and extended hexagonal close packed arrays, with spacings ranging from 40 nm and below to 1 μm. TCR signaling strength was monitored by measuring phosphorylated tyrosine (pY) intensity. T-cell adhesion was assessed by the number of cells bound to the arrays, as well as by the area of spread cells.Systematic variation of the spacing and cluster size of TCR binding sites in the arrays enabled determination of the minimum conditions that support T-cell signaling and the formation of the IS. TCR signaling increased with decreasing spacing below a threshold spacing ∼ 60 nm. For clusters with these spacings the formation of the stereotypic “bullseye” geometry that characterizes the immune synapse became evident, with ICAM-1 excluded to the periphery. In terms of stoichiometry, at least 4 TCR-binding sites (within ∼ 60 nm) were required for T-cell adhesion and spreading. Further, in the absence of ICAM-1 (or at low concentrations), a threshold density of agonist sites was found, above which the TCR apparently plays a dual role of immune activity and adhesion.

HLA‐A SNPs and amino acid variants are associated with nasopharyngeal carcinoma in Malaysian Chinese

Nasopharyngeal carcinoma (NPC) arises from the mucosal epithelium of the nasopharynx and is constantly associated with Epstein-Barr virus type 1 (EBV-1) infection. We carried out a genome-wide association study (GWAS) of 575,247 autosomal SNPs in 184 NPC patients and 236 healthy controls of Malaysian Chinese ethnicity. Potential association signals were replicated in a separate cohort of 260 NPC patients and 245 healthy controls. We confirmed the association of HLA-A to NPC with the strongest signal detected in rs3869062 (p = 1.73 × 10(-9)). HLA-A fine mapping revealed associations in the amino acid variants as well as its corresponding SNPs in the antigen peptide binding groove (p(HLA-A-aa-site-99) = 3.79 × 10(-8), p(rs1136697) = 3.79 × 10(-8)) and T-cell receptor binding site (p(HLA-A-aa-site-145) = 1.41 × 10(-4), p(rs1059520) = 1.41 × 10(-4)) of the HLA-A. We also detected strong association signals in the 5'-UTR region with predicted active promoter states (p(rs41545520) = 7.91 × 10(-8)). SNP rs41545520 is a potential binding site for repressor ATF3, with increased binding affinity for rs41545520-G correlated with reduced HLA-A expression. Multivariate logistic regression diminished the effects of HLA-A amino acid variants and SNPs, indicating a correlation with the effects of HLA-A*11:01, and to a lesser extent HLA-A*02:07. We report the strong genetic influence of HLA-A on NPC susceptibility in the Malaysian Chinese.

Molecular studies of anti-HLA-A2 using light-chain shuffling: a structural model for HLA antibody binding.

Human leukocyte antigen (HLA) A2 is one of the most immunodominant HLA antigens. Through a process of light-chain variable domain (VL) shuffling, we analyzed the VL domains' role in anti-HLA-A2/A28-binding site diversity. This was achieved by combining a VH3-30-encoded HLA-A2/A28-specific heavy-chain variable domain with 10(4) non-immune VL domains. Twelve HLA-A2/A28-specific antibodies were subsequently identified. VL gene analysis demonstrated an absence of Vlambda domains and that all have VkappaI-encoded light chains. The affinities correlated with the VkappaI gene present, with the seven highest affinity antibodies using Vkappa domains encoded by the O18 gene segment. A 300-fold difference in affinity was observed between the 12 antibodies, and homology modeling demonstrated a correlation between electrostatic surface potential of the antigen-binding site and affinity for HLA. Overlap between the T-cell receptor-binding site and that of the antibodies was indicated by inhibition of cytotoxic T-lymphocyte killing of peptide-pulsed target cells. A model of antibody binding to HLA-A2 suggested contact with both alpha helices of the HLA molecule, such that the antigen-binding site spans the peptide-binding groove. These data increase the understanding of antibody recognition of HLA and may facilitate the production of clonotypic antibodies with peptide-specific binding.

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.

The Spectral and Thermodynamic Properties of Staphylococcal Enterotoxin A, E, and Variants Suggest That Structural Modifications Are Important to Control Their Function

The superantigens staphylococcal enterotoxin A and E (SEA and SEE) can activate a large number of T-cells. SEA and SEE have approximately 80% sequence identity but show some differences in their biological function. Here, the two superantigens and analogues were characterized biophysically. SEE was shown to have a substantially higher thermal stability than SEA. Both SEA and SEE were thermally stabilized by 0.1 mM Zn(2+) compared with Zn(2+)-reduced conditions achieved using 1 mM EDTA or specific replacements that affect Zn(2+) coordination. The higher stability of SEE was only partly caused by the T-cell receptor (TCR) binding regions, whereas regions in the vicinity of the major histocompatibility complex class II binding sites affected the stability to a greater extent. SEE exhibited a biphasic denaturation between pH 5.0-6.5, influenced by residues in the TCR binding regions. Interestingly, enzyme-linked immunosorbent assay, isoelectric focusing, and circular dichroism analysis indicated that conformational changes had occurred in the SEA/E chimerical constructs relative to SEA and SEE. Thus, it is proposed that the Zn(2+) binding site is very important for the stability and potency of SEA and SEE, whereas residues in the TCR binding site have a substantial influence on the molecular conformation to control specificity and function.

Evolution of MHC-G in primates: a different kind of molecule for each group of species

When MHC-G molecules in primates (New World and Old World monkeys, Anthropoids and humans) were compared phylogenetically, very different evolutionary patterns within each species were found; their molecules did not have a straight forward and linear development throughout the postulated evolutionary pathway of primates. The earlier New World monkeys (South America) had relatively more alleles and the polymorphism was placed in the T-cell receptor (TcR), NK receptors and antigen binding sites; MHC-G probably works as a classical class I presenting molecule in these monkeys. MHC-G intron 2 from New World monkeys does not show the typical 23 bp deletion found in all other more recent primate species. Thus, it is possible that MHC-G molecules in New World monkeys belong to a different lineage than the MHC from higher primates. Another early lineage, Eurasian Old World monkeys, shows stop codons at exon 3: MHC-G proteins lacking the α2 domain may functionally suffice or otherwise reading-through stop-codon translational mechanisms may exist, as shown for other genes. Orangutans show lower (but significant) polymorphism than New World monkeys at NK, TcR and antigen binding regions; gorilla and chimpanzee show very low polymorphism. Humans only show three different HLA-G proteins with changes not affecting NK, TcR or antigen binding sites. It is observed that the more exposed the mother to allogeneic fetuses (polygamy), the less polymorphic HLA-G is observed within a given species. The data are concordant with the postulated immune inhibitory function for MHC-G in Old World monkeys, anthropoids and humans both at placental and inflammatory level.

Crystal Structure of an MHC Class I Presented Glycopeptide that Generates Carbohydrate-Specific CTL

T cell receptor (TCR) recognition of nonpeptidic and modified peptide antigens has been recently uncovered but is still poorly understood. Immunization with an H-2K b-restricted glycopeptide RGY8-6H-Gal 2 generates a population of cytotoxic T cells that express both α/β TCR, specific for glycopeptide, and γ/δ TCR, specific for the disaccharide, even on glycolipids. The crystal structure of K b/RGY8-6H-Gal 2 now demonstrates that the peptide and H-2K b structures are unaffected by the peptide glycosylation, but the central region of the putative TCR binding site is dominated by the extensive exposure of the tethered carbohydrate. These features of the K b/RGY8-6H-Gal 2 structure are consistent with the individual ligand binding preferences identified for the α/β and γ/δ TCRs and thus explain the generation of a carbohydrate-specific T cell response.

Molecular features of the hepatitis B virus nucleocapsid T-cell epitope 18-27: interaction with HLA and T-cell receptor.

The strength of the cytotoxic T lymphocyte (CTL) response is believed to influence the final outcome of hepatitis B virus (HBV) infection. Among the different CTL epitopes so far identified, the sequence 18-27 of the HBV nucleocapsid antigen is widely recognized by CTL of HLA-A2-positive patients with acute self-limited HBV infection, and represents the main component of a peptide-based therapeutic vaccine aimed at stimulating the antiviral CTL response in patients with chronic hepatitis B. In the present study, we further analyzed the features of this important HBV region by the following: 1) defining the contribution of individual residues of the epitope to the interaction with the T-cell receptor (TCR) and with the HLA-A0201 molecule; 2) assessing the antigenicity of this viral region in the context of the different HLA-A2 subtypes; and 3) testing whether this sequence can stimulate not only HLA-class I but also HLA class II restricted T-cell responses. A clear hierarchy was observed in the ability of individual residues to act as TCR or HLA binding sites. Furthermore, the sequence HBc18-27 was able to be recognized by specific CTL when presented in the context of different HLA-A2 subtypes. Finally, this HBV region was also found to stimulate HLA class II restricted T-cell responses. These data further increase the potential coverage and efficacy of therapeutic vaccines based on the HBc18-27 sequence.

Molecular features of the hepatitis B virus nucleocapsid T-cell epitope 18-27: Interaction with HLA and T-cell receptor

The strength of the cytotoxic T lymphocyte (CTL) response is believed to influence the final outcome of hepatitis B virus (HBV) infection. Among the different CTL epitopes so far identified, the sequence 18-27 of the HBV nucleocapsid antigen is widely recognized by CTL of HLA-A2-positive patients with acute self-limited HBV infection, and represents the main component of a peptide-based therapeutic vaccine aimed at stimulating the antiviral CTL response in patients with chronic hepatitis B. In the present study, we further analyzed the features of this important HBV region by the following: 1) defining the contribution of individual residues of the epitope to the interaction with the T-cell receptor (TCR) and with the HLA-A0201 molecule; 2) assessing the antigenicity of this viral region in the context of the different HLA-A2 subtypes; and 3) testing whether this sequence can stimulate not only HLA-class I but also HLA class II restricted T-cell responses. A clear hierarchy was observed in the ability of individual residues to act as TCR or HLA binding sites. Furthermore, the sequence HBc18-27 was able to be recognized by specific CTL when presented in the context of different HLA-A2 subtypes. Finally, this HBV region was also found to stimulate HLA class II restricted T-cell responses. These data further increase the potential coverage and efficacy of therapeutic vaccines based on the HBc18-27 sequence. (Hepatology 1997 Oct;26(4):1027-34)

Production of antisera using peptide conjugates.

AbstractSince an immunogen requires both an antigenic site and a T-cell receptor binding site, there is a minimum size necessary (1). Natural immunogens have a molecular weight >5000. Small molecules such as neuropeptides may be able to bind to the surface of B-cells, but do not stimulate an immune response. Such molecules are known as haptens. A hapten is an incomplete immunogen but can be made immunogenic by coupling to a suitable carrier molecule. There are a variety of different crosslinking agents utilized for the coupling of peptides to carrier proteins; examples of each type are covered in this chapter. In the case of larger neuropeptides, such as calcitonin gene-related peptide (CGRP), it is possible to stimulate an immune response by presenting the peptide together with a carrier such as polyvinyl-pyrrolidone without the need for conjugation. This method, described in Section 3.5., has proven to be useful for the author for a number of different peptides. Unfortunately, however, the success of this method compared with the responses to conjugated peptides is largely a matter of trial and error.KeywordsCoupling AgentCarrier ProteinFree Sulfhydryl GroupAntigenic ResponseLysyl ResidueThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Bacterial superantigen specificities of mouse T cell receptor V beta 20.

The study of the mouse T cell receptor (TcR) beta chain repertoire in BALB/c thymocytes led to the identification of the V beta 20 gene segment. The expression of V beta 20 estimated at the transcriptional level differs among mouse strains, suggesting clonal deletion. In the present study, we reconstituted by transfection functional TcR using the V beta 20 segment with different V alpha segments and studied the action of superantigen toxins. The V beta 20-transfectant T cells are activated by staphylococcal enterotoxins A and E (SEA and SEE) but not by the other tested toxins. The activation is dependent on the presence of cells expressing major histocompatibility complex class II molecules. Different HLA DR alleles can present the bacterial toxins, establishing that they interact with TcRV beta 20 as superantigens. Moreover, the V alpha domain associated with the V beta 20 domain has an influence on the response to these toxins. The fact that V beta 20 is recognized by SEA and SEE, although both toxins are known to interact with different sets of V beta, suggests the presence of different TcR binding sites on the toxins.

Residues defining Vβ specificity in staphylococcal enterotoxins

The three-dimensional structure of staphylococcal enterotoxin C2 has been determined at 2.7 A resolution by x-ray diffraction, while the structures of enterotoxins A and E have been modelled based on their sequence homology to other staphylococcal enterotoxins. The T-cell receptor-binding sites of staphylococcal enterotoxin (SE) B and SEC2 are compared and the stereochemical interactions likely to be responsible for their differing V beta specificities are identified. A similar comparison is made between SEA and SEE.

Identification of class II major histocompatibility complex and T cell receptor binding sites in the superantigen toxic shock syndrome toxin 1.

Superantigens, in association with class II major histocompatibility complex (MHC) molecules, activate T cells bearing particular beta chain variable domains of the T cell receptor (TCR). Unlike conventional peptide antigens, superantigens bind as intact proteins to TCR and MHC molecules outside their peptide binding sites. To characterize these interactions at the molecular level, random point mutations were generated in the gene encoding toxic shock syndrome toxin 1, a bacterial superantigen associated with toxic shock syndrome. Functionally impaired mutants were identified based on their lack of murine and human T cell stimulatory activities, and experiments analyzing binding to human histocompatibility leukocyte antigen-DR molecules differentiated residues involved in MHC from TCR binding. The results showed that the great majority of mutations are clustered in two distinct regions of the toxic shock syndrome toxin 1 molecule. The class II MHC binding site is located in the hydrophobic region of the NH2-terminal domain, and the TCR binding site is primarily in the major central groove of the COOH-terminal domain. These studies provide insight into the interactions necessary for superantigen-mediated disease in humans.

Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.

Crystal structure of the complex of rat neonatal Fc receptor with Fc.

The neonatal Fc receptor (FcRn) transports maternal immunoglobulin G (IgG) to the bloodstream of the newborn. FcRn is structurally similar to class I major histocompatibility complex (MHC) molecules, despite differences in the ligands they bind (the Fc portion of IgG and antigenic peptides, respectively). A low-resolution crystal structure of the complex between FcRn and Fc localizes the binding site for Fc to the side of FcRn, distinct from the tops of the alpha 1 and alpha 2 domains which serve as the peptide and T-cell receptor binding sites in class I molecules. FcRn binds to Fc at the interface between the Fc CH2 and CH3 domains, which contains several histidine residues that could account for the sharply pH-dependent FcRn/IgG interaction. A dimer of FcRn heterodimers observed in the co-crystals and in the crystals of FcRn alone could be involved in binding Fc, correlating with the 2:1 binding stoichiometry between FcRn and IgG (ref. 4) and suggesting an unusual orientation of FcRn on the membrane.

Crystal structure of staphylococcal enterotoxin B, a superantigen.

The three-dimensional structure of staphylococcal enterotoxin B, which is both a toxin and a super-antigen, has been determined to a resolution of 2.5 A. The unusual main-chain fold containing two domains may represent a general motif adopted by all staphylococcal enterotoxins. The T-cell receptor binding site encompasses a shallow cavity formed by both domains. The MHCII molecule binds to an adjacent site. Another cavity with possible biological activity was also identified.