T-cell receptor (TCR)-peptide specificity overrides affinity-enhancing TCR-major histocompatibility complex interactions.

αß T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short "foreign" peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.

Peptide length determines the outcome of TCR/peptide-MHCI engagement.

αß-TCRs expressed at the CD8(+) T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8(+) T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8(+) T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8(+) T-cell clonotypes.

T-cell receptor specificity maintained by altered thermodynamics.

The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nM to 270 µM). We used phage-display to produce a melanoma-specific TCR (α24ß17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nM) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24ß17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.

MUC1 limits Helicobacter pylori infection both by steric hindrance and by acting as a releasable decoy.

The bacterium Helicobacter pylori can cause peptic ulcer disease, gastric adenocarcinoma and MALT lymphoma. The cell-surface mucin MUC1 is a large glycoprotein which is highly expressed on the mucosal surface and limits the density of H. pylori in a murine infection model. We now demonstrate that by using the BabA and SabA adhesins, H. pylori bind MUC1 isolated from human gastric cells and MUC1 shed into gastric juice. Both H. pylori carrying these adhesins, and beads coated with MUC1 antibodies, induced shedding of MUC1 from MKN7 human gastric epithelial cells, and shed MUC1 was found bound to H. pylori. Shedding of MUC1 from non-infected cells was not mediated by the known MUC1 sheddases ADAM17 and MMP-14. However, knockdown of MMP-14 partially affected MUC1 release early in infection, whereas ADAM17 had no effect. Thus, it is likely that shedding is mediated both by proteases and by disassociation of the non-covalent interaction between the alpha- and beta-subunits. H. pylori bound more readily to MUC1 depleted cells even when the bacteria lacked the BabA and SabA adhesins, showing that MUC1 inhibits attachment even when bacteria cannot bind to the mucin. Bacteria lacking both the BabA and SabA adhesins caused less apoptosis in MKN7 cells than wild-type bacteria, having a greater effect than deletion of the CagA pathogenicity gene. Deficiency of MUC1/Muc1 resulted in increased epithelial cell apoptosis, both in MKN7 cells in vitro, and in H. pylori infected mice. Thus, MUC1 protects the epithelium from non-MUC1 binding bacteria by inhibiting adhesion to the cell surface by steric hindrance, and from MUC1-binding bacteria by acting as a releasable decoy.

Design, synthesis and evaluation of ß-lactam antigenic peptide hybrids; unusual opening of the ß-lactam ring in acidic media.

ß-Lactam peptides were envisioned as conformational constraints in antigenic peptides (APs). Three different ß-lactam tripeptides of varying flexibility were prepared in solution and incorporated in place of the central part of the altered melanoma associated antigenic peptide Leu(27)-Melan-A(26-35) using solid phase synthesis techniques. Upon TFA cleavage from the solid support, an unexpected opening of the ß-lactam ring occurred with conservation of the amide bond. After adaptation of the solid phase synthesis strategy, ß-lactam peptides were successfully obtained and both opened and closed forms were evaluated for their capacity to bind to the antigen-presenting class-I MHC HLA-A2 protein system. None of the closed ß-lactam peptides bound to HLA-A2, but their opened variants were shown to be moderate to good HLA-A2 ligands, one of them being even capable of stimulating a Melan-A-specific T cell line.

Synthesis and Biological Evaluation of Hapten-Clicked Analogues of The Antigenic Peptide Melan-A/MART-126(27L)-35.

A click-chemistry-based approach was implemented to prepare peptidomimetics designed in silico and made from aromatic azides and a propargylated GIGI-mimicking platform derived from the altered Melan-A/MART-126(27L)-35 antigenic peptide ELAGIGILTV. The CuI -catalyzed Huisgen cycloaddition was carried out on solid support to generate rapidly a first series of peptidomimetics, which were evaluated for their capacity to dock at the interface between the major histocompatibility complex class-I (MHC-I) human leucocyte antigen (HLA)-A2 and T-cell receptors (TCRs). Despite being a weak HLA-A2 ligand, one of these 11 first synthetic compounds bearing a p-nitrobenzyl-triazole side chain was recognized by the receptor proteins of Melan-A/MART-1-specific T-cells. After modification of the N and C termini of this agonist, which was intended to enhance HLA-A2 binding, one of the resulting seven additional compounds triggered significant T-cell responses. Thus, these results highlight the capacity of naturally circulating human TCRs that are specific for the native Melan-A/MART-126-35 peptide to cross-react with peptidomimetics bearing organic motifs structurally different from the native central amino acids.

Real time detection of peptide-MHC dissociation reveals that improvement of primary MHC-binding residues can have a minimal, or no, effect on stability.

The majority of known major histocompatibility complex class I (MHCI)-associated tumor-derived peptide antigens do not contain an optimal motif for MHCI binding. As a result, anchor residue-modified 'heteroclitic' peptides have been widely used in therapeutic cancer vaccination trials in order to enhance immune responsiveness. In general, the improved stability of these heteroclitic complexes has been inferred from their improved immunogenicity but has not been formally assessed. Here, we investigated the binding of 4 HLA A*0201-restricted tumor-derived peptides and their commonly used heteroclitic variants. We utilized a cell surface binding assay and a novel robust method for testing the durability of soluble recombinant pMHCI in real time by surface plasmon resonance. Surprisingly, we show that heteroclitic peptides designed with optimal MHC binding motifs do not always form pMHCs that are substantially more stable than their wildtype progenitors. These findings, combined with our recent discovery that TCRs can distinguish between wildtype peptides and those altered at a primary buried MHC anchor residue, suggest that altered TCR binding may account for a large part of the increased immune response that can be generated by anchor residue-modified ligands. Our results further highlight the fact that heteroclitic peptide-based immune interventions require careful evaluation to ensure that wildtype antigen specificity is maintained in vivo.