Lisa F. Boyd

T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity

The critical discriminatory event in the activation of T lymphocytes bearing alpha beta T cell receptors (TCRs) is their interaction with a molecular complex consisting of a peptide bound to a major histocompatibility complex (MHC)-encoded class I or class II molecule on the surface of an antigen-presenting cell. The kinetics of binding were measured of a purified TCR to molecular complexes of a purified soluble analog of the murine MHC class I molecule H-2Ld (sH-2Ld) and a synthetic octamer peptide p2CL in a direct, real-time assay based on surface plasmon resonance. The kinetic dissociation rate of the MHC-peptide complex from the TCR was rapid (2.6 x 10(-2) second-1, corresponding to a half-time for dissociation of approximately 27 seconds), and the kinetic association rate was 2.1 x 10(5) M-1 second-1. The equilibrium constant for dissociation was approximately 10(-7) M. These values indicate that TCRs must interact with a multivalent array of MHC-peptide complexes to trigger T cell signaling.

Enhanced Antigen-Specific Antitumor Immunity with Altered Peptide Ligands that Stabilize the MHC-Peptide-TCR Complex

T cell responsiveness to an epitope is affected both by its affinity for the presenting MHC molecule and the affinity of the MHC-peptide complex for TCR. One limitation of cancer immunotherapy is that natural tumor antigens elicit relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to these antigens. We report here that amino acid substitutions in a natural MHC class I-restricted tumor antigen that increase the stability of the MHC-peptide-TCR complex are significantly more potent as tumor vaccines. The improved immunity results from enhanced in vivo expansion of T cells specific for the natural tumor epitope. These results indicate peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor immunity through enhanced stimulation of specific T cells.

Measuring interactions of MHC class I molecules using surface plasmon resonance

To examine the molecular interactions between major histocompatibility complex (MHC)-encoded molecules and peptides, monoclonal antibodies (mAbs), or T cell receptors, we have developed model systems employing genetically engineered soluble MHC class I molecules (MHC-I), synthetic peptides, purified mAbs, and engineered solubilizable T cell receptors. Direct binding assays based on immobilization of one of the interacting components to the dextran modified gold biosensor surface of a surface plasmon resonance (SPR) detector have been developed for each of these systems. The peptide binding site of the MHC-I molecule can be sterically mapped by evaluation of a set of peptides immobilized through the thiol group of cysteine substitutions at each peptide position. Kinetic binding studies indicate that the MHC-I/peptide interaction is characterized by a low to moderate apparent kass (approximately 5000-60000 M-1 s-1) and very small kdis (approximately 10(-4)-10(-6) s-1) consistent with the biological requirement for a long cell surface residence time to permit engagement with T cell receptors. Several mAb directed against different MHC-I epitopes were examined, and kinetic parameters of their interaction with MHC molecules were determined. These showed characteristic moderate association rate constants and moderate dissociation rate constants (kass approximately 10(4)-10(6) M-1 s-1 and kdis approximately 10(-2)-10(-4) s-1), characteristic of many antibody/protein antigen interactions. The interaction of an anti-idiotypic anti-TCR mAb with its purified cognate TCR was of moderate affinity and revealed kinetic binding similar to that of the anti-MHC mAbs. The previously determined interaction of a purified T cell receptor with its MHC-I/peptide ligand is characterized by kinetic constants more similar to those of the antibody/antigen interaction than of the MHC-I/peptide interaction, but is remarkable for rapid dissociation rates (apparent kdis approximately 10(-2) s-1). Such binding studies of reactions involving the MHC-I molecules offer insight into the mechanisms responsible for the initial specific events required for the stimulation of T cells.

Interaction of the NK Cell Inhibitory Receptor Ly49A with H-2Dd:Identification of a Site Distinct from the TCR Site

Natural killer cell function is controlled by interaction of NK receptors with MHC I molecules expressed on target cells. We describe the binding of bacterially expressed Ly49A, the prototype murine NK inhibitory receptor, to similarly engineered H-2Dd. Despite its homology to C-type lectins, Ly49A binds independently of carbohydrate and Ca2+ and shows specificity for MHC I but not bound peptide. The affinity of the Ly49A/H-2Dd interaction as determined by surface plasmon resonance is from 6 to 26 microM at 25 degrees C and is greater by ultracentrifugation at 4 degrees C. Biotinylated Ly49A stains H-2Dd-expressing cells. Competition experiments indicate that the Ly49A and T cell receptor (TCR) binding sites on MHC I are distinct, suggesting complex regulation of cells that bear both TCR and NK cell receptors.

Mapping the Ligand of the NK Inhibitory Receptor Ly49A on Living Cells

We have used a recombinant, biotinylated form of the mouse NK cell inhibitory receptor, Ly49A, to visualize the expression of MHC class I (MHC-I) ligands on living lymphoid cells. A panel of murine strains, including MHC congenic lines, was examined. We detected binding of Ly49A to cells expressing H-2Dd, H-2Dk, and H-2Dp but not to those expressing other MHC molecules. Cells of the MHC-recombinant strain B10.PL (H-2u) not only bound Ly49A but also inhibited cytolysis by Ly49A+ effector cells, consistent with the correlation of in vitro binding and NK cell function. Binding of Ly49A to H-2Dd-bearing cells of different lymphoid tissues was proportional to the level of H-2Dd expression and was not related to the lineage of the cells examined. These binding results, interpreted in the context of amino acid sequence comparisons and the recently determined three-dimensional structure of the Ly49A/H-2Dd complex, suggest a role for amino acid residues at the amino-terminal end of the α1 helix of the MHC-I molecule for Ly49A interaction. This view is supported by a marked decrease in affinity of an H-2Dd mutant, I52 M, for Ly49A. Thus, allelic variation of MHC-I molecules controls measurable affinity for the NK inhibitory receptor Ly49A and explains differences in functional recognition in different mouse strains.

The Peptide-Receptive Transition State of MHC Class I Molecules: Insight from Structure and Molecular Dynamics

MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I–restricted CD8+ T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 310 helix that flips from an exposed “open” position in the PR transition state to a “closed” position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46–53 of murine H-2Ld MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.

Alternative processing of H-2Dd pre-mRNAs results in membrane expression of differentially phosphorylated protein products.

Two distinct mRNA species encoding the mouse major histocompatibility antigen H‐2Dd have been identified in BALB/c spleen cells as well as in cultured cell lines expressing this cell surface glycoprotein. The alternate transcripts of H‐2Dd arise from either removal or inclusion of exon VII (encoding I2) during pre‐mRNA processing. The relative levels of each kind of H‐2Dd transcript varied considerably between different cell types, and in all cells examined both forms of alloantigen were expressed on the cell membrane. Antigen derived from both types of transcript reacted with H‐2Dd‐specific monoclonal antibodies, whereas only protein lacking the 13 amino acids of I2 reacted with a specific antiserum raised against a predicted exon VI/VIII fusion peptide. Those H‐2Dd proteins translated from full length, but not smaller, transcripts were phosphorylated in resting and phorbol myristate acetate‐stimulated BALB/c spleen cells, suggesting that the major site of in vivo phosphorylation is within the highly conserved sequence encoded by exon VII. Thus alternative splicing of pre‐mRNA transcripts is a mechanism which leads to membrane expression of two forms of H‐2Dd, one of which lacks a major site of phosphorylation.

Crystal structure of a TAPBPR-MHC I complex reveals the mechanism of peptide editing in antigen presentation.

Two snapshots of the TAPBPR-MHC I complex Cytotoxic CD8+ T cells recognize infected and cancerous cells by scrutinizing the antigenic peptides presented by the major histocompatibility complex class I (MHC I). Peptide binding and exchange occurs in the endoplasmic reticulum in a sequence of events mediated by the chaperones tapasin and TAPBPR (see the Perspective by Cresswell). Thomas and Tampé resolved the crystal structure of the TAPBPR-MHC I editing complex by using a photocleavable high-affinity peptide to stabilize the MHC molecule. Jiang et al. crystalized MHC I molecules inhabited by truncated disulfide-linked peptides that still permit TAPBPR to bind. These complimentary snapshots elucidate the dynamic process by which chaperones stabilize the groove of peptide-free MHC I molecules. This helps MHC I sample peptide candidates and facilitates the generation of peptide repertoires enriched with high-affinity antigenic peptides. Science, this issue p. 1060, p. 1064; see also p. 992 Two different approaches yield complimentary structures of TAPBR in complex with MHC I. Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing

Significance This report explores the biochemical and structural basis of the interactions of TAP binding protein, related (TAPBPR), a tapasin homolog, with MHC-I molecules. TAPBPR associates with MHC-I molecules early in their biosynthesis and folding but is not part of the peptide-loading complex (PLC). Here, by examining the interactions of recombinant TAPBPR with peptide-free and peptide-complexed MHC-I molecules, we show that TAPBPR serves as a peptide editor. Structural comparison of TAPBPR with tapasin indicates the similarities of the two molecules and provides a basis for evaluating the steps of peptide loading. Understanding the molecular underpinnings of peptide loading of MHC-I by TAPBPR and tapasin has wide-ranging influence on our ability to modulate peptide loading for vaccine design and T-cell recognition. Peptide loading of major histocompatibility complex class I (MHC-I) molecules is central to antigen presentation, self-tolerance, and CD8+ T-cell activation. TAP binding protein, related (TAPBPR), a widely expressed tapasin homolog, is not part of the classical MHC-I peptide-loading complex (PLC). Using recombinant MHC-I molecules, we show that TAPBPR binds HLA-A*02:01 and several other MHC-I molecules that are either peptide-free or loaded with low-affinity peptides. Fluorescence polarization experiments establish that TAPBPR augments peptide binding by MHC-I. The TAPBPR/MHC-I interaction is reversed by specific peptides, related to their affinity. Mutational and small-angle X-ray scattering (SAXS) studies confirm the structural similarities of TAPBPR with tapasin. These results support a role of TAPBPR in stabilizing peptide-receptive conformation(s) of MHC-I, permitting peptide editing.

Structural basis of mouse cytomegalovirus m152/gp40 interaction with RAE1γ reveals a paradigm for MHC/MHC interaction in immune evasion

Natural killer (NK) cells are activated by engagement of the NKG2D receptor with ligands on target cells stressed by infection or tumorigenesis. Several human and rodent cytomegalovirus (CMV) immunoevasins down-regulate surface expression of NKG2D ligands. The mouse CMV MHC class I (MHC-I)–like m152/gp40 glycoprotein down-regulates retinoic acid early inducible-1 (RAE1) NKG2D ligands as well as host MHC-I. Here we describe the crystal structure of an m152/RAE1γ complex and confirm the intermolecular contacts by mutagenesis. m152 interacts in a pincer-like manner with two sites on the α1 and α2 helices of RAE1 reminiscent of the NKG2D interaction with RAE1. This structure of an MHC-I–like immunoevasin/MHC-I–like ligand complex explains the binding specificity of m152 for RAE1 and allows modeling of the interaction of m152 with classical MHC-I and of related viral immunoevasins.