Human leukocyte antigen (HLA) class II peptide flanking residues tune the immunogenicity of a human tumor-derived epitope.

CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR-pHLA-II interactions.

Exploring the nature of the H-bonds between the human class II MHC protein, HLA-DR1 (DRB*0101) and the influenza virus hemagglutinin peptide, HA306-318, using the quantum theory of atoms in molecules.

The nature of the H-bonds between the human protein HLA-DR1 (DRB*0101) and the hemagglutinin peptide HA306-318 has been studied using the Quantum Theory of Atoms in Molecules for the first time. We have found four H-bond groups: one conventional CO··HN bond group and three nonconventional CO··HC, π··HC involving aromatic rings and HN··HCaliphatic groups. The calculated electron density at the determined H-bond critical points suggests the follow protein pocket binding trend: P1 (2,311) >> P9 (1.109) > P4 (0.950) > P6 (0.553) > P7 (0.213) which agrees and reveal the nature of experimental findings, showing that P1 produces by a long way the strongest binding of the HLA-DR1 human protein molecule with the peptide backbone as consequence of the vast number of H-bonds in the P1 area and at the same time the largest specific binding of the peptide Tyr308 residue with aromatic residues located at the binding groove floor. The present results suggest the topological analysis of the electronic density as a valuable tool that allows a non-arbitrary partition of the pockets binding energy via the calculated electron density at the determined critical points.

The N-terminal region of photocleavable peptides that bind HLA-DR1 determines the kinetics of fragment release.

Major Histocompatibility Complex class II (MHC-II) molecules bind peptides and present them to receptors on CD4+ T cells as part of the immune system's surveillance of pathogens and malignancy. In the absence of peptide, MHC-II equilibrates between peptide-receptive and peptide-averse conformations. The conversion between these forms has been postulated to be important in regulating cellular antigen presentation but has been difficult to study. In order to generate the MHC-II molecule HLA-DR1 in the peptide-receptive form, we designed and tested a series of photocleavable peptides that included the UV-sensitive 3-amino-3-(2-nitrophenyl)-propionate amino acid analog. They were intended to bind tightly to the HLA-DR1 MHC molecule, but to generate low-affinity fragments after UV exposure that would be released to yield HLA-DR1 in the peptide-receptive conformation. We were able to identify photocleavable peptides that bound tightly to HLA-DR1 and generated the peptide-receptive conformation after UV exposure. However, slow release of photocleaved peptide fragments from the binding site limited the rate of binding of an incoming labeled peptide and complicated kinetic measurements of the individual steps of the overall peptide binding reaction. Modification of the N-terminal region of the photocleavable peptide to reduce MHC-II pocket or H-bonding interactions allowed for generation of the peptide receptive form immediately after UV exposure with peptide fragments neither retained within the site nor interfering with binding of an incoming peptide. However this was achieved only at the expense of a substantial reduction in overall peptide binding affinity, and these peptides had such weak interaction with HLA-DR1 that they were easily exchanged by incoming peptide without UV exposure. These results show that photocleavable peptides can be used to generate peptide-receptive HLA-DR1 and to facilitate peptide exchange in generation of specific peptide-MHC-II complexes, but that usage of these peptides for kinetic studies can be constrained by slow fragment release.

The ADAMTS131239-1253 peptide is a dominant HLA-DR1-restricted CD4+ T-cell epitope.

Acquired thrombotic thrombocytopenic purpura is a rare and severe disease characterized by auto-antibodies directed against "A Disintegrin And Metalloproteinase with Thrombospondin type 1 repeats, 13th member" (ADAMTS13), a plasma protein involved in hemostasis. Involvement of CD4+ T cells in the pathogenesis of the disease is suggested by the IgG isotype of the antibodies. However, the nature of the CD4+ T-cell epitopes remains poorly characterized. Here, we determined the HLA-DR-restricted CD4+ T-cell epitopes of ADAMTS13. Candidate T-cell epitopes were predicted in silico and binding affinities were confirmed in competitive enzyme-linked immunosorbent assays. ADAMTS13-reactive CD4+ T-cell hybridomas were generated following immunization of HLA-DR1 transgenic mice (Sure-L1 strain) and used to screen the candidate epitopes. We identified the ADAMTS131239-1253 peptide as the single immunodominant HLA-DR1-restricted CD4+ T-cell epitope. This peptide is located in the CUB2 domain of ADAMTS13. It was processed by dendritic cells, stimulated CD4+ T cells from Sure-L1 mice and was recognized by CD4+ T cells from an HLA-DR1-positive patient with acute thrombotic thrombocytopenic purpura. Interestingly, the ADAMTS131239-1253 peptide demonstrated promiscuity towards HLA-DR11 and HLA-DR15. Our work paves the way towards the characterization of the ADAMTS13-specific CD4+ T-cell response in patients with thrombotic thrombocytopenic purpura using ADAMTS131239-1253-loaded HLA-DR tetramers.

Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide-MHC complexes.

Measurements of thermal stability by circular dichroism (CD) spectroscopy have been widely used to assess the binding of peptides to MHC proteins, particularly within the structural immunology community. Although thermal stability assays offer advantages over other approaches such as IC50 measurements, CD-based stability measurements are hindered by large sample requirements and low throughput. Here we demonstrate that an alternative approach based on differential scanning fluorimetry (DSF) yields results comparable to those based on CD for both class I and class II complexes. As they require much less sample, DSF-based measurements reduce demands on protein production strategies and are amenable for high throughput studies. DSF can thus not only replace CD as a means to assess peptide/MHC thermal stability, but can complement other peptide-MHC binding assays used in screening, epitope discovery, and vaccine design. Due to the physical process probed, DSF can also uncover complexities not observed with other techniques. Lastly, we show that DSF can also be used to assess peptide/MHC kinetic stability, allowing for a single experimental setup to probe both binding equilibria and kinetics.

The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity.

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin ß4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.

Matched Peptides: Tuning Matched Molecular Pair Analysis for Biopharmaceutical Applications.

Biopharmaceuticals hold great promise for the future of drug discovery. Nevertheless, rational drug design strategies are mainly focused on the discovery of small synthetic molecules. Herein we present matched peptides, an innovative analysis technique for biological data related to peptide and protein sequences. It represents an extension of matched molecular pair analysis toward macromolecular sequence data and allows quantitative predictions of the effect of single amino acid substitutions on the basis of statistical data on known transformations. We demonstrate the application of matched peptides to a data set of major histocompatibility complex class II peptide ligands and discuss the trends captured with respect to classical quantitative structure-activity relationship approaches as well as structural aspects of the investigated protein-peptide interface. We expect our novel readily interpretable tool at the interface of cheminformatics and bioinformatics to support the rational design of biopharmaceuticals and give directions for further development of the presented methodology.

Evaluating the Role of HLA-DM in MHC Class II-Peptide Association Reactions.

Ag presentation by MHC class II (MHC II) molecules to CD4(+) T cells plays a key role in the regulation of the adaptive immune response. Loading of antigenic peptides onto MHC II is catalyzed by HLA-DM (DM), a nonclassical MHC II molecule. The mechanism of DM-facilitated peptide loading is an outstanding problem in the field of Ag presentation. In this study, we systemically explored possible kinetic mechanisms for DM-catalyzed peptide association by measuring real-time peptide association kinetics using fluorescence polarization assays and comparing the experimental data with numerically modeled peptide association reactions. We found that DM does not facilitate peptide association by stabilizing peptide-free MHC II against aggregation. Moreover, DM does not promote transition of an inactive peptide-averse conformation of MHC II to an active peptide-receptive conformation. Instead, DM forms an intermediate with MHC II that binds peptide with faster kinetics than MHC II in the absence of DM. In the absence of peptides, interaction of MHC II with DM leads to inactivation and formation of a peptide-averse form. This study provides novel insights into how DM efficiently catalyzes peptide loading during Ag presentation.

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.

Disruption of hydrogen bonds between major histocompatibility complex class II and the peptide N-terminus is not sufficient to form a human leukocyte antigen-DM receptive state of major histocompatibility complex class II.

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306-318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.

HLA-DO as the optimizer of epitope selection for MHC class II antigen presentation.

Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing.

Crystal structure of the HLA-DM-HLA-DR1 complex defines mechanisms for rapid peptide selection.

HLA-DR molecules bind microbial peptides in an endosomal compartment and present them on the cell surface for CD4 T cell surveillance. HLA-DM plays a critical role in the endosomal peptide selection process. The structure of the HLA-DM-HLA-DR complex shows major rearrangements of the HLA-DR peptide-binding groove. Flipping of a tryptophan away from the HLA-DR1 P1 pocket enables major conformational changes that position hydrophobic HLA-DR residues into the P1 pocket. These conformational changes accelerate peptide dissociation and stabilize the empty HLA-DR peptide-binding groove. Initially, incoming peptides have access to only part of the HLA-DR groove and need to compete with HLA-DR residues for access to the P2 site and the hydrophobic P1 pocket. This energetic barrier creates a rapid and stringent selection process for the highest-affinity binders. Insertion of peptide residues into the P2 and P1 sites reverses the conformational changes, terminating selection through DM dissociation.

Minimal conformational plasticity enables TCR cross-reactivity to different MHC class II heterodimers.

Successful immunity requires that a limited pool of αß T-cell receptors (TCRs) provide cover for a vast number of potential foreign peptide antigens presented by 'self' major histocompatibility complex (pMHC) molecules. Structures of unligated and ligated MHC class-I-restricted TCRs with different ligands, supplemented with biophysical analyses, have revealed a number of important mechanisms that govern TCR mediated antigen recognition. HA1.7 TCR binding to the influenza hemagglutinin antigen (HA(306-318)) presented by HLA-DR1 or HLA-DR4 represents an ideal system for interrogating pMHC-II antigen recognition. Accordingly, we solved the structure of the unligated HA1.7 TCR and compared it to both complex structures. Despite a relatively rigid binding mode, HA1.7 T-cells could tolerate mutations in key contact residues within the peptide epitope. Thermodynamic analysis revealed that limited plasticity and extreme favorable entropy underpinned the ability of the HA1.7 T-cell clone to cross-react with HA(306-318) presented by multiple MHC-II alleles.

Peptide linkage to the α-subunit of MHCII creates a stably inverted antigen presentation complex.

Class II proteins of the major histocompatibility complex (MHCII) typically present exogenous antigenic peptides to cognate T cell receptors of CD4-T lymphocytes. The exact conformation of peptide-MHCII complexes (pMHCII) can vary depending on the length, register and orientation of the bound peptide. We have recently found the self-peptide CLIP (class-II-associated invariant chain-derived peptide) to adopt a dynamic bidirectional binding mode with regard to the human MHCII HLA-DR1 (HLA, human leukocyte antigen). We suggested that inversely bound peptides could activate specific T cell clones in the context of autoimmunity. As a first step to prove this hypothesis, pMHC complexes restricted to either the canonical or the inverted peptide orientation have to be constructed. Here, we show that genetically encoded linkage of CLIP and two other antigenic peptides to the HLA-DR1 α-chain results in stable complexes with inversely bound ligands. Two-dimensional NMR and biophysical analyses indicate that the CLIP-bound pMHC(inv) complex (pMHC(inv), inverted MHCII-peptide complex) displays high thermodynamic stability but still allows for the exchange against higher-affinity viral antigen. Complemented by comparable data on a corresponding ß-chain-fused canonical HLA-DR1/CLIP complex, we further show that linkage of CLIP leads to a binding mode exactly the same as that of the corresponding unlinked constructs. We suggest that our approach constitutes a general strategy to create pMHC(inv) complexes. Such engineering is needed to create orientation-specific antibodies and raise T cells to study phenomena of autoimmunity caused by isomeric pMHCs.

Human CD4+ T cell response to human herpesvirus 6.

Following primary infection, human herpesvirus 6 (HHV-6) establishes a persistent infection for life. HHV-6 reactivation has been associated with transplant rejection, delayed engraftment, encephalitis, muscular dystrophy, and drug-induced hypersensitivity syndrome. The poor understanding of the targets and outcome of the cellular immune response to HHV-6 makes it difficult to outline the role of HHV-6 in human disease. To fill in this gap, we characterized CD4 T cell responses to HHV-6 using peripheral blood mononuclear cell (PBMC) and T cell lines generated from healthy donors. CD4(+) T cells responding to HHV-6 in peripheral blood were observed at frequencies below 0.1% of total T cells but could be expanded easily in vitro. Analysis of cytokines in supernatants of PBMC and T cell cultures challenged with HHV-6 preparations indicated that gamma interferon (IFN-γ) and interleukin-10 (IL-10) were appropriate markers of the HHV-6 cellular response. Eleven CD4(+) T cell epitopes, all but one derived from abundant virion components, were identified. The response was highly cross-reactive between HHV-6A and HHV-6B variants. Seven of the CD4(+) T cell epitopes do not share significant homologies with other known human pathogens, including the closely related human viruses human herpesvirus 7 (HHV-7) and human cytomegalovirus (HCMV). Major histocompatibility complex (MHC) tetramers generated with these epitopes were able to detect HHV-6-specific T cell populations. These findings provide a window into the immune response to HHV-6 and provide a basis for tracking HHV-6 cellular immune responses.

Conformational lability in the class II MHC 310 helix and adjacent extended strand dictate HLA-DM susceptibility and peptide exchange.

HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 3(10) helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.

Affinity maturation of human CD4 by yeast surface display and crystal structure of a CD4-HLA-DR1 complex.

Helper T-cell activation generally requires the coreceptor CD4, which binds MHC class II molecules. A remarkable feature of the CD4-MHC class II interaction is its exceptionally low affinity, which ranges from K(D) = ∼200 µM to >2 mM. Investigating the biological role of the much lower affinity of this interaction than those of other cell-cell recognition molecules will require CD4 mutants with enhanced binding to MHC class II for testing in models of T-cell development. To this end, we used in vitro-directed evolution to increase the affinity of human CD4 for HLA-DR1. A mutant CD4 library was displayed on the surface of yeast and selected using HLA-DR1 tetramers or monomers, resulting in isolation of a CD4 clone containing 11 mutations. Reversion mutagenesis showed that most of the affinity increase derived from just two substitutions, Gln40Tyr and Thr45Trp. A CD4 variant bearing these mutations bound HLA-DR1 with K(D) = 8.8 µM, compared with >400 µM for wild-type CD4. To understand the basis for improved affinity, we determined the structure of this CD4 variant in complex with HLA-DR1 to 2.4 Å resolution. The structure provides an atomic-level description of the CD4-binding site on MHC class II and reveals how CD4 recognizes highly polymorphic HLA-DR, -DP, and -DQ molecules by targeting invariant residues in their α2 and ß2 domains. In addition, the CD4 mutants reported here constitute unique tools for probing the influence of CD4 affinity on T-cell activation and development.

Bidirectional binding of invariant chain peptides to an MHC class II molecule.

T-cell recognition of peptides bound to MHC class II (MHCII) molecules is a central event in cell-mediated adaptive immunity. The current paradigm holds that prebound class II-associated invariant chain peptides (CLIP) and all subsequent antigens maintain a canonical orientation in the MHCII binding groove. Here we provide evidence for MHCII-bound CLIP inversion. NMR spectroscopy demonstrates that the interconversion from the canonical to the inverse alignment is a dynamic process, and X-ray crystallography shows that conserved MHC residues form a hydrogen bond network with the peptide backbone in both orientations. The natural catalyst HLA-DM accelerates peptide reorientation and the exchange of either canonically or inversely bound CLIP against antigenic peptide. Thus, noncanonical MHC-CLIP displays the hallmarks of a structurally and functionally intact antigen-presenting complex.

High-throughput engineering and analysis of peptide binding to class II MHC.

Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.