Immunologic hierarchy,class II MHC promiscuity,and epitope spreading of a melanoma helper peptide vaccine

Immunization with a combination melanoma helper peptide (6MHP) vaccine has been shown to induce CD4⁺ T cell responses, which are associated with patient survival. In the present study, we define the relative immunogenicity and HLA allele promiscuity of individual helper peptides and identify helper peptide-mediated augmentation of specific CD8⁺ T cell responses. Thirty-seven participants with stage IIIB-IV melanoma were vaccinated with 6MHP in incomplete Freund’s adjuvant. The 6MHP vaccine is comprised of 6 peptides representing melanocytic differentiation proteins gp100, tyrosinase, Melan-A/MART-1, and cancer testis antigens from the MAGE family. CD4⁺ and CD8⁺ T cell responses were assessed in peripheral blood and in sentinel immunized nodes (SIN) by thymidine uptake after exposure to helper peptides and by direct interferon-γ ELIspot assay against 14 MHC class I-restricted peptides. Vaccine-induced CD4⁺ T cell responses to individual epitopes were detected in the SIN of 63 % (22/35) and in the peripheral blood of 38 % (14/37) of participants for an overall response rate of 65 % (24/37). The most frequently immunogenic peptides were MAGE-A3_281–295 (49 %) and tyrosinase_386–406 (32 %). Responses were not limited to HLA restrictions originally described. Vaccine-associated CD8⁺ T cell responses against class I-restricted peptides were observed in 45 % (5/11) of evaluable participants. The 6MHP vaccine induces both CD4⁺ and CD8⁺ T cell responses against melanoma antigens. CD4⁺ T cell responses were detected beyond reported HLA-DR restrictions. Induction of CD8⁺ T cell responses suggests epitope spreading and systemic activity mediated at the tumor site.     

A peptide filtering relation quantifies MHC class I peptide optimization

Major Histocompatibility Complex (MHC) class I molecules enable cytotoxic T lymphocytes to destroy virus-infected or cancerous cells, thereby preventing disease progression. MHC class I molecules provide a snapshot of the contents of a cell by binding to protein fragments arising from intracellular protein turnover and presenting these fragments at the cell surface. Competing fragments (peptides) are selected for cell-surface presentation on the basis of their ability to form a stable complex with MHC class I, by a process known as peptide optimization. A better understanding of the optimization process is important for our understanding of immunodominance, the predominance of some T lymphocyte specificities over others, which can determine the efficacy of an immune response, the danger of immune evasion, and the success of vaccination strategies. In this paper we present a dynamical systems model of peptide optimization by MHC class I. We incorporate the chaperone molecule tapasin, which has been shown to enhance peptide optimization to different extents for different MHC class I alleles. Using a combination of published and novel experimental data to parameterize the model, we arrive at a relation of peptide filtering, which quantifies peptide optimization as a function of peptide supply and peptide unbinding rates. From this relation, we find that tapasin enhances peptide unbinding to improve peptide optimization without significantly delaying the transit of MHC to the cell surface, and differences in peptide optimization across MHC class I alleles can be explained by allele-specific differences in peptide binding. Importantly, our filtering relation may be used to dynamically predict the cell surface abundance of any number of competing peptides by MHC class I alleles, providing a quantitative basis to investigate viral infection or disease at the cellular level. We exemplify this by simulating optimization of the distribution of peptides derived from Human Immunodeficiency Virus Gag-Pol polyprotein.     

Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design

Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.     

Hydrogen bond integrity between MHC class II molecules and bound peptide determines the intracellular fate of MHC class II molecules.

MHC class II molecules associate with peptides through pocket interactions and the formation of hydrogen bonds. The current paradigm suggests that the interaction of side chains of the peptide with pockets in the class II molecule is responsible for the formation of stable class II-peptide complexes. However, recent evidence has shown that the formation of hydrogen bonds between genetically conserved residues of the class II molecule and the main chain of the peptide contributes profoundly to peptide stability. In this study, we have used I-A(k), a class II molecule known to form strong pocket interactions with bound peptides, to probe the general importance of hydrogen bond integrity in peptide acquisition. Our studies have revealed that abolishing hydrogen bonds contributed by positions 81 or 82 in the beta-chain of I-A(k) results in class II molecules that are internally degraded when trafficked through proteolytic endosomal compartments. The presence of high-affinity peptides derived from either endogenous or exogenous sources protects the hydrogen bond-deficient variant from intracellular degradation. Together, these data indicate that disruption of the potential to form a complete hydrogen bond network between MHC class II molecules and bound peptides greatly diminishes the ability of class II molecules to bind peptides. The subsequent failure to stably acquire peptides leads to protease sensitivity of empty class II molecules, and thus to proteolytic degradation before export to the surface of APCs.     

The quality control of MHC class I peptide loading

The assembly of major histocompatibility complex (MHC) class I molecules is one of the more widely studied examples of protein folding in the endoplasmic reticulum (ER). It is also one of the most unusual cases of glycoprotein quality control involving the thiol oxidoreductase ERp57 and the lectin-like chaperones calnexin and calreticulin. The multistep assembly of MHC class I heavy chain with beta(2)-microglobulin and peptide is facilitated by these ER-resident proteins and further tailored by the involvement of a peptide transporter, aminopeptidases, and the chaperone-like molecule tapasin. Here we summarize recent progress in understanding the roles of these general and class I-specific ER proteins in facilitating the optimal assembly of MHC class I molecules with high affinity peptides for antigen presentation.     

The I-Ag7 MHC class II molecule linked to murine diabetes is a promiscuous peptide binder

Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common alpha-chain with I-Ad but has a peculiar beta-chain. As with most beta-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position beta57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the beta-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.     

Functional screening of a retroviral peptide library for MHC class I presentation

We have used retroviral vector technology to develop a method for functional screening of combinatorial peptide libraries expressed inside mammalian cells with the ultimate goal of identifying new drug targets. The method was validated in a library screening experiment based on antigen presentation of small peptides. A library encoding SIXNXEKX-peptides, where X designates randomised positions corresponding to major histocompatibility (MHC) class I anchor residues, was generated in a retroviral vector. The library was transduced into a population of antigen presenting cells (APCs) known to mediate MHC class I restricted presentation of the SIINFEKL peptide. The cellular library was screened by using an antigen presentation assay in which a T cell hybridoma recognising the MHC class I/SIINFEKL peptide complex was employed. Using this experimental model, we identified two positive cellular clones both encoding SIINFEKL peptides with identical codon usage. This number corresponded well to the expected frequency of SIINFEKL in the library. The lack of identification of other peptides capable of activating the T-hybridoma supports previous findings of a high degree of specificity at the level of peptide-loading of MHC-molecules. The result further demonstrates the potential of using combinatorial libraries for functional screening and selection of effector peptides stably expressed in mammalian cells.     

MHC Class I/peptide interactions: Binding specificity and kinetics

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran-modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three-dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (k_d) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured k_d values for antigenic peptide/class I interactions at 25°C are in the range of ca 10^?4–10^?6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid-phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.     

Monoclonal antibody detection of a major self peptide. MHC class II complex.

MHC class I and class II molecules transport foreign and self peptides to the cell surface and present them to T lymphocytes. Detection of these peptide:MHC complexes has thus far been limited to analysis of the response of a T cell. Previously, we showed that a mAb, Y-Ae, reacts with 10 to 15% of class II molecules on peripheral B lymphocytes and on cells in the thymus medulla but not thymus cortex in mice that express both I-Ab and I-Eb molecules. Elsewhere, we show that Y-Ae detects a self E alpha peptide bound to I-Ab molecules. Data presented here suggest that the antibody binds over the peptide binding groove of class II molecules, and, like a TCR, appears to recognize both the self peptide and polymorphic class II residues. In addition to B lymphocytes, the Y-Ae determinant is expressed at comparable levels on other APC, including macrophages and dendritic cells. Finally, the antibody does not react with invariant chain-associated class II complexes, thus providing direct evidence that invariant chain:class II complexes and peptide:class II complexes are mutually exclusive. These data provide further evidence that immunologic self is of limited complexity, and have important implications for T cell selection, self tolerance, and autoreactivity.     

MAPPP: MHC class I antigenic peptide processing prediction

MAPPP is a bioinformatics tool for the prediction of potential antigenic epitopes presented on the cell surface by major histocompatibility complex class I (MHC I) molecules to CD8 positive T lymphocytes. It combines existing predictions for proteasomal cleavage with peptide anchoring to MHC I molecules.     

A three-step kinetic mechanism for peptide binding to MHC class II proteins

Peptide binding reactions of class II MHC proteins exhibit unusual kinetics, with extremely slow apparent rate constants for the overall association (<100 M(-)(1) s(-)(1)) and dissociation (<10(-)(5) s(-)(1)) processes. Various linear and branched pathways have been proposed to account for these data. Using fluorescence resonance energy transfer between tryptophan residues in the MHC peptide binding site and aminocoumarin-labeled peptides, we measured real-time kinetics of peptide binding to empty class II MHC proteins. Our experiments identified an obligate intermediate in the binding reaction. The observed kinetics were consistent with a binding mechanism that involves an initial bimolecular binding step followed by a slow unimolecular conformational change. The same mechanism is observed for different peptide antigens. In addition, we noted a reversible inactivation of the empty MHC protein that competes with productive binding. The implications of this kinetic mechanism for intracellular antigen presentation pathways are discussed.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

Comparison of rat MHC class I antigens by peptide mapping

Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.Aⁿ, RT1.A^u, and RT1.E^u were used for immunoprecipitation of antigens biosynthetically radiolabeled with¹⁴C- or³H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (Aⁿ and A^u) differed by 30%, whereas the products of two different loci in the same haplotype (A^u and E^u) differed only by 1–3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in A^u have been substituted by an arginine and probably by a tyrosine residue, respectively, in E^u. The high degree of homology between the products of theA andE loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.We dedicate this publication to Professor Paul Doty on the occasion of his sixty-fifth birthday     

Major histocompatibility complex (MHC)-encoded HAM2 is necessary for antigenic peptide loading onto class I MHC molecules.

The mutant murine lymphoma cell line RMA-S is unable to present endogenous antigens due to its inability to efficiently assemble class I major histocompatibility complex molecules and antigenic peptides. Therefore, it has been suggested that RMA-S cells are defective either in peptide generation or in peptide transport into the endoplasmic reticulum, where class I major histocompatibility complex molecule assembly is believed to occur. As proteasomes and the putative peptide transporters HAM1 and HAM2 have been implicated in class I antigen processing, we have investigated their expression in RMA-S and its wild-type counterpart RMA. Both proteasomes and HAM1 proteins are expressed at similar levels and show identical subcellular distributions in the two cell lines. However, only one copy of the HAM2 gene is present in RMA-S cells, and it contains a point mutation that leads to a premature stop codon. Thus, the HAM2 protein is absent from RMA-S cells. These data demonstrate that HAM2 is essential for peptide loading onto class I molecules.     

A revised model for invariant chain-mediated assembly of MHC class II peptide receptors

The enormous number of allelic MHC class II glycoproteins provides the immune system with a large set of heterodimeric receptors for the binding of pathogen-derived peptides. How do inherited allo- or isotypic subunits of MHC class II combine to produce such a variety of functional peptide receptors? We propose a new mechanism in which pairing of matched MHC class II alpha- and beta-subunits is coordinated by the invariant chain chaperone. The assembly is proposed to occur in a sequential fashion, with a matched beta-chain being selected by the alpha-chain-invariant chain 'scaffold' complex that is formed first. This sequential assembly is a prerequisite for subsequent intracellular transport of the alpha-chain-invariant chain-beta-oligomer and its maturation into a functional peptide receptor.     

Polymorphic peptide transporters in MHC class I monomorphic Syrian hamster

We have already shown that in species with highly polymorphic major histocompatibility complex (MHC) class I molecules (human, mouse) no functional polymorphism of the peptide transporters TaP1 and TAP2 is detectable (Lobigs and Müllbacher 1993).Investigating the antigen-presentation machinery of the class I MHC I expression via recombinant vaccinia viruses MHC class I expression via recombinant vaccinia viruses (VV) we found that six hamster cell lines fall into two phenotypic classes. Four cell lines (HaK, FF, MF-2, and HT-1) showed no defect in expressing four different H2 class I molecules (K^K, K^d, K^b, D^d) and the appropriate VV peptide recognized by mouse VV-immune cytotoxic T (Tc) cells on the cell surface. Two cell lines (BHK-21 and NIL-2) expressed D^d and K^b in association with VV peptides as recognized by VV-immune, H2-restricted Tc cells but not K^k and K^d. However, K^d was expressed on the cell surface, as shown by fluorescence-activated cell sorter (FACS) analysis and alloreactive Tc-cell recogniction. K^k is only surface-expressed in these two cell lines when superinfected with two VV recombinants encoding rat TAP1 (VV-mtp1) and TAP2 (VV-mtp2). Superinfection with VV-mtpl and VV-mtp2 rendered both cell lines, after infection with either VV-K^k or V-K^d, susceptible to lysis by either K^k-orK^d- restricted VV-immune Tc cells. Thus Syrian hamster cell lines express functionally polymorphic peptide transporters.The TAP2 gene from FF cells was cloned and sequenced; comparison with human, mouse, and rat TAP2 sequences show 78%, 88% and 87% similarity, respectively.     

A peptide binding motif for I-Eg7, the MHC class II molecule that protects E alpha-transgenic nonobese diabetic mice from autoimmune diabetes.

The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due to a deletion in the E alpha gene promoter. E alpha-transgenic NOD mice express the E alpha E beta g7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7-restricted T cell epitope corresponding to the sequence 4-13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and E alpha-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in E alpha-transgenic NOD mice.     

Cooperativity during the formation of peptide/MHC class II complexes

To generate an effective immune response, class II major histocompatibility complex molecules (MHCII) must present a diverse array of peptide ligands for recognition by T lymphocytes. Peptide/MHCII complexes are stabilized by hydrophobic anchoring of peptide side chains to pockets in the MHCII protein and the formation of hydrogen bonds to the peptide backbone. Many current models of peptide/MHCII association assume an additive and independent contribution of the interactions between major MHCII pockets and corresponding side chains in the peptide. However, significant conformational rearrangements occur in both the peptide and MHCII during binding. Therefore, we hypothesize that peptide binding to MHCII could be viewed as a folding process in which both molecules cooperate to produce the final conformation. To directly test this hypothesis, we adapt a serial mutagenesis strategy to study cooperativity in the interaction of the human MHCII HLA-DR1 and a peptide derived from influenza hemagglutinin. Substitutions in either the peptide or HLA-DR1 that are predicted to interfere with hydrogen bond formation show cooperative effects on complex stability and affinity. Substitution of a peptide side chain that provides a hydrophobic contact also contributes to the cooperative effect, suggesting a role for all energetic sources in the folding process. We propose that cooperativity throughout the peptide-binding groove reflects the folding of segments of the MHCII molecule into helices around the peptide with a concomitant folding of the peptide into a polyproline helix. The implications of cooperativity for peptide/MHCII structure and epitope selection are discussed.     

Kinetics of peptide binding to the class II MHC protein I-Ek

Class II MHC glycoproteins bind short (7-25 amino acid) peptides in an extended type II polyproline-like conformation and present them for immune recognition. Because empty MHC is unstable, measurement of the rate of the second-order reaction between peptide and MHC is challenging. In this report, we use dissociation of a pre-bound peptide to generate the active, peptide-receptive form of the empty class II MHC molecule I-Ek. This allows us to measure directly the rate of reaction between active, empty I-Ek and a set of peptides that vary in structure. We find that all peptides studied, despite having highly variable dissociation rates, bind with similar association rate constants. Thus, the rate-limiting step in peptide binding is minimally sensitive to peptide side-chain structure. An interesting complication to this simple model is that a single peptide can sometimes bind to I-Ek in two kinetically distinguishable conformations, with the stable peptide-MHC complex isomer forming much more slowly than the less-stable one. This demonstrates that an additional free-energy barrier limits the formation of certain specific MHC-peptide complex conformations.