Interaction of antigenic peptides with MHC class I molecules on living cells studied by photoaffinity labeling.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of antigenic peptides with murine MHC class I molecules on living cells. Photoreactive derivatives were prepared by N-terminal amidation with iodo, 4-azido salicylic acid of the Kd restricted Plasmodium berghei circumsporozoite (P.b. CS) peptide 253-260 (YIPSAEKI) and the Db-restricted Adenovirus 5 early region 1A (Ad5 E1A) peptide 234-243 (SGPSNTPPEI). As assessed in functional competition experiments, both peptide derivatives retained the specific binding activity of the parental peptides for Kd or Dd, respectively. The P.b. CS photoprobe specifically labeled Kd molecules on P815 (H-2d) cells, but failed to label RMA (H-2b) cells. Conversely, the Ad5 E1A photoprobe specifically labeled Db molecules on RMA cells, but failed to label P815 cells. When the two photoprobes were tested on a panel of Con A-activated spleen cells expressing 10 different H-2 haplotypes, significant photoaffinity labeling was observed only on H-2d cells with the P.b. CS photoprobe and on H-2b cells with the Ad5 E1A photoprobe. Labeling of cell-associated Kd or Db molecules with the photoprobes was specifically inhibited by antigenic peptides known to be presented by the same class I molecule. Photoaffinity labeling of Kd with the P.b. CS photoprobe was used to study the dynamics of peptide binding on living P815 cells. Binding increased steadily with the incubation period (up to 8 h) at 37 degrees C and at ambient temperature, but was greatly reduced (greater than 95%) at 0 to 4 degrees C or in the presence of ATP synthesis inhibitors. The magnitude of the labeling was twofold higher at room temperature than at 37 degrees C. In contrast, binding to isolated Kd molecules in solution rapidly reached maximal binding, particularly at 37 degrees C. Dissociation of the photoprobe from either cell-associated or soluble Kd molecules was similar, with a half time of approximately 1 h at 37 degrees C, whereas the complexes were long-lived at 4 degrees C in both instances.     

Structural prediction of peptides binding to MHC class I molecules

Peptide binding to class I major histocompatibility complex (MHCI) molecules is a key step in the immune response and the structural details of this interaction are of importance in the design of peptide vaccines. Algorithms based on primary sequence have had success in predicting potential antigenic peptides for MHCI, but such algorithms have limited accuracy and provide no structural information. Here, we present an algorithm, PePSSI (peptide-MHC prediction of structure through solvated interfaces), for the prediction of peptide structure when bound to the MHCI molecule, HLA-A2. The algorithm combines sampling of peptide backbone conformations and flexible movement of MHC side chains and is unique among other prediction algorithms in its incorporation of explicit water molecules at the peptide-MHC interface. In an initial test of the algorithm, PePSSI was used to predict the conformation of eight peptides bound to HLA-A2, for which X-ray data are available. Comparison of the predicted and X-ray conformations of these peptides gave RMSD values between 1.301 and 2.475 A. Binding conformations of 266 peptides with known binding affinities for HLA-A2 were then predicted using PePSSI. Structural analyses of these peptide-HLA-A2 conformations showed that peptide binding affinity is positively correlated with the number of peptide-MHC contacts and negatively correlated with the number of interfacial water molecules. These results are consistent with the relatively hydrophobic binding nature of the HLA-A2 peptide binding interface. In summary, PePSSI is capable of rapid and accurate prediction of peptide-MHC binding conformations, which may in turn allow estimation of MHCI-peptide binding affinity.     

Exploration of peptides bound to MHC class I molecules in melanoma

Advancements in high‐resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83–87% of these were 8–11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA‐type binding prediction for 10 078 9/10 mer peptides assigned 88–95% to a patient‐specific HLA subtype, revealing a disparity in strength of predicted binding. HLA‐B*27‐specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.     

Peptide presentation by MHC class I molecules

The presentation of peptides by class I histocompatibility molecules plays a central role in the cellular immune response to virally infected or transformed cells. The main steps in this process include the degradation of both self and 'foreign' proteins to short peptides in the cytosol, translocation of peptides into the lumen of the endoplasmic reticulum, binding of a subset of peptides to assembling class I molecules and expression of class-I-peptide complexes at the cell surface for examination by cytotoxic T cells. A molecular understanding of most of these steps is emerging, revealing a remarkable coordination between the processes of peptide translocation, delivery and binding to class I molecules.     

Human major histocompatibility complex (MHC) class I molecules with disulfide traps secure disease-related antigenic peptides and exclude competitor peptides

The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, beta(2)m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.     

Differential stability of antigenic MHC class I-restricted synthetic peptides

Various synthetic peptides recognized as Ag by CTL in the context of MHC class I molecules were tested for stability in vitro and in vivo. Peptide inactivation in vitro was quantitated by titrating the amount of peptide required to sensitize target cells for lysis by specific CTL clones. The degree of inactivation after overnight incubation at 37 degrees C varied widely among a series of antigenic peptides. Some were nearly unaffected, whereas others lost activity by more than 100-fold or even 10,000-fold. However, no correlation was found between susceptibility to serum inactivation and antigenic potency as measured in short term cytolytic assays. No inactivation occurred at 4 degrees C, or at 37 degrees C in the absence of serum, under the conditions used. Serum inactivation most likely involved proteolysis because it could be inhibited by protease inhibitors. Moreover, presumed cleavage products of a radiolabeled susceptible peptide could be visualized by TLC. In vivo, the persistence of the antigenic activity of the injected peptides, either in extracellular fluids or on tumor target cells growing in an ascites form, correlated with the degree of stability found for the peptides in vitro. The differential stability of synthetic peptides may have important consequences for attempts to manipulate the development of an immune response in vivo.     

Neuronal cells are deficient in loading peptides onto MHC class I molecules.

Virally infected neurons avoid destruction by cytotoxic T lymphocytes (CTLs) by failing to express major histocompatibility complex (MHC) class I molecules. Like neurons in vivo and in primary culture, the OBL21 neuronal cell line expressed barely detectable levels of MHC class I molecules. This correlated with very low levels of mRNAs for the MHC class I heavy chains (alpha C). OBL21 cells also fail to provide MHC class I molecules with the peptides necessary for their efficient assembly and transport to the cell surface. This function can be restored by treatment with interferon-gamma (IFN-gamma). The mRNA for peptide transporters HAM1 and HAM2 was not detectable in OBL21 neuronal cells, but was induced by IFN-gamma treatment. Hence, the ability of neurons to evade CTL-mediated killing results from expression at low levels of the MHC class I alpha C, the peptide transporters HAM1 and HAM2, and possibly other genes of the peptide-loading machinery.     

A comparative analysis of viral peptides presented by contemporary human and chimpanzee MHC class I molecules

Genetic factors such as the MHC influence the immunocompetence of an individual. MHC genes are the most polymorphic genes in primates, which is often interpreted as an adaptation to establish good T cell responses to a wide range of (evolving) pathogens. Chimpanzee MHC (Patr) genes are less polymorphic than human MHC (HLA) genes, which is surprising because chimpanzee is the older species of the two and is therefore expected to display more variation. To quantify the effect of the reduced polymorphism, we compared the peptide binding repertoire of human and chimpanzee MHC molecules. Using a peptide-MHC binding predictor and proteomes of >900 mammalian viruses, we show that, at the population level, the total peptide binding repertoire of Patr-A molecules is ~36% lower than that of their human counterparts, whereas the reduction of the peptide binding repertoire of the Patr-B locus is only 15%. In line with these results, different Patr-A molecules turn out to have largely overlapping peptide binding repertoires, whereas the Patr-B molecules are more distinct from each other. This difference is somewhat less apparent at the individual level, where we found that only 25% of the viruses are significantly better presented by "simulated" humans with heterozygous HLA-A and -B loci. Taken together, our results indicate that the Patr-B molecules recovered more after the selective sweep, whereas the Patr-A locus shows the most signs of the selective sweep with regard to its peptide binding repertoire.     

Reduced KIR2DL1 recognition of MHC class I molecules presenting phosphorylated peptides

As initially described by K. Karre and colleagues in the missing self hypothesis, cells expressing self-MHC class I proteins are protected from NK cells attack. In contrast, reduction in the expression of MHC class I molecules due to viral infection or tumor transformation result in the killing of these "abnormal" cells by NK cells via NK-activating receptors. Thus, NK killing of target cells is determined by both negative signals coming from MHC class I proteins and by positive signals derived from the activating ligands. The bound peptide in MHC class I play an important role in the balanced recognition of NK cells. The peptide stabilizes the MHC complex and interacts directly with the NK inhibitory receptors, thus participating in the determination of the fate of the target cells. In this study we demonstrate that posttranslational modifications such as phosphorylation of the presented peptide altered the ability of NK cells to recognize MHC class I molecules. By using a consensus peptide (QYDDAVYKL) that binds HLA-Cw4 in which different positions in the bound peptide were modified by serine phosphorylation, we observed a reduction in KIR2DL1 binding that led to decreased protection from NK killing. Therefore, it might be possible that alteration in the phosphorylation pattern during tumor transformation or viral infection may result in less inhibition and, consequently, improved NK cell killing.     

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.     

Phosphorylated peptides can be transported by TAP molecules, presented by class I MHC molecules, and recognized by phosphopeptide-specific CTL.

CTL recognize short peptide fragments presented by class I MHC molecules. In this study, we examined the effect of phosphorylation on TAP transport, binding to class I MHC molecules, and recognition by CTL of peptide fragments from known phosphorylated oncogene proteins or virus phosphoproteins. We show that phosphopeptides can be efficiently transported from the cytosol to the endoplasmic reticulum by the TAP. Furthermore, we show that phosphorylation can have a neutral, negative, or even a positive effect on peptide binding to class I MHC. Finally, we have generated phosphopeptide-specific CTL that discriminate between the phosphorylated and the nonphosphorylated versions of the peptide. We conclude that phosphopeptide-specific CTL responses are likely to constitute a subset of the class I MHC-restricted CTL repertoire in vivo.     

Constitutive internalization of murine MHC class I molecules

The total number of cell surface glycoprotein molecules at the plasma membrane results from a balance between their constitutive internalization and their egress to the cell surface from intracellular pools and/or biosynthetic pathway. Constitutive internalization is net result of constitutive endocytosis and endocytic recycling. In this study we have compared spontaneous internalization of murine major histocompatibility complex (MHC) class I molecules (K(d), D(d), full L(d), and empty L(d)) after depletion of their egress to the cell surface (Cycloheximide [CHX], brefeldin A [BFA]) and internalization after external binding of monoclonal antibody (mAb). MHC class I alleles differ regarding their cell surface stability, kinetics, and in the way of internalization and degradation. K(d) and D(d) molecules are more stable at the cell surface than L(d) molecules and, thus, constitutively internalized more slowly. Although the binding of mAbs to cell surface MHC class I molecules results in faster internalization than depletion of their egress, it is still slow and, thereby, can serve as a model for tracking of MHC class I endocytosis. Internalization of fully conformed MHC class I molecules (K(d), D(d), and L(d)) was neither inhibited by chlorpromazine (CP) (inhibitor of clathrin endocytosis), nor with filipin (inhibitor of lipid raft dependent endocytosis), indicating that fully conformed MHC class I molecules are internalized via the bulk pathway. In contrast, internalization of empty L(d) molecules was inhibited by filipin, indicating that non-conformed MHC class I molecules require intact cholesterol-rich membrane microdomains for their constitutive internalization. Thus, conformed and non-conformed MHC class I molecules use different endocytic pathways for constitutive internalization.     

Structure-based prediction of binding peptides to MHC class I molecules: application to a broad range of MHC alleles

Specific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T-cells. Prediction of MHC-binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T-cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide-MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623-644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361-369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.     

Endoplasmic reticulum aminopeptidase associated with antigen processing regulates quality of processed peptides presented by MHC class I molecules

Effective immune surveillance by CD8 T cells depends on the presentation of diverse peptides by MHC class I (pMHC I) molecules on the cell surface. The pMHC I repertoire is shaped in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP). The ERAAP activity is required for producing peptides of appropriate length for generating optimal pMHC I. Paradoxically, ERAAP also inhibits generation of certain peptides such as the SVL9 (SSVVGVWYL) peptide encoded by the H13(a) histocompatibility gene and presented by D(b) MHC by an unknown mechanism. In this study, we show that the presentation of the SVL9-D(b) complex is inhibited when other peptides compete for binding D(b). Conversely, improving the binding of SVL9 peptide to D(b) suppresses the inhibition. Interestingly, the inhibitory effect of competitor peptides is observed only when ERAAP is expressed in the same cells. Thus, ERAAP, in concert with MHC I molecules, regulates the quality of processed peptides presented on the cell surface.     

Rethinking peptide supply to MHC class I molecules

The notion that peptides bound to MHC class I molecules are derived mainly from newly synthesized proteins that are defective, and are therefore targeted for immediate degradation, has gained wide acceptance. This model, still entirely hypothetical, has strong intuitive appeal and is consistent with some experimental results, but it is strained by other findings, as well as by established and emerging concepts in protein quality control. While not discounting defectiveness as a driving force for the processing of some proteins, we propose that MHC-class-I-restricted epitopes are derived mainly from nascent proteins that are accessed by the degradation machinery prior to any assessment of fitness, and we outline one way in which this could be accomplished.     

Transfer of GRP94(Gp96)-associated peptides onto endosomal MHC class I molecules

GRP94 (gp96)-associated peptides can elicit cellular immune responses, an activity thought to reflect the presence of a cell surface receptor (CD91) on antigen-presenting cells that mediates GRP94 internalization and trafficking to an amenable site for peptide transfer to major histocompatibility complex class I molecules. We report that GRP94 internalized by receptor-mediated endocytosis is trafficked to a Rab5a, CD1 and transferrin-negative, Fc receptor and major histocompatibility complex class I-positive endocytic compartment. Receptor-internalized GRP94 did not access the endoplasmic reticulum of antigen-presenting cells. To identify the site of re-presentation of GRP94-associated peptides, kinetic analyses were performed utilizing GRP94-OVA (SIINFEKL) peptide complexes, with peptide re-presentation assayed with the K^b-SIINFEKL-specific MAb, 25-D1.16. Analyses of the kinetics of re-presentation of GRP94-associated peptides, under conditions in which de novo synthesis of major histocompatibility complex class I molecules was inhibited, identified a post-endoplasmic reticulum compartment, accessed by mature major histocompatibility complex class I, as the predominant site of GRP94-associated peptide exchange onto major histocompatibility complex class I.     

Assembly of MHC class I molecules analyzed in vitro

Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.     

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.