Impact of peptides on the recognition of HLA class I molecules by human HLA antibodies

MHC class I molecules expressed on cell surfaces are composed of H chain, beta2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.     

Biochemical complexity of serum HLA class I molecules

Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around M _r 44 000, 40 000, and 35 000–37 000. HLA-A24-positive individuals are distinguished by higher serum levels of M _r 44 000 and 40 000 class I-like molecules than those found in HLA-A24-negative individuals. The M _r 44 000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The M _r 35 000 and 37 000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum M _r 44 000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The M _r 40 000 molecules do not contain a TM region. M _r 40 000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.Abbreviations used in this paper B2m beta-2 microglobulin - BSA bovine serum albumin - EBV-BLCL Epstein-Barr virus-transformed B lymphoblastoid cell line - mAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Cytotoxic T lymphocyte recognition of secreted HLA class I molecules

The cytolytic responses of DBA/2 mice against syngeneic transfected P815 mastocytoma cells expressing either membrane-associated (HLA-Cw3) or -secreted hybrid (HLA-Cw3 x H-2 Q10b) molecules were compared. In spite of the absence of serologically detectable hybrid molecules on their plasma membrane, cells secreting these molecules elicited a CTL response similar to that of cells expressing the membrane associated HLA-Cw3 molecules, in terms of both MHC-restriction and peptide specificity. Together with the observation that syngeneic mice were capable of rejecting the injected secreting cells, these results imply that secreted HLA class I molecules can function as minor histocompatibility Ag and suggest that processing of both the membrane-bound and the -secreted forms of a protein may follow common or overlapping pathways.     

ERp57 interacts with conserved cysteine residues in the MHC class I peptide-binding groove

The oxidoreductase ERp57 is a component of the major histocompatibility complex (MHC) class I peptide-loading complex. ERp57 can interact directly with MHC class I molecules, however, little is known about which of the cysteine residues within the MHC class I molecule are relevant to this interaction. MHC class I molecules possess conserved disulfide bonds between cysteines 101-164, and 203-259 in the peptide-binding and alpha3 domain, respectively. By studying a series of mutants of these conserved residues, we demonstrate that ERp57 predominantly associates with cysteine residues in the peptide-binding domain, thus indicating ERp57 has direct access to the peptide-binding groove of MHC class I molecules during assembly.     

Prediction of promiscuous peptides that bind HLA class I molecules

Promiscuous T-cell epitopes make ideal targets for vaccine development. We report here a computational system, MULTIPRED, for the prediction of peptide binding to the HLA-A2 supertype. It combines a novel representation of peptide/MHC interactions with a hidden Markov model as the prediction algorithm. MULTIPREDis both sensitive and specific, and demonstrates high accuracy of peptide-binding predictions for HLA-A*0201, *0204, and *0205 alleles, good accuracy for *0206 allele, and marginal accuracy for *0203 allele. MULTIPREDreplaces earlier requirements for individual prediction models for each HLA allelic variant and simplifies computational aspects of peptide-binding prediction. Preliminary testing indicates that MULTIPRED can predict peptide binding to HLA-A2 supertype molecules with high accuracy, including those allelic variants for which no experimental binding data are currently available.     

Exon shuffling in vivo can generate novel HLA class I molecules

The human class I histocompatibility molecule HLA-Aw69 has serological and structural properties which suggested it was a hybrid of the allelic products HLA-A2 and HLA-Aw68. We have now isolated three genes for HLA-Aw69 and one gene for HLA-Aw68. The sequences of exons encoding the entire extracellular portion of the molecule and of intron 2 have been determined. Their comparison with the published sequence of HLA-A2 proves that HLA-Aw69 is a hybrid molecule with complete identity to HLA-Aw68 in the alpha 1 domain and with HLA-A2 in the alpha 2 and alpha 3 domains. This comparison also localised regions involved in the epitopes recognised by monoclonal antibodies. The three HLA-Aw69 genes obtained from unrelated individuals of diverse ethnic backgrounds are identical. All results are consistent with HLA-Aw69 having arisen by a single reciprocal recombination event between the HLA-Aw68 and HLA-A2 genes somewhere in a region of 86 bp about the 3' donor splice site of exon 2. Estimates of the silent mutation rate in HLA genes suggest this event occurred not more than 330 000 years ago. Intra-allelic reciprocal recombination thus represents a further mechanism in addition to gene conversion for the generation of novel class I histocompatibility alleles.     

Amyloid precursor-like protein 2 association with HLA class I molecules

Amyloid precursor-like protein 2 (APLP2) is a ubiquitously expressed protein. The previously demonstrated functions for APLP2 include binding to the mouse major histocompatibility complex (MHC) class I molecule H-2K^d and down regulating its cell surface expression. In this study, we have investigated the interaction of APLP2 with the human leukocyte antigen (HLA) class I molecule in human tumor cell lines. APLP2 was readily detected in pancreatic, breast, and prostate tumor lines, although it was found only in very low amounts in lymphoma cell lines. In a pancreatic tumor cell line, HLA class I was extensively co-localized with APLP2 in vesicular compartments following endocytosis of HLA class I molecules. In pancreatic, breast, and prostate tumor lines, APLP2 was bound to the HLA class I molecule. APLP2 was found to bind to HLA-A24, and more strongly to HLA-A2. Increased expression of APLP2 resulted in reduced surface expression of HLA-A2 and HLA-A24. Overall, these studies demonstrate that APLP2 binds to the HLA class I molecule, co-localizes with it in intracellular vesicles, and reduces the level of HLA class I molecule cell surface expression.     

Disulfide bond engineering to trap peptides in the MHC class I binding groove

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells-particularly when the antigenic peptide has relatively weak affinity for the MHC.     

A geometric study of the amino acid sequence of class I HLA molecules

 HLA class I alleles are studied by representing them in a metric space where each dimension corresponds to each one of the amino acid positions. Their similarity in reference to their ability to present peptides to T cells is then evaluated by calculating the correlation matrix between the amino-acid-composition tables (or binding affinity tables) for the sets of peptides presented by each allele. This correlation matrix is considered an empirical similarity matrix between HLA alleles, and is modeled in terms of possible structures defined in the metric space of HLA class I amino acid sequences. These geometric structures are adequate models of the peptide-binding data currently available. The following clusters of HLA class I molecules are identified in reference to their ability to present peptides: Cluster I) HLA-A3/ HLA-A11/ HLA-A31/ HLA-A33/ HLA-A68; Cluster II) HLA-B35/ HLA-B51/ HLA-B53/ HLA-B54/ HLA-B7; and Cluster III) HLA-A29/ HLA-B61/HLA-B44; the last cluster showing possible similarities between alleles from different loci. In modeling these natural clusters, the geometric structures with more predictive power confirm the importance of those positions in the peptide-binding groove, particularly those in the B pocket. In addition, other positions (46, 79, 113, 131, 144, and 177) appeared to bear some relevance in determining which peptides can be presented by which HLA alleles. Received: 20 January 1998 / Revised: 30 March 1998     

Soluble HLA class I molecules: biological significance and clinical implications

Soluble class I human leukocyte antigens (sHLAs) have been detected in serum, sweat, lymphatic fluid, urine and cerebrospinal fluid. Their biological function has, however, remained a puzzle. The physiological concentration of sHLA varies more than tenfold depending on the phenotype of the individual, and is significantly upregulated in various diseases and during inflammation. This suggests that sHLAs might serve as a marker of pathological changes. Recent experiments have shown that, in vitro, sHLAs can modulate T-cell reactivity and induce cell-activated apoptosis, implicating sHLAs in the induction and maintenance of peripheral tolerance. Therefore, sHLAs have the therapeutic potential to induce tolerance to transplants.     

Differential transport requirements of HLA and H-2 class I glycoproteins

Transport of human and mouse major histocompatibility complex class I glycoproteins has been examined in a transport deficient B-lymphoblastoid cell line × T-lymphoblastoid cell line (B-LCL × T-LCL) hybrid, 174 × CEM. T2 (T2). This cell line expresses no detectable endogenous HLA-B5 and reduced levels of HLA-A2 on its surface although these molecules are synthesized. In order to study this defect further, either HLA-Bw58 or HLA-B7 genomic clones were transfected into T2. Metabolic labeling and immune precipitation demonstrated biosynthesis of the Bw58 or 137 glycoprotein. However, like the endogenous HLA-B5 molecule, neither HLA-Bw58 nor HLA-B7 was expressed at the cell surface. The cloned genes were properly expressed on the surface of C1R, a control B-LCL. To determine if mouse class I alleles had the same transport requirements as the human class I glycoproteins, either mouse H-2D ^p or H-2K ^b class I genes were introduced into T2. Surprisingly, the H-2 class I glycoproteins were transported to the cell surface normally. These data suggest a fundamental difference between human and mouse histocompatibility antigens in their requirements for intracellular transport.     

Lectin-deficient calnexin is capable of binding class I histocompatibility molecules in vivo and preventing their degradation

Calnexin is a membrane-bound lectin of the endoplasmic reticulum (ER) that binds transiently to newly synthesized glycoproteins. By interacting with oligosaccharides of the form Glc(1)Man(9)GlcNAc(2), calnexin enhances the folding of glycoprotein substrates, retains misfolded variants in the ER, and in some cases participates in their degradation. Calnexin has also been shown to bind polypeptides in vivo that do not possess a glycan of this form and to function in vitro as a molecular chaperone for nonglycosylated proteins. To test the relative importance of the lectin site compared with the polypeptide-binding site, we have generated six calnexin mutants defective in oligosaccharide binding using site-directed mutagenesis. Expressed as glutathione S-transferase fusions, these mutants were still capable of binding ERp57, a thiol oxidoreductase, and preventing the aggregation of a nonglycosylated substrate, citrate synthase. They were, however, unable to bind Glc(1) Man(9)GlcNAc(2) oligosaccharide and were compromised in preventing the aggregation of the monoglucosylated substrate jack bean alpha-mannosidase. Two of these mutants were then engineered into full-length calnexin for heterologous expression in Drosophila cells along with the murine class I histocompatibility molecules K(b) and D(b) as model glycoproteins. In this system, lectin site-defective calnexin was able to replace wild type calnexin in forming a complex with K(b) and D(b) heavy chains and preventing their degradation. Thus, at least for class I molecules, the lectin site of calnexin is dispensable for some of its chaperone functions.     

HLA class I binding of HBZ determines outcome in HTLV-1 infection

CD8(+) T cells can exert both protective and harmful effects on the virus-infected host. However, there is no systematic method to identify the attributes of a protective CD8(+) T cell response. Here, we combine theory and experiment to identify and quantify the contribution of all HLA class I alleles to host protection against infection with a given pathogen. In 432 HTLV-1-infected individuals we show that individuals with HLA class I alleles that strongly bind the HTLV-1 protein HBZ had a lower proviral load and were more likely to be asymptomatic. We also show that in general, across all HTLV-1 proteins, CD8(+) T cell effectiveness is strongly determined by protein specificity and produce a ranked list of the proteins targeted by the most effective CD8(+) T cell response through to the least effective CD8(+) T cell response. We conclude that CD8(+) T cells play an important role in the control of HTLV-1 and that CD8(+) cells specific to HBZ, not the immunodominant protein Tax, are the most effective. We suggest that HBZ plays a central role in HTLV-1 persistence. This approach is applicable to all pathogens, even where data are sparse, to identify simultaneously the HLA Class I alleles and the epitopes responsible for a protective CD8(+) T cell response.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

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.     

Messenger RNA-electroporated dendritic cells presenting MAGE-A3 simultaneously in HLA class I and class II molecules

An optimal anticancer vaccine probably requires the cooperation of both CD4(+) Th cells and CD8(+) CTLs. A promising tool in cancer immunotherapy is, therefore, the genetic modification of dendritic cells (DCs) by introducing the coding region of a tumor Ag, of which the antigenic peptides will be presented in both HLA class I and class II molecules. This can be achieved by linking the tumor Ag to the HLA class II-targeting sequence of an endosomal or lysosomal protein. In this study we compared the efficiency of the targeting signals of invariant chain, lysosome-associated membrane protein-1 (LAMP1) and DC-LAMP. Human DCs were electroporated before or after maturation with mRNA encoding unmodified enhanced green fluorescent protein (eGFP) or eGFP linked to various targeting signals. The lysosomal degradation inhibitor chloroquine was added, and eGFP expression was evaluated at different time points after electroporation. DCs were also electroporated with unmodified MAGE-A3 or MAGE-A3 linked to the targeting signals, and the presentation of MAGE-A3-derived epitopes in the context of HLA class I and class II molecules was investigated. Our data suggest that proteins linked to the different targeting signals are targeted to the lysosomes and are indeed presented in the context of HLA class I and class II molecules, but with different efficiencies. Proteins linked to the LAMP1 or DC-LAMP signal are more efficiently presented than proteins linked to the invariant chain-targeting signal. Furthermore, DCs electroporated after maturation are more efficient in Ag presentation than DCs electroporated before maturation.     

Sequence-specific minor groove binding by bis-benzimidazoles: water molecules in ligand recognition

The binding of two symmetric bis-benzimidazole compounds, 2,2-bis-[4′-(3″-dimethylamino-1″-propyloxy)phenyl]-5,5-bi-1H-benzimidazole and its piperidinpropylphenyl analog, to the minor groove of DNA, have been studied by DNA footprinting, surface plasmon resonance (SPR) methods and molecular dynamics simulations in explicit solvent. The footprinting and SPR methods find that the former compound has enhanced affinity and selectivity for AT sequences in DNA. The molecular modeling studies have suggested that, due to the presence of the oxygen atom in each side chain of the former compound, a water molecule is immobilized and effectively bridges between side chain and DNA base edges via hydrogen bonding interactions. This additional contribution to ligand–DNA interactions would be expected to result in enhanced DNA affinity, as is observed.     

Identification of five different Patr class I molecules that bind HLA supertype peptides and definition of their peptide binding motifs

We have sequenced the Pan troglodytes class I (Patr) molecules from three common chimpanzees and expressed them as single molecules in a class I-deficient cell line. These lines were utilized to obtain purified class I molecules to define the peptide binding motifs associated with five different Patr molecules. Based on these experiments, as well as analysis of the predicted structure of the B and F polymorphic MHC pockets, we classified five Patr molecules (Patr-A*0101, Patr-B*0901, Patr-B*0701, Patr-A*0602, and Patr-B*1301) within previously defined supertype specificities associated with HLA class I molecules (HLA-A3, -B7, -A1, and -A24 supertypes). The overlap in the binding repertoire between specific HLA and Patr class I molecules was in the range of 33 to 92%, depending on the particular Patr molecule as assessed by the binding of HIV-, hepatitis B virus-, and hepatitis C virus-derived epitopes. Finally, live cell binding assays of nine chimpanzee-derived B cell lines demonstrated that HLA supertype peptides bound to Patr class I molecules with frequencies in the 20-50% range.