Lectin purified human class I MHC-derived peptides: evidence for presentation of glycopeptides in vivo.

Previously, using synthetic glycopeptides carrying a natural cytosolic type of monosaccharide O-beta-linked N-acetylglucosamine (GlcNAc) glycosylation of serine residues, we have shown that glycopeptides act as suitable substrates for TAP-mediated transport into the endoplasmic reticulum (ER), and that they bind efficiently to class I major histocompatibility complex (MHC) molecules and can elicit glycopeptide-specific cytotoxic T-lymphocyte (CTL) responses in mice. Recently, we have reported that peptides presented by human class I MHC molecules in vivo encompass a small but significant amount of peptides which seem to be carrying O-beta-linked monosaccharide GlcNAc. In the present report we provide further evidence that glycosylated peptides are indeed presented by class I MHC molecules in vivo. Thus, peptides derived from HLA-A*0201 were purified by wheat germ agglutinin (WGA) lectin affinity chromatography as previously described. Subsequently, the peptides contained in the WGA-eluate were subjected to sequence analysis by Edman degradation. It was found that the peptides derived from HLA-A*0201 which had been retained by the O-GlcNAc-binding lectin WGA did indeed carry a HLA-A*0201 binding motif. Furthermore, using an enzymatic labeling procedure we present evidence that the HLA-A*0201-derived peptides which bind to the WGA lectin are glycosylated with terminal GlcNAc residues. Together, these data provide further evidence for the natural presentation by human class I MHC of glycopeptides carrying terminal O-GlcNAc residues in vivo.

Poor correspondence between predicted and experimental binding of peptides to class I MHC molecules.

Naturally processed peptides presented by class I major histocompatibility complex (MHC) molecules display a characteristic allele specific motif of two or more essential amino acid side chains, the so-called peptide anchor residues, in the context of an 8-10 amino acid long peptide. Knowledge of the peptide binding motif of individual class I MHC molecules permits the selection of potential peptide antigens from proteins of infectious organisms that could induce protective T-cell-mediated immunity. Several methods have been developed for the prediction of potential class I MHC binding peptides. One is based on a simple scanning for the presence of primary peptide anchor residues in the sequence of interest. A more sophisticated technology is the utilization of predictive computer algorithms. Here, we have analyzed the experimental binding of 84 peptides selected on the basis of the presence of peptide binding motifs for individual class I MHC molecules. The actual binding was compared with the results obtained when analyzing the same peptides by two well-known, publicly available computer algorithms. We conclude that there is no strong correlation between actual and predicted binding when using predictive computer algorithms. Furthermore, we found a high number of false-negatives when using a predictive algorithm compared to simple scanning for the presence of primary anchor residues. We conclude that the peptide binding assay remains an important step in the identification of cytotoxic T lymphocyte (CTL) epitopes which can not be substituted by predictive algorithms.

Peptides spanning the junctional region of both the abl/bcr and the bcr/abl fusion proteins bind common HLA class I molecules.

The Philadelphia (Ph) chromosome, resulting from the t(9;22) translocation, is characteristic of chronic myeloid leukemia (CML). As a result of this translocation, two novel chimeric genes are generated and the bcr/abl and abl/bcr fusion proteins expressed. The bcr/abl fusion mRNA is present in all CML patients, whereas the reciprocal abl/bcr fusion mRNA is detectable in about 80% of the Ph+ CML patients. These fusion proteins may undergo enzymatic degradation in the cytosol and give rise to MHC class I restricted peptide epitopes originating from the junctional regions of the translocation products, which thus may serve as novel tumor specific antigens. Previously, other groups have tested peptides corresponding to the junctional region of the bcr/abl protein for their binding capacity to HLA class I molecules and have identified a few candidate epitopes. Peptides originating from the abl/bcr fusion protein have on the other hand so far been neglected, for no apparent reason. We have now extended these studies to include also the reciprocal abl/bcr translocation product by testing a large panel of synthetic peptides corresponding to the junctional regions of both the abl/bcr and the bcr/abl fusion proteins for their ability to stabilize HLA class I molecules. We find that the abl/bcr translocation product may be an even more important source of CML specific peptide antigens and together the junctional sequences of both these proteins contain peptide sequences which bind efficiently to a number of HLA molecules (HLA-A1, -A2, -A3, -A11, -B7, -B27, -B35) and thus may serve as candidate CML specific tumor antigens.

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.

An assay for peptide binding to HLA-Cw*0102.

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) molecules is based on the ability of peptides to stabilize MHC class I molecules synthesized by transporter associated with antigen processing (TAP)-deficient cell. The TAP-deficient cell line T2 has previously been used in the assembly assay to analyze peptide binding to HLA-A*0201 and -B*5101. In this study, we have extended this technique to assay for peptides binding to endogenous HLA-Cw*0102 molecules. We have analyzed the peptide binding of 20 peptides with primary anchor motifs for HLA-Cw*0102. One-third of the peptides analyzed bound with high affinity, half of the peptides examined did not bind, whereas the remaining peptides displayed intermediate binding activity. Interest in HLA-C molecules has increased significantly in recent years, since it has been shown that HLA-C molecules both can present peptides to cytotoxic T lymphocytes (CTL) and in addition are able to inhibit natural killer (NK)-mediated lysis.

Presentation of cytosolic glycosylated peptides by human class I major histocompatibility complex molecules in vivo.

Antigens presented by class I major histocompatibility complex (MHC) molecules for recognition by cytotoxic T lymphocytes consist of 8-10-amino-acid-long cytosolic peptides. It is not known whether posttranslationally modified peptides are also presented by class I MHC molecules in vivo. Many different posttranslational modifications occur on cytoplasmic proteins, including a cytosolic O-beta-linked glycosylation of serine and threonine residues with N-acetylglucosamine (GlcNAc). Using synthetic glycopeptides carrying the monosaccharide O-beta-GlcNAc substitution on serine residues, we have shown that glycopeptides bind efficiently to class I MHC molecules and elicit a glycopeptide-specific cytotoxic T lymphocyte response in mice. In this study, we provide evidence that peptides presented by human class I MHC molecules in vivo encompass a small, significant amount of glycopeptides, constituting up to 0.1% of total peptide. Furthermore, we find that carbohydrate structures present on glycopeptides isolated from class I MHC molecules are dominated by the cytosolic O-beta-GlcNAc substitution, and synthetic peptides carrying this substitution are efficiently transported by TAP (transporter associated with antigen presentation) into the endoplasmic reticulum. Thus, in addition to unmodified peptides, posttranslationally modified cytosolic peptides carrying O-beta-linked GlcNAc can be presented by class I MHC molecules to the immune system.

Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand.

MUC1 peptide epitopes associated with five different H-2 class I molecules.

We have previously described the induction of murine CD8+ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [Kb (three epitopes), Dd, Ld and Kk] were closely related, all containing the amino acids PDTRPAP. For Db, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low 'affinity', compared with known high-affinity peptides. CD8+ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (Kb) or lost (Db) depending on the epitope.

An improved assembly assay for peptide binding to HLA-B*2705 and H-2K(k) class I MHC molecules.

The assembly assay for peptide binding to class I major histocompatibility complex (MHC) is based on the ability to stabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional transporter associated with antigen presentation (TAP) and as a result accumulate empty, unstable class I molecules in the ER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide during lysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B * 2705 and H-2Kk are unusually stable in their peptide-receptive state making them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHC class I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptide binding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence of peptide and incubated at 4 degrees C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heated to a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique to assay peptide binding to HLA-B * 2705, as well as to the murine allele H-2Kk which also displays a stable phenotype when transfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methods in terms of lower background signal and higher recovery of peptide bound molecules.

Induction of HLA-A2-restricted CTLs to the mucin 1 human breast cancer antigen.

HLA-A*0201/Kb transgenic mice were immunized with oxidized mannan-mucin 1 (MUC1) as a fusion protein (containing five repeats of the 20-amino-acid MUC1 VNTR (variable number of tandem repeats) that generated highly active CD8+ CTLs to MUC1 peptides. In a direct CTL assay, the MUC1 peptides could be presented specifically by both the transgenic murine HLA-A*0201/Kb and human HLA-A*0201 molecules. The 9-mer MUC1 peptide sequences (APDTRPA and STAPPAHGV) were presented by HLA-A*0201, although they did not contain L at P2 and L/V at P9, the preferred motifs; as a consequence, the binding was of relatively low affinity when compared with a high affinity-binding HIV peptide (ILKEPVHGV). In addition, when mice were immunized separately with the HLA-A*0201-binding peptides (STAPPAHGV or APDTRPAP-containing peptides-keyhole limpet hemocyanin-mannan), direct lysis of MCF-7 (HLA-A*0201+, MUC1+) also occurred. The findings are of interest for tumor immunotherapy, particularly as the CTLs generated to low affinity-binding peptides were highly active and could specifically lyse an HLA-A*0201+ human breast cancer cell line without further in vitro stimulation. The findings demonstrate that the range of peptides that can generate CTLs is broader than formerly considered.

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition.

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2Db using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind Db. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to Db. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the Db pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited Db-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all Db-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, Db-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to Db, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.

[The cell mediated immune response]. / Det cellemedierede immunrespons.

T-lymphocytes are the most important regulatory cells of the immune system and mediate a number of crucial effector functions. The T-lymphocyte antigen receptor recognises peptide antigen bound to so-called Major Histocompatibility Complex (MHC) molecules on the surface of the target cell. Activation of CD8-positive cytotoxic T-lymphocytes leads to lysis of the antigen presenting cell, whereas activation of CD4-positive T-helper lymphocytes results in release of regulatory and stimulatory cytokines. In this review, the results of recent years' research in the function of the cell mediated immune system is presented, followed by an overview of the role played by the mediated immune system in protection against infectious disease, cancer, or as a cause of immune-mediated pathology and autoimmunity.

Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines against which they were raised.

We have raised CD8+ cytotoxic T lymphocytes (CTL) from three Epstein-Barr virus-seropositive donors by incubating peripheral blood lymphocytes with irradiated autologous B95.8-strain EBV-transformed B lymphoblastoid cells (LCL). However, to detect lysis in a standard 51Cr release assay of the LCL against which these CTL were raised, superinfection with recombinant vaccinia expressing the appropriate EBV protein or incubation with the peptide epitope was necessary. The untreated LCL were not lysed, even though Western blotting demonstrated that they expressed the EBV antigens containing the CTL epitopes. We have found CTL of this phenotype that are restricted by human leukocyte antigen-A2, -A3, -B7, or -B39, and which recognize the EBV latent proteins, EBV nuclear antigen (EBNA)-3A, EBNA-3C, or terminal protein. During these experiments, we identified a new human leukocyte antigen-A3-restricted EBNA-3A epitope, residues 603-611, RLRAEAGVK. We raised a spontaneous LCL, transformed by endogenous EBV, from one donor, but this was also not lysed. For at least one of the epitopes, CTL from another donor lysed the LCL without superinfection or addition of peptides. We conclude that the CTL were unable to achieve a high enough avidity of interaction with untreated LCL to trigger effector function, although the LCL were able to stimulate them to grow in vitro for up to 4 mo. To assess whether a small percentage of the LCL might possess a higher antigen density, we used an assay of tumor necrosis factor release from a CTL clone, which was able to detect antigen-bearing cells representing only 1% of a stimulating LCL population. Nevertheless, the untreated autologous LCL line failed to stimulate tumor necrosis factor release.

Binding of human collectins (SP-A and MBP) to influenza virus.

Collectins are a group of soluble proteins each of which has collagenous domains and non-collagenous globular domains, the latter containing the consensus residues found in C-type lectins. Members of the collectin family are the serum proteins mannan-binding protein (MBP), conglutinin, CL-43, and the lung-associated proteins surfactant protein A (SP-A) and surfactant protein D (SP-D). MBP and conglutinin have been shown previously to bind to influenza viruses and to inhibit the infectivity and haemagglutinating activity of influenza viruses. We report here that the lung protein SP-A, like MBP, can bind to influenza virus (strain A/X31) through its lectin domain and inhibit the virus-mediated agglutination of red cells. The binding of SP-A or MBP to influenza virus was saturable, concentration-dependent, and required the presence of Ca2+ ions. Ligand-blot analysis, using MBP as ligand, of the virus lysate indicated that MBP binds to a 68 kDa viral species. The 68 kDa species was isolated to homogeneity and was shown to be the viral neuraminidase. The purified 68 kDa species inhibited the binding of both MBP and SP-A to influenza virus.

Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes.

Cytotoxic T cells (CTL) recognize short peptide epitopes presented by class I glycoproteins encoded by the major histocompatibility complex (MHC). It is not yet known whether peptides containing posttranslationally modified amino acids can also be recognized by CTL. To address this issue, we have studied the immunogenicity and recognition of a glycopeptide carrying an O-linked N-acetylglucosamine (GlcNAc) monosaccharide-substituted serine residue. This posttranslational modification is catalyzed by a recently described cytosolic glycosyltransferase. We show that glycosylation does not affect peptide binding to MHC class I and that glycopeptides can elicit a strong CTL response that is glycopeptide specific. Furthermore, glycopeptide recognition by cytotoxic T cells is dependent on the chemical structure of the glycan as well as its position within the peptide.

Complement activation upon binding of mannan-binding protein to HIV envelope glycoproteins.

OBJECTIVE: Retroviruses can activate the complement system in the absence of antibodies, and the purpose of this study was to examine whether the serum collection, mannan-binding protein (MBP), could mediate such complement activation. DESIGN: Virus envelope proteins gp120 and gp110 from HIV-1 and HIV-2 were incubated in microtitre wells coated with anti-gp120 or anti-gp110 antibodies. After further incubation with serum, complement activation was measured as deposition of complement factor C4 and C3 onto the wells. Deposited C4 and C3 were detected with enzyme-labelled antibodies. Normal human serum depleted of endogenous lectins by affinity chromatography was used as the complement source. Serum from C1q-deficient patients was used in some experiments. Complement activation was then assessed with and without prior addition of MBP to the wells. Complement activation was also correlated with the quantity of endogenous MBP in a number of normal sera. RESULTS: Complement activation by HIV envelope glycoproteins was found to be mediated by the binding of MBP to carbohydrates on natural envelope protein produced in virus-infected cells, as well as on glycosylated recombinant envelope proteins produced in insect cells. Non-glycosylated recombinant envelope proteins produced in Escherichia coli did not induce this type of complement activation. CONCLUSIONS: Activation of the classical complement pathway by retrovirus envelope proteins can be initiated by the binding of MBP to carbohydrate side chains of envelope glycoproteins.

Studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein A and comparison with two other collectins: mannan-binding protein and conglutinin.

The surfactant-associated protein A (SP-A) belongs to the collectin family, a group of C-type lectins encompassing also surfactant-associated protein D, mannan-binding protein (MBP) and conglutinin. These proteins all have carbohydrate-recognition domains joined to collagen stalks. It seems likely that SP-A, like MBP and conglutinin, may mediate anti-microbial activity through binding to carbohydrates on the microorganisms and collectin receptors on phagocytic cells. We have studied the influence of carbohydrates on the binding of SP-A, MBP and conglutinin to mannan in an enzyme-linked lectin-binding assay. All sugars were of D-configuration, except fucose of which both L- and D-configurations were tested. The order of inhibiting potency on the binding of SP-A was: N-acetylmannosamine > L-fucose, maltose > glucose > mannose. The following sugars were non-inhibitory: galactose, D-fucose, glucosamine, mannosamine, galactosamine, N-acetylglucosamine, and N-acetylgalactosamine. The best inhibitor of MBP was N-acetylglucosamine. Otherwise MBP showed a selectivity similar to that of SP-A. Conglutinin binding was inhibited by all the sugars examined except N-acetylgalactosamine. For conglutinin, as for MBP, the best inhibitor was N-acetylglucosamine. Normal human SP-A, alveolar-proteinosis SP-A purified by ion-exchange chromatography, and alveolar-proteinosis SP-A purified by n-butanol extraction showed no difference in sugar selectivity. The influence of pH and of the calcium concentration was also examined. Organic solvent-extracted SP-A from patients suffering from alveolar proteinosis and normal SP-A showed different sensitivity profiles.