The epitopes for some antiphospholipid antibodies are adducts of oxidized phospholipid and β2 glycoprotein 1 (and other proteins)

Circulating autoantibodies to phospholipids (aPLs), such as cardiolipin (CL), are found in patients with antiphospholipid antibody syndrome (APS). We recently demonstrated that many aPLs bound to CL only after it had been oxidized (OxCL), but not to a reduced CL analogue that could not undergo oxidation. We now show that the neoepitopes recognized by some aPLs consist of adducts formed between breakdown products of oxidized phospholipid and associated proteins, such as β2 glycoprotein 1 (β2GP1). Addition of human β2GP1, polylysine, native low-density lipoprotein, or apolipoprotein AI to OxCL-coated wells increased the anticardiolipin antibody (aCL) binding from APS sera that first had been diluted so that no aCL binding to OxCL could be detected. No increase in aCL binding was observed when these proteins were added to wells coated with reduced CL. The ability of β2GP1, polylysine, or low-density lipoprotein to be a “cofactor” for aCL binding to OxCL was greatly reduced when the proteins were methylated. Incubation of β2GP1 with oxidized 1-palmitoyl-2-linoleyl-[1-14C]-phosphatidylcholine (PC), but not with dipalmitoyl-[1-14C]-PC, led to formation of covalent adducts with β2GP1 recognized by APS sera. These data suggest that the reactive groups of OxCL, such as aldehydes generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with β2GP1 (and other proteins) and that these are epitopes for aCLs. Knowledge that the epitopes recognized by many aPLs are adducts of oxidized phospholipid and associated proteins, including β2GP1, may give new insights into the pathogenic events underlying the clinical manifestations of APS.

Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195–212- and p259–271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.

Superactivation of an immune response triggered by oligomerized T cell epitopes

The peptides bound to class II major histocompatibility complex (MHC) molecules extend out both ends of the peptide binding groove. This structural feature provided the opportunity to design multivalent polypeptide chains that cross-link class II MHC molecules through multiple, repetitive MHC binding sites. By using recombinant techniques, polypeptide oligomers were constructed that consist of up to 32 copies of an HLA-DR1-restricted T cell epitope. The epitope HA306–318, derived from influenza virus hemagglutinin, was connected by 12- to 36-aa long spacer sequences. These oligomers were found to cross-link soluble HLA-DR1 molecules efficiently and, upon binding to the MHC molecules of a monocyte line, to trigger signal transduction indicated by the enhanced expression of some cell surface molecules. A particularly strong effect was evident in the T cell response. A hemagglutinin-specific T cell clone recognized these antigens at concentrations up to three to four orders of magnitude lower than that of the peptide or the hemagglutinin protein. Both signal transduction in the monocyte and the proliferative response of the T cell were affected greatly by the length of the oligomer (i.e., the number of repetitive units) and the distance of the epitopes within the oligomer (spacing). Thus, the formation of defined clusters of T cell receptor/MHC/peptide antigen complexes appears to be crucial for triggering the immune response and can be used to enhance the antigenicity of a peptide antigen by oligomerizing the epitope.

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: Evidence that oxidation-specific epitopes mediate macrophage recognition

Apoptosis is recognized as important for normal cellular homeostasis in multicellular organisms. Although there have been great advances in our knowledge of the molecular events regulating apoptosis, much less is known about the receptors on phagocytes responsible for apoptotic cell recognition and phagocytosis or the ligands on apoptotic cells mediating such recognition. The observations that apoptotic cells are under increased oxidative stress and that oxidized low-density lipoprotein (OxLDL) competes with apoptotic cells for macrophage binding suggested the hypothesis that both OxLDL and apoptotic cells share oxidatively modified moieties on their surfaces that serve as ligands for macrophage recognition. To test this hypothesis, we used murine monoclonal autoantibodies that bind to oxidation-specific epitopes on OxLDL. In particular, antibodies EO6 and EO3 recognize oxidized phospholipids, including 1-palmitoyl 2-(5-oxovaleroyl) phosphatidylcholine (POVPC), and antibodies EO12 and EO14 recognize malondialdehyde-lysine, as in malondialdehyde-LDL. Using FACS analysis, we demonstrated that each of these EO antibodies bound to apoptotic cells but not to normal cells, whereas control IgM antibodies did not. Confocal microscopy demonstrated cell-surface expression of the oxidation-specific epitopes on apoptotic cells. Furthermore, each of these antibodies inhibited the phagocytosis of apoptotic cells by elicited peritoneal macrophages, as did OxLDL. In addition, an adduct of POVPC with BSA also effectively prevented phagocytosis. These data demonstrate that apoptotic cells express oxidation-specific epitopes—including oxidized phospholipids—on their cell surface, and that these serve as ligands for recognition and phagocytosis by elicited macrophages.

Dependence of lymphopenia-induced T cell proliferation on the abundance of peptide/ MHC epitopes and strength of their interaction with T cell receptors

Factors that affect naïve T cell proliferation in syngeneic lymphopenic hosts were investigated. 2C T cell receptor (TCR) transgenic T cells lacking both CD8 and CD4 survived but hardly proliferated. Proliferation of CD8+ 2C cells was proportional to the abundance of cognate peptide/MHC complexes and was severely inhibited by injection of anti-CD8 antibody. Weakly reactive self-peptides slightly enhanced CD8+ 2C cell proliferation whereas a potent agonist peptide promoted much more rapid proliferation, but inflammation-stimulating adjuvant had only a small effect on the rate of cell proliferation. The findings suggest that under uniform lymphopenic conditions, the widely different rates of proliferation of T cells expressing various TCR, or the same TCR in the presence or absence of CD8, reflect the strength of interaction between TCR and MHC associated with particular self-peptides.

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.

CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production

NY-ESO-1 is a tumor-specific shared antigen with distinctive immunogenicity. Both CD8+ T cells and class-switched Ab responses have been detected from patients with cancer. In this study, a CD4+ T cell line was generated from peripheral blood mononuclear cells of a melanoma patient and was shown to recognize NY-ESO-1 peptides presented by HLA-DP4, a dominant MHC class II allele expressed in 43–70% of Caucasians. The ESO p157–170 peptide containing the core region of DP4-restricted T cell epitope was present in a number of tumor cell lines tested and found to be recognized by both CD4+ T cells as well as HLA-A2-restricted CD8+ T cells. Thus, the ESO p157–170 epitope represents a potential candidate for cancer vaccines aimed at generating both CD4+ and CD8+ T cell responses. More importantly, 16 of 17 melanoma patients who developed Ab against NY-ESO-1 were found to be HLA-DP4-positive. CD4+ T cells specific for the NY-ESO-1 epitopes were generated from 5 of 6 melanoma patients with NY-ESO-1 Ab. In contrast, no specific DP4-restricted T cells were generated from two patients without detectable NY-ESO-1 Ab. These results suggested that NY-ESO-1-specific DP4-restricted CD4+ T cells were closely associated with NY-ESO-1 Ab observed in melanoma patients and might play an important role in providing help for activating B cells for NY-ESO-1-specific Ab production.

Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.

Peptide analogs to pathogenic epitopes of the human acetylcholine receptor alpha subunit as potential modulators of myasthenia gravis.

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy.

Discontinuous epitopes of hepatitis B surface antigen derived from a filamentous phage peptide library.

The structure of the small hepatitis B virus surface antigen (HBsAg) was investigated by epitope mapping of four anti-HBsAg monoclonal antibodies (mAbs). Amino acid sequences of epitopes were derived from affinity-enrichment experiments (biopanning) using a filamentous phage peptide library. The library consists of 10(9) different clones bearing a 30-residue peptide fused to gene III. Sequence homologies between peptides obtained from panning the library against the antibodies and the native HBsAg sequence allowed for precise description of the binding regions. Three of four mAbs were found to bind to distinct discontinuous epitopes between amino acid residues 101 and 207 of HBsAg. The fourth mAb was demonstrated to bind to residues 121-124. The sequence data are supported by ELISA assays demonstrating the binding of the HBsAg-specific peptides on filamentous phage to mAbs. The sequence data were used to map the surface of HBsAg and to derive a topological model for the alpha-carbon trace of the 101-207 region of HBsAg. The approach should be useful for other proteins for which the crystal structure is not available but a representative set of mAbs can be obtained.

Antibodies against specific extracellular epitopes of the glucagon receptor block glucagon binding.

Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.

Identification of the galactose-adherence lectin epitopes of Entamoeba histolytica that stimulate tumor necrosis factor-alpha production by macrophages.

The 170-kDa subunit of the galactose-adherence lectin (Gal-lectin) of Entamoeba histolytica mediates adherence to human colonic mucins and intestinal epithelium as a prerequisite to amebic invasion. The Gal-lectin is an immunodominant molecule and a protective antigen in the gerbil model of amebiasis. Tumor necrosis factor alpha (TNF-alpha) produced by activated macrophages enhances nitric oxide-dependent cytotoxicity in host defense against E. histolytica. The purpose of this study was to identify the Gal-lectin epitopes which stimulate TNF-alpha production by macrophages. Murine bone marrow-derived macrophages (BMMs) exposed to Gal-lectin (100-500 ng/ml) stimulated stable expression of TNF-alpha mRNA (8-fold increase) and TNF-alpha production similar to that of lipopolysaccharide-stimulated cells (100 ng/ml). Polyclonal anti-lectin serum specifically inhibited TNF-alpha mRNA induction in response to the Gal-lectin but not to lipopolysaccharide. Anti-lectin monoclonal antibodies 8C12, H85 and 1G7, which recognize nonoverlapping epitopes of the cysteine-rich region of the 170-kDa heavy subunit, inhibited both amebic adherence to mammalian cells and Gal-lectin-stimulated TNF-alpha mRNA expression by BMMs,but monoclonal antibody 7F4 did neither. As these inhibitory antibodies map to amino acids 596-1082 of the 170-kDa Gal-lectin, our results have identified the functional region that mediates amebic adherence and TNF-alpha mRNA induction in BMMMs; thus, this region of the Gal-lectin is a subunit vaccine candidate.

Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity.

Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development.