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PubMed1998

Characterization of anti-mouse Fc gamma RII single-chain Fv fragments derived from human phage display libraries.

BACKGROUND: Few antibodies are available to study the function of the Fc gamma RII murine immunoglobulin receptor. Human phage display libraries represent a potential source of single-chain Fv (sFv) to facilitate the study of the Fc gamma RII murine immunoglobulin receptor. OBJECTIVES: To isolate human sFv specific for mouse Fc gamma RII. STUDY DESIGN: Two human phage display libraries were selected for reactivity to mouse Fc gamma RII. Those human anti-mouse Fc gamma RII sFv that were derived from the libraries were characterized with respect to kinetics, cellular binding, epitope specificity and amino acid sequence. RESULTS: Nine anti-mouse Fc gamma RII sFv molecules were isolated from two human phage display libraries (Marks et al., J Mol Biol 1991;222:581-597; Sheets et al., Proc Natl Acad Sci USA, in press). Surface plasmon resonance (SPR) analysis revealed that the human anti-mouse Fc gamma RII sFv had off-rates ranging from 10(-2) to 10(-3) s-1, with KD values calculated to range between 10(-7) and 10(-9) M. The binding of the FITC-labeled human anti-mouse Fc gamma RII sFv to mouse peritoneal neutrophils was not detected by flow cytometry, due to the rapid off-rates of these monomeric proteins. However, when the human anti-mouse Fc gamma RII sFv were coated on yellow-green latex particles, all of the human sFv were found to specifically bind to mouse peritoneal neutrophils. Deglycosylation of mouse Fc gamma RII did not diminish the binding of these sFv, suggesting that the sFv molecules recognize a polypeptide epitope on murine Fc gamma RII. In contrast, denaturation of mouse Fc gamma RII dramatically reduced the binding of the human sFv, suggesting that the epitopes are conformational. Sequence analysis of the human anti-mouse Fc gamma RII sFv revealed a high degree of structural similarity among the nine sFv. The DP73 VH gene segment was utilized by four of the nine sFv, while seven of the nine sFv contained the DPL16 V lambda gene segment. The sequence similarities between these sFv suggested that several of the human sFv may recognize a common epitope on mouse Fc gamma RII. Epitope mapping studies demonstrated that eight of the nine human anti-mouse Fc gamma RII sFv recognized overlapping epitopes. All of these human anti-mouse Fc gamma RII sFv competed with the 2.4G2 rat monoclonal anti-mouse Fc gamma RII/III antibody for binding with mouse Fc gamma RII, suggesting that the targeted epitopes reside in or near the Fc binding pocket of mouse Fc gamma RII. CONCLUSIONS: The availability of novel sFv recognizing mouse Fc gamma RII will facilitate the study of receptor triggering events. Such sFv may prove useful to engage murine Fc gamma RII for targeted cytotoxicity or immunization strategies.

Amino Acid Sequence↗
PubMed1998

A functional epitope on P-selectin that supports binding of P-selectin to P-selectin glycoprotein ligand-1 but not to sialyl Lewis X oligosaccharides.

P-selectin mediates the adhesion of leukocytes to activated platelets and endothelial cells. To characterize the functional domains of P-selectin for ligand recognition, we established nine hybridoma cell lines secreting anti-rat P-selectin mAb. Among them, the mAb C215 bound both rat and human P-selectins, and inhibited binding of rat and human P-selectins to P-selectin glycoprotein ligand-1 (PSGL-1) from HL-60 cells. In contrast, mAb C215 failed to inhibit the binding of rat and human P-selectin-IgG to sialyl Lewis X (sLe(x)) oligosaccharides. Epitope mapping of mAb C215 using synthetic decapeptides revealed that mAb C215 binds specifically to an eight-residue epitope that spans amino acids 76-83 of rat P-selectin, a region completely conserved by human P-selectin. Synthetic peptides containing the mAb C215 epitope inhibited binding of P-selectin to PSGL-1, but not to sLe(x) oligosaccharides, suggesting that the C215 epitope on P-selectin may directly interact with a particular site on the PSGL-1 core protein essential for interaction with P-selectin, such as sulfated tyrosine residues. Our results suggest the presence of two ligand recognition sites on P-selectin necessary for binding to PSGL-1--one recognizes sLe(x), while the other recognises the PSGL-1 core protein.

Amino Acid Sequence↗
PubMed2000

Multiple epitopes from the Mycobacterium tuberculosis ESAT-6 antigen are recognized by antigen-specific human T cell lines.

A synthetic-peptide approach was used to map epitope regions of the Mycobacterium tuberculosis 6-kDa early secreted antigen target (ESAT-6) by testing human CD4(+) T cell lines for secretion of IFN-gamma in response to recombinant ESAT-6 (rESAT-6) and overlapping 20-mer peptides covering the antigen sequence. The results demonstrate that all of the ESAT-6 peptides screened were able to induce IFN-gamma secretion from one or more of the T cell lines tested. Some of the individual T cell lines showed the capacity to respond to all peptides. Human leukocyte antigen (HLA-DR) typing of the donors showed that rESAT-6 was presented to T cells in association with multiple HLA-DR molecules. The results suggest that frequent recognition of the M. tuberculosis ESAT-6 antigen by T cells from patients with tuberculosis is due to the presence of multiple epitopes scattered throughout the ESAT-6 sequence.

Amino Acid Sequence↗
PubMed2004

The C-terminal antibody binding domain of Candida albicans mp58 represents a protective epitope during candidiasis.

The 58-kDa surface mannoprotein of Candida albicans (mp58) elicits strong antibody responses during infection. Epitope mapping with sera from patients with candidiasis and control individuals indicated the presence of multiple IgG-reactive continuous epitopes on the protein, expanding both the amino- and carboxy-terminal domains and several internal regions. These immunoreactive regions were similar to the ones previously identified using sera from immunized animals. Two of the epitopic regions (including the C-terminal domain) showed increased reactivity with antibodies present in sera from patients with candidiasis as compared to control individuals. Patients who survived the infection displayed increased antibody reactivity towards the C-terminal epitope as compared to those succumbing to candidiasis. A monoclonal antibody directed towards this epitopic region conferred protection in serum therapy experiments in a murine model of hematogenously disseminated candidiasis. Together, these observations indicate the carboxy-terminal antibody binding domain of C. albicans mp58 represents a protective epitope during candidiasis.

Amino Acid Sequence↗
PubMed2003

Advances in B-cell epitope analysis of autoantigens in connective tissue diseases.

The characterization of autoantibody specificities in rheumatic diseases is important in both diagnostic and basic research areas. Identification of the epitopes recognized by autoantibodies and their clinical and biological significance is not a trivial task. Epitopes may range in complexity from simple linear sequences of amino acids to complex quaternary structures. In addition to this structural complexity the frequency with which an autoantigen and its epitopes are recognized in a patient population may be useful in diagnosis, defining disease subgroups, and may offer information on disease prognosis. In this review recent advances in the epitope mapping of autoantigens in connective tissue diseases are discussed, with particular emphasis placed on the methodologies used to identify epitopes and the classification of the structural features of epitopes. To illustrate the identification of epitope structure, clinically relevant autoantigens, including CENP-A, PM/Scl-100, fibrillarin, filaggrin, Ro-52, and dsDNA, are discussed as examples of each type of epitope.

Amino Acid Sequence↗
PubMed1993

Immunochemical mapping of domains in human interleukin 4 recognized by neutralizing monoclonal antibodies.

Human interleukin 4 is a highly pleiotropic cytokine secreted by activated T cells that exerts multiple biological effects on B and T lymphocytes and other cell types. Elucidation of structure-function relations was accomplished by epitope mapping of a panel of monoclonal antibodies and by mutagenesis of selected amino acid residues. Epitope mapping of these monoclonal antibodies was achieved through binding studies with recombinant human interleukin 4 (rhuIL-4), proteolytic fragments produced by digestion with Staphylococcus aureus V8 protease and synthetic peptides derived from the sequence of the parent molecule. Monoclonal antibodies 25D2, 35F2, and 11B4 neutralized the in vitro T-cell proliferation activity of rhuIL-4 and also prevented binding of rhuIL4 to its cell surface receptor. These antibodies recognized sequences 104-129, 70-92, and 61-82, respectively. These regions comprise the BC loop/helix C (residues 61-92) and helix D (residues 104-129). A nonneutralizing monoclonal antibody (1A2) recognized a nonoverlapping region (residues 43-59) comprising almost entirely helix B. Mutagenesis of a cluster of residues within helix C showed that at least three residues (K84, R88, and N89) were potentially involved in receptor recognition. The existence of two distinct nonneighboring binding domains in the three-dimensional structure of rhuIL-4 provided preliminary evidence for a model of receptor interaction involving the formation of a ternary complex consisting of two molecules of the extracellular portion of the receptor and one molecule of rhuIL-4.

Antibodies, Monoclonal↗
PubMed2006

Vaccination of cattle with TickGARD induces cross-reactive antibodies binding to conserved linear peptides of Bm86 homologues in Boophilus decoloratus.

Vaccines based on recombinant Bm86 gut antigen from Boophilus microplus are a useful component of integrated control strategies against B. microplus infestations of cattle. The capacity of such vaccines to control heterologous infestations by two African tick species was investigated. The mean weight of engorged female ticks and mean egg mass per tick were significantly reduced in B. decoloratus infestations, but there was no effect of the vaccine against adult Rhipicephalus appendiculatus. We cloned, sequenced and expressed two Bm86 homologues (Bd86) from B. decoloratus. Amino acid sequence identity between Bd86 homologues (Bd86-1 and Bd86-2) and Bm86 was 86% and 85%, respectively, compared to 93% identity between the variants. Native Bd86 protein in B. decoloratus tick mid-gut sections and recombinant Bd86-1 reacted strongly with sera from TickGARD vaccinated cattle. TickGARD can therefore protect against a heterologous tick species with multiple antigen sequences. Epitope mapping using sera from TickGARD-vaccinated cattle identified two linear peptides conserved between the Bd86 homologues and Bm86. These epitopes represent candidate synthetic peptide vaccines for control of Boophilus spp. and the pathogens transmitted by these tick vectors.

Animals↗
PubMed1998

Establishment of epitope-defined monoclonal antibodies with specificity for fibroblast growth factor receptor types 1 and 2.

The development of specific antibody probes for characterizing the expression of the family of 4 fibroblast growth factor receptor (FGFR) types has been difficult because of their close homology to each other and high degree of evolutionary conservation. Of the existing anti-FGFR monoclonal antibodies (MAbs), there are few that are useful for staining paraffin-embedded tissues. We have raised MAbs against human FGFR1 and FGFR2 in both rats and mice using bacterial recombinant receptor fusion proteins as immunogens. We used peptide epitope mapping to characterize the immune sera and the selected MAbs. Immunized animals were selected that displayed the broadest reactivity against epitopes unique to the immunizing receptor type. We produced FGFR1 specific MAbs that bind epitopes in immunoglobulin domain I (Ig-I) and FGFR2 specific MAbs that bind epitopes in Ig-I, Ig-II, and the acid box. The specificity of the antibodies was demonstrated by ELISA and immunoblot analysis of purified recombinant FGFR1 and FGFR2 extracellular domains produced both in E. coli and in eucaryotic cells. Based on the lack of epitope homology, these MAbs would not be expected to cross-react with FGFR3 or FGFR4. We isolated MAbs that bound to paraffin embedded tissue and immunoblots of recombinant receptor. These epitope-defined MAbs can distinguish between members of the FGF receptor family and should be useful as tools for assessing FGF receptor expression in a variety of normal and diseased tissues.

Amino Acid Sequence↗
PubMed2005

Both core and terminal glycosylation alter epitope expression in thyrotropin and introduce discordances in hormone measurements.

Thyroid-stimulating hormone (TSH) is routinely measured in blood to diagnose thyroid disorders using immunoassays. This study used recombinant TSH (recTSH) as a source of hormonal compound exhibiting a serum-type glycosylation and putatively reflecting physiopathological alterations in TSH polymorphism. Mass spectrometry revealed that in recTSH, both subunits display high-molecular-size glycoforms compared to the pituitary hormone (pitTSH), indicating more complex glycosylation. To determine how changes in TSH glycosylation may affect epitope expression, comparative epitope mapping of rec- and pitTSH was carried out using a panel of ten hormone-specific monoclonal antibodies. Three common epitopes, I, II and III, were identified as common to both preparations and allowed the design of six assays as I/II, II/I, I/III, III/I, II/III, and III/II. Highly sialylated recTSHs were produced by enzymatic remodeling to mimic the hormone circulating in blood and revealed limited expression of epitope I, but enhanced recognition of epitope II. Fractionation on a lentil lectin-Sepharose column allowed selection of non-fucosylated recTSH, thought to be associated with primary hypothyroidism. Recognition of epitope I was not modified by TSH core fucosylation, while epitope III expression was increased in non-fucosylated glycoforms. Taken together, our findings demonstrate that changes in both core and terminal glycosylation alter epitope expression in TSH and thereby induce highly variable antibody recognition, resulting in significant discordances among hormone measurements.

Blood Chemical Analysis↗
PubMed1990

[Monoclonal antibodies to non-assembled alpha and beta chain of human chorionic gonadotropin (hCG)].

Using a panel of monoclonal antibodies (MCA) to human chorionic gonadotropin (hCG) and its alpha and beta subunits and additional MCA to the non-assembled, free alpha and beta chains which were produced in the course of the present experiments, it was possible to extend the previously established epitope map of hCG. Two MCAs turned out to be specific for the free alpha chain of the glycoprotein hormones (GPH) and, thus, did not react with holo hCG. Two other MCA recognized two epitopes (beta 6 and beta 7) on the free form of the hCG beta only. Again, no holo hormone cross-reaction was observed. Whereas previously only nine antigenic hCG determinants (3 alpha, 4 beta, 2c) had been demonstrated, it was now possible to distinguish fourteen epitopes (5 alpha, 5 beta, 4c). In addition, epitope maps were established for the non-assembled, free subunits of hCG. These comprise six epitopes on the alpha chain (alpha 1- alpha 6) and seven on the beta chain (beta 1- beta 7). Both so far generally accepted premises of Judith Vaitukaitis, claiming the beta chain of the GPH to be immunologically species-specific, the beta chain to be immunologically hormone-specific, were clearly disproven by demonstrating inter- and intra-species cross-reaction of some MCA. Based on the immunological topography of the holo hCG molecule and its free subunits, highly specific and sensitive immuno-enzymometric assays (IEMA) with predictable specificities were designed, measuring either non-assembled subunits alone or in combination with the intact hormones.

Antibodies, Monoclonal↗
PubMed1999

Association of antibiotic treatment-resistant Lyme arthritis with T cell responses to dominant epitopes of outer surface protein A of Borrelia burgdorferi.

OBJECTIVE: To explore further the association of antibiotic treatment-resistant Lyme arthritis and T cell reactivity with outer surface protein A (OspA) of Borrelia burgdorferi, including the identification of T cell epitopes associated with this treatment-resistant course. METHODS: The responses of peripheral blood and, if available, synovial fluid lymphocytes to B burgdorferi proteins, fragments, and synthetic peptides, as determined by proliferation assay and interferon-gamma production, were compared in 16 patients with treatment-responsive and 16 with treatment-resistant Lyme arthritis. RESULTS: The maximum severity of joint swelling correlated directly with the response to OspA. Moreover, the only significant difference between patients with treatment-resistant and treatment-responsive arthritis was in reactivity with N-terminal and C-terminal fragments of OspA, OspA1 (amino acids [aa] 16-106), and OspA3 (aa 168-273). Epitope mapping showed that 14 of the 16 patients with treatment-resistant arthritis had responses to OspA peptides (usually 4 or 5 epitopes), whereas only 5 of the 16 patients with treatment-responsive arthritis had reactivity with these peptides (usually 1 or 2 epitopes) (P = 0.003). Patients with HLA-DRB1 alleles associated with treatment-resistant arthritis were more likely to react with peptide 15 (aa 154-173) and, to a lesser degree, with peptide 21 (aa 214-233) than patients with other alleles, whereas the responses to other epitopes were similar in both groups. CONCLUSION: The maximum severity of joint swelling and the duration of Lyme arthritis after antibiotic treatment are associated with T cell responses to specific epitopes of OspA.

Adolescent↗
PubMed2004

Autoantigenicity of DFS70 is restricted to the conformational epitope of C-terminal alpha-helical domain.

Autoantibodies against DFS70 (Dense Fine Speckles 70) are found in 30% of Japanese atopic dermatitis patients, and less frequently in patients with other diseases. We have recently reported that they are also seen in 11% of hospital workers, but in only approximately 2% of patients with systemic rheumatic disease. In this study, in order to investigate the possible pathological role of anti-DFS70 antibodies, fine epitope mapping was carried out using 93 anti-DFS70 autoantibody-positive sera. Immunoblotting using overlapping peptides failed to reveal major linear epitopes. Western blotting using various truncated proteins showed a strikingly uniform epitope distribution on a suspected tertiary structure expressed by DFS70(349-435). Some sera showed reactivity only in an immunoprecipitation assay using an in vitro translated DFS70. Circular dichroism analysis revealed that DFS(349-435) contains an approximately 40% alpha-helical conformation, while an overlapping, non-antigenic peptide is composed of random coiled structures. The skewed single major epitope enabled us to establish a highly quantitative ELISA for the epitope region. Antibody titers showed no significant differences between the diseased group and healthy individuals. We propose that anti-DFS70 antibody may be a natural autoantibody, which might modify or reflect the inflammatory process of various disorders.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Genetic immunization and comprehensive screening approaches in HLA-A2 transgenic mice lead to the identification of three novel epitopes in hepatitis C virus NS3 antigen.

Interferon-gamma (IFN gamma)-producing CD8+ T cells have been shown to play a key role in the control or eradication of hepatitis C virus (HCV) infections. In particular, T cells specific of the non-structural protein 3 (NS3) are often associated with control of viremia. The aim of the study was to identify novel HLA-A2 restricted CD8+ T cell epitopes specific of NS3 using a combination of comprehensive approaches. HLA-A2.1 transgenic mice were immunized with a DNA vaccine optimized for NS3 specific epitope presentation and induced CD8+ T cell reactivity was screened using 42 algorithm-predicted peptides as well as a library of 78 overlapping 15-mer peptides spanning the whole protein. Three epitopes mapping within the NS3 protease (GLL: aa 1038-1047) or helicase (ATL: aa 1260-1268 and TLH: aa 1617-1625) were identified. These epitopes, which display similar and high in vitro binding capacities to soluble HLA-A2 molecules, are able to induce either cytotoxic T lymphocytes (CTL) and/or IFN gamma-producing T cells. Comparative in vitro target cell sensitization studies revealed a higher immunogenicity of the GLL peptide as compared with both ATL and TLH peptides. This peptide was capable to recall in vitro HCV-specific IFN gamma and IL-10-producing T cells from peripheral blood mononuclear cells (PBMC) of chronically infected patients. These data increase the pool of NS3-specific CD8+ T cell epitopes available to analyze HCV associated immunity and could contribute to the design and evaluation of candidate vaccines.

Amino Acid Sequence↗
PubMed1996

Human self-reactive T cell clones expressing identical T cell receptor beta chains differ in their ability to recognize a cryptic self-epitope.

Recognition of self-antigens by T lymphocytes is a central event in autoimmunity. Understanding of the molecular interactions between T cell receptors (TCR) and self-epitopes may explain how T cells escape thymic education and initiate an autoimmune reaction. We have studied five human in vivo activated T cell clones specific for the region 535-551 of human thyroid peroxidase (TPO) established from a Graves' patient. Three clones (37, 72, and 73) expressed identical TCR beta and alpha chains rearranging V beta 1.1 and V alpha 15.1, and were considered sister clones. Clone 43 differed from clone 37 and its sisters in the J alpha region only. Clone NP-7 expressed V beta 6.5 but rearranged two in-frame TCR alpha chain, both using the V alpha 22.1 segment. Fine epitope mapping using nested peptides showed that clones using identical TCR beta chains, identical V alpha, but a different J alpha recognized distinct, nonoverlapping epitopes in the TPO 535-551 region. This finding shows that a different J alpha region alone leads to a heterogeneous pattern of recognition. This indicates that the "restricted" TCR V region usage sometimes found in autoimmune diseases may not always correspond to identical epitope recognition. To confirm that clones 37 (and its sisters) and 43 recognize different epitopes, the T cell clones were stimulated with a TPO-transfected autologous Epstein-Barr virus (EBV) cell line (TPO-EBV) that presents TPO epitopes afer endogenous processing. Only clone 37 and its sisters recognizes the TPO-EBV cell line, suggesting that the epitope recognized by clone 43 is not presented upon endogenous processing. We have shown that thyroid epithelial cells (TEC), the only cells that produce TPO, express HLA class II molecules in Graves' disease and can act as an antigen-presenting cells, presenting TPO after endogenous processing to autoantigen-reactive T cell clones. We tested, therefore, whether autologous TEC induced the same pattern of stimulation as TPO-EBV; T cell clone 37 recognizes the TEC, whereas it is stimulated poorly by the TPO loaded to autologous peripheral blood mononuclear cells (PBMC). Clone 43, which fails to recognize the TPO-EBV, also fails to recognize the TEC, but is activated by exogenous TPO presented by autologous PBMC. These results show that exogenous versus endogenous processing in vivo generates a different TPO epitope repertoire, producing a "cryptic" epitope (epitope not always available for recognition). Our findings define a route by which human self-reactive T cells may escape thymic selection and become activated in vivo, thus possibly leading to autoimmunity.

Amino Acid Sequence↗
PubMed2000

Preparation and characterization of mabs against different epitopes of CD226 (PTA1).

Recently the platelet and T-cell activation antigen 1 (PTA1) was assigned as CD226 at the 7th Conference and Workshop on Human Leukocyte Differentiation antigens (HLDA). PTA1 is mainly expressed on activated T cells, natural killer (NK) cells, platelets and stimulated endotheliocytes, and involved in the differentiation of cytotoxic T lymphocytes (CTL) and NK, as well as platelet activation and aggregation. We raised hybridomas secreting monoclonal antibodies (MAbs) to PTA1 by using the natural PTA1 as immunogen, which was purified from platelets via affinity chromatography. These MAbs, designated FMU1, FMU2, FMU3, FMU4, FMU5, FMU6 and FMU7, could recognize PTA1 cDNA transfected COS7 cells detected by flow cytometry (FCM), and also react with both natural PTA1 and PTA1/Ig fusion protein in indirect enzyme-linked immunoadsorbent assay (ELISA). The biosensor epitope mapping assay showed that the seven MAbs, together with previous PTA1-specific MAbs Leo A1 and New E1, could bind seven distinct epitopes of PTA1, respectively. The panel of MAbs might be new powerful tools to study the structure-function relationship of PTA1 molecule, and to search for the ligand of PTA1.

Animals↗
PubMed2004

Induction of broad-based immune response against HIV-1 subtype C gag DNA vaccine in mice.

Prevention of HIV infection through an effective vaccine is need of the hour as per the AIDS pandemic scene, particularly in the developing world. Here we report the work done with gag gene construct pJWgagprotease49587 from HIV-1subtype C Indian strain. The construct pJWgagprotease49587 was tested positive for expression in COS-7 cells by p24 antigen capture ELISA, immunoblotting and by transmission electron microscopy that revealed virus like particle formation. Immunogenicity studies showed induction of good lymphoproliferative and cytotoxic (CTL) responses in Balb/c mice. The cytokine repertoire elicited showed a TH1 type of immune response. In an epitope mapping study, IFNgamma secretion by spleen cells from immunized mice was observed to seven peptides from different regions of gag. Recognition of multiple epitopes demonstrates elicitation of a broad based immune response against gag following immunization with the construct. In view of the high propensity of escape mutant induction during the course of HIV infection, it is encouraging to use immunogens eliciting viable immune responses to a broad spectrum of epitopes. Hence the construct pJWgagprotease49587 is a good candidate for immunogenecity testing in nonhuman primates as a probable vaccine candidate.

AIDS Vaccines↗
PubMed1998

The dominant epitope of Borrelia garinii outer surface protein C recognized by sera from patients with neuroborreliosis has a surface-exposed conserved structural motif.

Epitope mapping of outer surface protein C (OspC) by using sera from patients with neuroborreliosis led to the identification of one single major immunodominant epitope within the C-terminal 10 amino acid residues. Peptide binding studies and alanine replacement scanning of the C-terminal decapeptide, PVVAESPKKP, revealed a critical role for the PKKP sequence and its terminal carboxyl group for the binding of immunoglobulin M (IgM) antibodies from patients with Lyme borreliosis. Electron microscopy of antibody-labeled spirochetes indicated that the C-terminal region is exposed on the surface of the spirochete. Based on homology to proteins of known function, this region most probably adopts a polyproline II-like helix, which is found in surface-exposed structures involved in protein-protein interactions. This structural motif is highly conserved in Borrelia species causing Lyme borreliosis and subjected to purifying selection. We suggest that the abundance of the C-terminal region of OspC on the surface of B. burgdorferi allows a multimeric high-avidity interaction between the spirochete and surface Igs on B cells. The resulting cross-linking of surface Igs on B cells may induce a T-cell-independent B-cell activation without IgM-to-IgG switching, thus explaining the lack of IgG antibodies to OspC in neuroborreliosis.

Adolescent↗
PubMed1995

Peptide-selected T cell lines from myasthenia gravis patients and controls recognize epitopes that are not processed from whole acetylcholine receptor.

To study pathogenic T helper cells in myasthenia gravis (MG) reacting against the acetylcholine receptor (AChR), we have previously selected five CD4+ T cell lines/clones from MG patients (or healthy controls) against full-length recombinant human AChR alpha subunit (alpha 1-437); these can all recognize AChR solubilized from human muscle. Recently, T cells selected with pooled AChR subunit synthetic peptides have shown greater heterogeneity than above. Hoping to validate that, we have characterized three MG and six control T cell lines selected with pooled peptides (averaging 33 residues long) covering the alpha subunit sequence; recurring responses to three particular peptides each showed preferred HLA class II restrictions--p75-115/DR4, p138-167/DR4, and p309-344/DR3 (or DR52a). However, none of three lines from MG patients recognized p138-167--even one from a previous responder to this epitope in full-length alpha 1-437; otherwise they resembled those from controls. Moreover, no peptide-selected line responded significantly to whole AChR, alpha 1-437, or even to shorter polypeptides sharing one terminus with the peptide, suggesting specificity for epitopes not naturally processed by APCs from blood. Of 20 sublines maintained with individual peptides, at least 10 responded to independently synthesized overlapping sequences, but four others depended on contaminants in the original peptides. A single line did recognize one longer polypeptide, but only after tryptic digestion; the processing of this cryptic epitope was evidently the limiting factor here rather than its concentration or the T cell sensitivity. Therefore, while synthetic peptides are essential for mapping epitopes, assessment of the pathogenic MG T cell repertoire requires full-length Ag processed naturally.

Amino Acid Sequence↗