Antigenic properties and processing requirements of 65-kilodalton mannoprotein, a major antigen target of anti-Candida human T-cell response, as disclosed by specific human T-cell clones

T-cell-mediated immunity is known to play a central role in the host response to Candida albicans. T-cell clones are useful tools for the exact identification of fungal T-cell epitopes and the processing requirements of C. albicans antigens. We isolated human T-cell clones from an HLA-DRB1*1101 healthy donor by using an antigenic extract (MP-F2) of the fungus. Specific clones were T-cell receptor alpha/beta and CD4(+)/CD8(-) and showed a T-helper type 1 cytokine profile (production of gamma interferon and not interleukin-4). The large majority of these clones recognized both the natural (highly glycosylated) and the recombinant (nonglycosylated) 65-kDa mannoprotein (MP65), an MP-F2 minor constituent that was confirmed to be an immunodominant antigen of the human T-cell response. Surprisingly, most of the clones recognized two synthetic peptides of different MP65 regions. However, the peptides shared the amino acid motif IXSXIXXL, which may be envisaged as a motif sequence representing the minimal epitope recognized by these clones. Three clones recognized natural and pronase-treated MP65 but did not detect nonglycosylated, recombinant MP65 or the peptides, suggesting a possible role for polysaccharides in T-cell recognition of C. albicans. Finally, lymphoblastoid B-cell lines were efficient antigen-presenting cells (APC) for recombinant MP65 and peptides but failed to present natural, glycosylated antigens, suggesting that nonprofessional APC might be defective in processing highly glycosylated yeast proteins. In conclusion, this study provides the first characterization of C. albicans-specific human T-cell clones and provides new clues for the definition of the cellular immune response against C. albicans.     

Chemically unambiguous peptide immunogen: preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system.

We described a novel and simple approach to prepare chemically unambiguous peptide immunogen using the multiple antigen peptide (MAP) approach. This approach requires the conjugation of two purified components: a chloroacetylated oligomeric lysine core matrix and a synthetic peptide containing cysteine at either the carboxyl or amino terminus. The resulting MAP is structurally unambiguous and contains a quantifiable amount of peptide antigens. Furthermore, this method also provides a flexible strategy to link a peptide antigen to the core matrix at the desirable orientation to mimic the native molecule. The carboxyl fragment 43-50 of human transforming growth factor alpha (TGF alpha) was used as a test model for this approach. Antipeptide antibodies did not recognize the "reverse immunogen" in which the peptide was attached to the MAP core matrix at a reverse orientation. To determine the specificity of the antibodies, we used two series of point-substituted TGF alpha analogs containing either alanine or the corresponding D-amino acid replacement to map the antigenic site. The alanine analogs were used to determine the contribution of the side chain while the D-amino acid analogs were used to determine the importance of backbone conformation. The antigen site was found to consist of four residues (Asp47-Leu48-Leu49-Ala50) at the distal end of the peptide-MAP conjugate. The results provide a clear explanation for the specificity of the antipeptide antibodies and their failure to recognize the "reverse immunogen" since the distal and the flexible end of the peptide-MAP construct constitutes the antigenic site. Furthermore, our results also suggests a strategy of placing the antigenic portion of a short-peptide at the distal end in the MAP approach to prepare immunogen.     

Recognition of CMV pp65 protein antigen by human CD4 T-cell lines induced with an immunodominant peptide pool

Cellular immunity against cytomegalovirus (CMV) is essential for recovery from infection and control of viral latency. In immunocompromised hosts, this balance between CMV and cellular immunity is lost. Accordingly, restoration of the CD8 compartment specific for CMV is beneficial for immunocompromised patients. It is clear that CMV-specific CD4 cells provide helper functions facilitating long-term persistence of CD8 cells. Considering the dearth of data on CMV-specific T-helper cells, we investigated the CD4 responses to the immunodominant protein pp65 to define antigenic peptides. Such peptides were pooled and used to generate long-term T-cell lines. The lines were responsive to CMV and pp65. T cells were selected with individual peptides to produce monospecific lines for accurate definition of fine epitope specificity and to confirm human leukocyte antigen HLA-DR restriction. Furthermore, these lines lost alloreactivity, suggesting that they can be generated from the allodonor for adoptive immunoreconstitution of stem cell graft recipients.     

Antigenic cross-reactivity and functional inhibition by antibodies to Clostridium difficile toxin A, Streptococcus mutans glucan-binding protein, and a synthetic peptide.

A 10-amino-acid repeating sequence of the hemagglutinating portion of Clostridium difficile toxin A has been synthesized and used to produce antisera in rabbits. Antipeptide antibody inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. Immunoblot analysis with the antipeptide antibody revealed cross-reactivity with native toxin, a recombinant protein containing the toxin A repeats, and a glucan-binding protein from Streptococcus mutans whose primary structure has repeating amino acid motifs similar to those of the synthetic peptide. A polyclonal antibody against the glucan-binding protein, which cross-reacted with purified toxin A, also inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity. We recently identified toxin A and the glucan-binding protein as members of a novel family of clostridial and streptococcal binding proteins based on conserved repeating amino acid motifs at the C-terminal region of the molecules. This study provides immunological and functional evidence of the predicted relationship between toxin A and the glucan-binding protein and further implicates the repeating subunits as ligand-binding domains in this family of proteins.     

A T-cell response to the anti-arthritic drug penicillamine in the mouse: requirements for generation of the drug-derived antigen.

Mice primed with the anit-arthritic drug D-penicillamine (DP) developed DP-specific T cells in the draining lymph nodes (DLN) which responded to drug-haptenated stimulator cells, but not to untreated control cells nor to free drug, in in vitro proliferation assays. The responder cells were CD4+ and the response was major histocompatibility complex (MHC) class II restricted. The conditions required to generate efficient stimulator cells for in vitro proliferation assays were investigated. Drug-haptenated syngeneic spleen cells, but not thymocytes, were able to stimulate T cells from DP-sensitized mice. However, prolonged incubations of spleen cells with DP were required to generate the drug-derived T-cell antigen. Further experiments revealed that the generation of a DP-derived antigenic determinant for T cells did not require intracellular processing, as stimulator cells pretreated with fixative or lysosomotropic agents before drug haptenation were as effective as untreated DP-haptenated cells in stimulating the responder cells to proliferative in vitro. These findings show that the protein-reactive drug DP can generate a cellular antigen that is capable of stimulating a T-cell response. Furthermore, the generation of this antigen appears to bypass conventional antigen processing, suggesting perhaps a direct chemical modification of cell surface molecules that are involved in immune recognition. This process may underlie adverse reactions to DP that are believed to be mediated by the cellular immune system.     

Detection of an antigen related to systemic Candida infection using a monoclonal antibody conjugated to colloidal gold

Previously we showed it was possible to detect antigenemia associated with systemic candidiasis using an anti-C. albicans monoclonal antibody conjugated to colloidal gold. The technique being used, known as Immuno-Gold-Silver staining (IGSS), is applied to serum dots on cellulose nitrate. It is very simple in practice, the results of the reaction being visible with the naked eye. The diagnostic value of IGSS has been compared, on the one hand, with that of the anti-Candida antibody detection by co-counterimmunoelectrophoresis and indirect immunofluorescence, on the other hand with that of the antigen detection using the Cand-Tec commercial test. The specificity and sensitivity of these methods have been established in relation to sera of 79 subjects shared out into 4 groups: sound-subjects, patients having developed systemic candidiasis following surgery, leukemic patients apparently uninfected with Candida and leukemic patients suffering from systemic candidiasis. The IGSS which is slightly less specific than the Cand Tec makes it possible to diagnose a much greater number of infections. Selected bioclinical observations show that there exists complementarity between the detection tests of antibodies and those of antigens and that it is possible to attribute a prognosis value to antigenemia detected with the IGSS dot method.     

Antigenic determinants of horse cytochrome c for lymphocyte transformation: identification of an immunodominant peptide.

Guinea pigs immunized with horse cytochrome c produce no measurable antibody but exhibit delayed hypersensitivity skin tests and T lymphocytes which undergo antigen dependent DNA synthesis. Analysis of the antigenicity of the cyanogen bromide peptides of horse cytochrome c revealed that peptide 81–104 is the immunodominant peptide as peptides 1–65 and 66–80 are inactive. In addition, peptide 81–104 was very immunogenic in guinea pigs, much more so than either intact cytochrome c or peptide 1–65. Peptide 66–80 was not immunogenic. Peptide 81–104 was therefore found to be antigenically and immunogenically the immunodominant peptide of horse cytochrome c.     

An HLA-DRw53-restricted T-cell epitope from a novel Mycobacterium leprae protein antigen important to the human memory T-cell repertoire against M. leprae.

Cellular immunity mediated by T cells plays a major role in protection against intracellular infections, including leprosy, a chronic disease caused by Mycobacterium leprae. In this work, we describe CD4+ T-cell clones, isolated from healthy humans immunized with M. leprae, which recognize a novel M. leprae protein antigen previously isolated from a lambda gt11 DNA expression library. On the basis of the deduced primary structure of the carboxyl-terminal part of the antigen, we have used a synthetic-peptide approach to exactly define the T-cell epitope recognized. Importantly, major histocompatibility complex restriction studies showed that the epitope is presented by an HLA-DRw53 molecule which is frequently expressed in many populations. In addition, we have demonstrated that a long-term cell-mediated immunity response against the peptide epitope is present after immunization with M. leprae. In conclusion, the M. leprae T-cell epitope described here fulfills the primary criteria for subunit vaccine candidates against leprosy.     

Maleimide-thiol coupling of a bioactive peptide to an elastin-like protein polymer

Recombinant elastin-like protein (ELP) polymers display several favorable characteristics for tissue repair and replacement as well as drug delivery applications. However, these materials are derived from peptide sequences that do not lend themselves to cell adhesion, migration, or proliferation. This report describes the chemoselective ligation of peptide linkers bearing the bioactive RGD sequence to the surface of ELP hydrogels. Initially, cystamine is conjugated to ELP, followed by the temperature-driven formation of elastomeric ELP hydrogels. Cystamine reduction produces reactive thiols that are coupled to the RGD peptide linker via a terminal maleimide group. Investigations into the behavior of endothelial cells and mesenchymal stem cells on the RGD-modified ELP hydrogel surface reveal significantly enhanced attachment, spreading, migration and proliferation. Attached endothelial cells display a quiescent phenotype.     

Type-specific opsonic antibodies evoked with a synthetic peptide of streptococcal M protein conjugated to polylysine without adjuvant.

A chemically synthesized copy (S-CB7) of a fragment (35 amino acid residues) of type 24 streptococcal M protein was covalently linked to polylysine with carbodiimide and injected subcutaneously into rabbits without adjuvant. Although the primary immune responses as measured by enzyme-linked immunosorbent assays at biweekly intervals were weak, the secondary responses as measured by both enzyme-linked immunosorbent assays and opsonophagocytic assays were as high as those obtained previously in rabbits immunized with the peptide conjugate emulsified in complete Freund adjuvant. Injection of murabutide, a synthetic muramyl dipeptide derivative of bacterial peptidoglycan, with the initial immunizing dose of peptide conjugate had no apparent effect on the secondary immune responses. These results indicate that protective immune responses may be raised against polylysine conjugates of chemically synthesized peptide copies of streptococcal M protein without adjuvant.     

T-cell immunity to peptide epitopes of liver-stage antigen 1 in an area of Papua New Guinea in which malaria is holoendemic.

Liver-stage antigen 1 (LSA1) is one of several pre-erythrocytic antigens considered for inclusion in a multiantigen, multistage subunit vaccine against falciparum malaria. We examined T-cell proliferation and cytokine responses to peptides corresponding to amino acids 84 to 107, 1813 to 1835, and 1888 to 1909 of LSA1 in asymptomatic adults living in an area of Papua New Guinea where malaria is holoendemic. Whereas T cells from North Americans never exposed to malaria did not respond to any of the peptides, those from 52 of 55 adults from the area where malaria is endemic had vigorous proliferation responses to one or more of the LSA1 peptides (mean stimulation indices of 6.8 to 7.2). Gamma interferon (IFN-gamma) production driven by LSA1 peptides ranged from 34 to more than 3,500 pg/2 x 10(6) cells, was derived primarily from CD8+ cells, and was dissociated from T-cell proliferation. The frequencies of IFN-gamma response to the amino acid 1819 to 1835 and 1888 to 1909 peptides were significantly greater than that to the amino acid 84 to 107 peptide (87 and 88% versus 33% of subjects; P < 0.0001). In contrast to proliferation and IFN-gamma, interleukin 4 (IL-4) and/or IL-5 responses to LSA1 peptides were detected in only 18% of the subjects. These data show that T-cell immunity to epitopes in the N- and C-terminal regions of LSA1 are common in persons living in this area of Papua New Guinea where malaria is endemic. The dominance of type 1 CD8 cell IFN-gamma responses is consistent with a role for this T-cell population in immunity to liver-stage Plasmodium falciparum in humans.     

利用gp41蛋白的合成肽抗原检测HIV—1抗体

采用固相多肽合成技术对人免疫缺损病毒-1型(HIV-1)gp41蛋白的一段代表优势抗原表位的22肽进行了化学合成,经反相高效液相层析(HPLC)和多肽序列测定表明,合成肽成品均质性良好,其氨基酸序列与设计相符。利用该合成肽作为包被抗原,以间接 ELISA 法检测 HIV-1血清抗体,其特异性、灵敏性和重复性均达到与国外生产的 gp41合成肽抗原相同的水平.     

Extracellular heat shock protein 90 plays a role in translocating chaperoned antigen from endosome to proteasome for generating antigenic peptide to be cross-presented by dendritic cells

Extracellular heat shock protein can deliver associated antigens into the MHC class I presentation pathway of antigen-presenting cells, a process called cross-presentation, thus inducing antigen-specific CD8(+) T-cell responses; however, the precise mechanism for intracellular antigen translocation and the processing pathway has not been fully elucidated. Here we demonstrate that cross-presentation of extracellular Hsp90-ovalbumin (OVA) protein complexes to specific CD8(+) T cells involves both classical proteasome-transporter-associated antigen processing (TAP)-dependent and TAP-independent-endosomal pathways. Using confocal microscopy, we found that the internalized extracellular Hsp90 and OVA co-localized with cytosolic proteasomes. When anti-Hsp90 mAb was introduced to dendritic cells (DCs), we observed that the co-localization of internalized Hsp90-chaperoned OVA and proteasomes was abolished, resulting in the inhibition of TAP-dependent cross-presentation of OVA. Thus, extracellular Hsp90 may play a pivotal role for the translocation of chaperoned antigens for proteasomal degradation in the cytosol. In contrast, OVA chaperoned by Hsp90 was not presented by MHC class II molecules in vitro or in vivo, although the antigen was exogenously loaded onto DCs. Our data indicate that extracellular Hsp90 might be essential for the translocation of chaperoned antigens from the extracellular milieu into cytosol, resulting in proteasomal degradation for cross-presentation.     

Antibody response to a protein antigen (ovalbumin) in dissociated spleen cell cultures from primed mice: Evidence for a suppressive effect of antigen

A system is described with which an antibody response to soluble chicken ovalbumin can be obtained in dissociated spleen cell cultures from primed mice. This was assayed by determining specific anti-ovalbumin plaque-forming cells (PFC). Only IgG-producing cells (indirect PFC) were seen.

The mice received alum-precipitated ovalbumin i.p. and cultures were set up either late in the primary splenic response (23–27 days after injection) or near the peak of the response (10–14 days). With both groups secondary responses were seen in the presence of antigen on the fourth day of culture. In the second group PFC were present in the cultures at a high level initially. The number of PFC decreased over the first 2 days, the rate of decrease being greater in the presence of antigen.

When mice were primed with alum-precipitated ovalbumin together with B. pertussis as adjuvant, responses could not now be obtained in culture. With mice taken late in the primary response (23–27 days), a considerable number of PFC were present in the cultures initially. The number decreased throughout the 4-day period and the decline was accelerated by antigen. With mice taken near the peak of the response (10–14 days) no PFC were seen in culture, even on the first day, in the presence or absence of antigen. The first phenomenon, suppression by antigen, is considered to be of general significance since it may also be seen in cultures from mice receiving no B. pertussis as described. It is suggested that the second phenomenon is due to adverse culture conditions resulting from the white cell mobilization initiated by B. pertussis; after 23–27 days conditions have recovered to a point where suppression but not the secondary response can occur. The suppressive effect of antigen could be related to antigenic competition or tolerance.

     

Detection of protein S deficiency: a new functional assay compared to an antigenic technique

Congenital protein S (PS) deficiency is associated with increased risk of venous thrombosis. To investigate the possibility of automating PS testing with decreased turnaround time, a clotting-based functional protein S assay was evaluated and compared to an antigenic method. Samples were collected from 126 patients within 5 days of their first acute cerebral infarction, from 62 controls and from 47 consecutive samples for thrombophilia investigation. The normal range for the clotting-based kit, calculated from the results of 20 healthy controls, was 62-136% (mean +/- 2 SD). Intra- and inter-assay co-efficients of variation were < 3.0 and 10.0% respectively. There was no significant correlation between the two methods (r = 0.30, P > 0.05). Two patients had low PS antigen results with normal functional levels. Both techniques were used to compare a further group of 53 patients with defined abnormalities which included nine antigenic protein S deficiencies, five protein C deficient patients, 10 patients with a lupus anticoagulant (LA), 17 Factor V Leiden (FVL) heterozygotes, two FVL homozygotes and 10 patients on therapeutic levels of heparin. In this group we found that four of nine antigenic PS deficient patients had normal functional PS levels. The test was susceptible to the FVL mutation with four of 17 FVL heterozygotes and both of two FVL homozygotes giving low levels. One of five protein C-deficient patients also had a low functional PS result with a normal antigenic level. Normal results were obtained by both methods for all of the LA and patients on therapeutic heparin. We concluded that the automated protein S clotting assay was rapid and simple to perform but appeared to be influenced by factors other than PS deficiency. Results need to be interpreted with caution but may be useful as part of a full thrombophilia investigation.     

Triggering of the lethal hit in human cytotoxic T lymphocytes: a functional role for a 103-kDa T cell-specific activation antigen

A monoclonal antibody (CB.1) is described that defines a new triggering signal for human cytotoxic T lymphocytes (CTL). The antibody precipitates a 103-kDa surface antigen from activated normal human T cells. The antigen is undetectable or present in only low amounts on resting T lymphocytes but its expression increases strongly after activation and proliferation on T4+ and T8+ T lymphocytes. Binding of antibody CB.1 to CTL results in triggering of the lethal hit. This induction of cytotoxicity is dependent on cross-linking of CTL and an Fc receptor-bearing target cell with CB.1 and requires Ca2+ like antigen-specific triggering. CB.1-induced triggering can be specifically inhibited by binding of antibodies to the T8 or T4 molecules on T8+ or T4+ CTL.     

Identification and functional characterization of guinea-pig CD4: antibody binding transduces a negative signal on T-cell activation.

The guinea-pig (gp) CD4 protein was identified using the rat monoclonal antibody (mAb) H155 derived from an interspecies hybrid. The hybrid was obtained after immunization of rats with purified guinea-pig T lymphocytes and fusion to a mouse myeloma line. The mAb H155 reacted with a subpopulation (60-70%) of nylon-wool-purified guinea-pig T cells. The majority of thymocytes also expressed the H155 antigen. Immunohistological staining showed that predominantly the cortical thymocytes were H155-positive, whereas a part of the medullary cells did not express the antigen. After cell-surface radioiodination of T cell lines, the mAb H155 immunoprecipitated a molecule of about 55,000 MW both under reducing and non-reducing conditions. The antigen recognized by mAb H155 on guinea-pig T cells resembles the human or mouse CD4 antigen. Depletion experiments in combination with functional studies further supported this assumption. In proliferation assays mAb H155 inhibited T-cell responses in vitro. Both the antigen- and alloantigen-induced T-cell activation were impaired in the continuous presence of mAb H155. In addition, mAb H155 inhibited both the mitogen-induced T-cell activation and T-cell proliferation induced by mitogenic mAb and phorbol myristate acetate (PMA). Binding of the mAb H155 to the CD4 molecule might therefore transduce a negative signal on T-cell activation.