A monoclonal antibody against an epitope common to HLA-B locus antigens

A monoclonal antibody G4 that appears to be directed against a determinant common to akl HLA-B locus antigens is described. This antibody reacted with a large panel of B and T lymphocytes and cell lines, but it did not react with two lines that do not serologically express HLA antigens (Daudi and K562) and two lines that expressed A-locus but not B-locus antigens (8402 and HPBMLT). The F(ab′)₂ fragment of G4 blocked B-locus but not A-locus HLA alloantisera. By immunoprecipitation and SDS-polyacrylamide gel electrophoresis G4 reacted with a dimer consisting of a heavy chain of 44,000 daltons and a light chain of 12,7000 daltons.     

Antigenic relationship of SV40 early proteins to purified large T polypeptide.

Rabbit antiserum was produced against SV40 large T antigen purified by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. This antiserum immunoprecipitated both large T and small t antigens, and reacted with SV40 T, U, and S antigens by immunofluorescence. these data establish the antigenic relatedness of all the known SV40 early gene products, with the exception of transplantation antigen activity, and confirm the virus-specific nature of each. The reactivity of the anti-T polypeptide serum was compared with the specificities of T-reactive antisera produced by different methods, including conventional tumor-bearing hamster sera, rabbit antiserum directed against whole-cell SDS-lysates of SV40-transformed rabbit kidney cells, and high-titer ascites fluid from hamsters in which ascites was induced by injection of SV40-transformed hamster ascites cells. Each of the antisera was reactive in all of the tests for SV40-induced early antigens, but the relative reactivity toward each protein varied considerably. It is postulated that the differences in reactivity to small t antigen and U antigen represent differences in the immune response of individual animals to the amino and carboxyl termini of the large T antigen polypeptide, respectively. Antiserum produced against the SDS-denatured large T polypeptide exhibited the highest reactivity to both small t and U antigenic sites relative to its reactivity against intranuclear large T antigen.     

Systemic lupus erythematosus sera antilymphocyte reactivity: detection of antibodies to Tac-antigen positive T cell lines.

Sera obtained from patients with systemic lupus erythematosus (SLE) were tested for their reactivity to cell lines derived from cutaneous T-cell lymphoma (CTCL), or adult T cell leukaemia (ATL), and with other cell lines, by indirect immunofluorescence method. Approximately one half of SLE sera reacted with the surface antigens of HUT-102 cells, a cell line from CTCL, which constitutively expresses Tac antigen. The titre tended to be higher in the active than in the inactive stage. These positive sera also reacted with other neoplastic or normal T cell lines having Tac antigen. SLE sera reacting with HUT-102 surface antigens were further examined for their reactivities to Tac antigen, the putative IL-2 receptor, using HUT-102 or ATL-2. Pretreatment with anti-Tac monoclonal antibody partially blocked the reactivities to HUT-102 surface antigens in nine of 15 SLE sera tested. The binding of 125I-labelled anti-Tac monoclonal antibody was displaced by the addition of sera from six of 15 SLE patients. In addition, nine of the 15 SLE sera could inhibit the binding of 125I-labelled IL-2 to ATL-2 cells. These results suggested that some of SLE sera contained antibodies against the IL-2 receptor.     

Di-allelic alloantigenic systems on subsets of T cells

Sera obtained from multiparous women and some of other origin contain antibodies which react with antigens on T cell subsets. These antibodies recognize two distinct diallelic systems, one of which is mainly present on T gamma cells while the other is present on T mu cells. The sera that reacted with the T gamma cells formed a pattern consistent with that of a diallelic system which we have called TCA system with alleles TCA 1 and TCA 2; the sera which reacted with the T mu cells formed a pattern consistent with another diallelic system, independent from TCA, which we have designated the TCB system, with alleles TCB 1 and TCB 2.     

Properties of simian virus 40 and BK virus tumor antigens form productively infected and transformed cells.

Simian virus 40 (SV40)-specific tumor antigen (T Ag) was immunoprecipitated from extracts of productively infected cells and virus-transformed cells and had the same molecular weight of approximately 97,000 as measured by acrylamide gel electrophoresis. In addition to T Ag proteins of that size, lower molecular weight forms were immunoprecipitated with anti-T serum. Experiments using the protease inhibitor l-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) indicated that nearly all of the smaller SV40 T Ag polypeptides were artifacts of the extraction procedure. The BK virus-specific T Ag was immunoprecipitated from extracts of infected human cells and coelectrophoresed with SV40 T Ag (97,000 daltons).     

Identification of tissue-specific antigens and concanavalin A receptors on rat epidermal cells using a radio-immunoprecipitation method.

Iodination with lactoperoxidase - 125I- - H2O2 was used to label surface components of rat epidermal cells. Lysis of the cells in non-idet P40 resulted in the solubilization of tissue-specific antigens and of concanavalin A receptors. These specificities were demonstrated using a radio-immunoprecipitation method. The tissue-specific antigens were recognized using absorbed rabbit anti-rat epidermal cell sera (Lloyd & Darnule 1974); they reacted with two low molecular weight components in the lysate (9,000 and 12,000 daltons). Concanavalin A reacted with three major components. Two had high molecular weights (75,000 and 95,000 daltons). The possibility that one of these components was radioiodinated lactooperoxidase, which would have reacted with concanavalin A, was disproved. Another component (which occasionally appeared as two peaks) was similar in size to the species detected by the tissue-specific antisera. Their non-identity was, however, demonstrated by the finding that the two specificities could be precipitated independently of each other.     

Immunofluorescence of simian adenovirus 7 (SA7)--specific antigens in infected LLC-MK 2 cells and SA7 transformed cells

Antigens synthesized in LLC-MK₂ cells infected with SA7 were described using indirect immunofluorescence. When cells were reacted with sera from tumor-bearing hamsters, “T” antigens were observed as early as 10 hr post-infection (PI). In certain cells a second type of exclusively intranuclear fluorescence was observed at 16 hr PI. Specific antiviral sera reacted with viral structural antigens approximately 15–16 hr PI. Sera from hamsters immunized with density gradient-purified virus reacted with both “T” antigens and viral antigens.     

A monoclonal antibody reactive with a second epitope of the 67,000-dalton human T cell antigen

Hybridomas were produced against the T-cell CLL derived-cell line, SKW3, by the fusion of hyperimmune spleen cells with P3 myeloma cells. One clone, designated DU-SKW3-1, was shown to produce a murine IgG2b antibody reactive with an antigen expressed on normal thymocytes and peripheral blood T cells. This antigen was not detected on human B cells, erythrocytes, monocytes, granulocytes, or platelets. D-SKW3-1 also reacted with T-ALL, T-CLL, and B-CLL cells, but did not react with common ALL or acute myelocytic or monocytic leukemias. Immunoprecipitation of lactoperoxidase-iodinated, detergent-solubilized PBL demonstrated that DU-SKW3-1 reacted with a protein with an apparent mass of 67,000 daltons (p67), which had identical mobility to the antigen precipitated by L17F12, Cocapping experiments suggested that DU-SKW3-1 and L17F12 detected the same molecule: however, DU-SKW3-1 was unable to block the binding of L17F12. In addition, DU-SKW3-1 reacted with the T lymphocytes of both the great apes and old world monkeys, in contrast to L17F12 and two other p67 monoclonals, T101 and 10.2, which reacted only with the cells of the great apes. This data suggests that DU-SKW3-1 may react with a second, less phylogenetically restricted epitope on the p67 T cell-/CLL-associated molecule.     

Serological and functional characterization of human T cell subsets

Two different specific anti-human T cell sera were studied. One was raised against fetal thymocytes (anti-HTY) and the other against human cultured T cells (anti-CTC) grown in the presence of conditioned medium from phytohaemagglutinin-stimulated peripheral blood mononuclear leucocytes (PBL). These sera, after various absorptions, reacted in microcytotoxicity assays against T cells, but not with B and null cells in the peripheral blood of both normal donors and patients with non-T leukaemias. A clear distinction between the labelling density of T versus B cells was also documented with the fluorescence-activated cell-sorter (FACS) analyses. These two sera were further absorbed on T cells from different sources with the aim of obtaining reagents specific for T cell subsets. Two reagents resulting from these absorptions were found to react with subsets of T-PBL. (1) Anti-HTY, after absorption with cells of a T-chronic lymphocyte leukaemia (T-CLL) expressing receptors for the Fc portion of IgG, reacted with thymocytes, 40–50% of normal T-PBL and only with those T cells that were not inhibited by theophylline in their ability to rosette with sheep erythrocytes. When PBL were preincubated with the absorbed serum and complement, the remaining cells had markedly diminished lymphoproliferative responses to lectins and in mixed lymphocyte culture (MLC), but they still responded well to tetanus toxoid (t.tox.). (2) Anti-CTC, after absorption with the MOLT-4 cell line reacted with about 30% of T-PBL and with the majority of the T-CLL cells with Fc receptors, as documented by cytotoxicity and FACS analyses. When normal PBL were preincubated with this absorbed serum and complement, the remaining cells had enhanced lymphoproliferative responses to lectins and especially to t.tox. and in MLC. Thus, these antisera, obtained following some rather simple absorption steps, were able to divide human T cells into two major and distinct subpopulations.     

Detection of B-Cell-Specific Alloantibodies in Pregnancy Sera in the Lymphocytotoxicity and the Indirect Immunofluorescence Techniques

Two hundred and eight pregnancy sera were tested for the presence of antibodies specific for lymphocyte sub-populations by using the isolated B and T lymphocytes from the women's mating partners. This was done by the microlymphocytotoxicity and the indirect immunofluorescence techniques. Five sera (2.5%) reacted exclusively with B lymphocytes and sixty-three sera (30.2%) reacted with both B and T lymphocytes; none of the sera was specific for T cells. Several sera, reacting with both B and T lymphocytes, were absorbed with platelets and this procedure revealed nine additional antiseraa specific for B lymphocyte antigens. Specificity studies on a panel of forty-eight HLA-ABCD typed individuals indicated that most antisera possibly defined new B-cell antigens. Family studies established that the antigens defined by these antiser were coded for by genes in the Major Histocompatibility Complex.     

Autoantibodies to a regulatory T cell subset in human ageing.

Anti-T cell autoantibodies were detected in some aged humans. Non-immunoglobulin-bearing (Ig-) cells were isolated from the peripheral blood of normal human donors by negative selection through Ficoll, using sheep erythrocytes coated with rabbit anti-human Ig. The Ig- cells were then reacted with sera from 83 individuals ranging in age from 60 to 99 years; 36% of the serum samples were noticeably reactive with the Ig- cells (average reactivity 28%). The peripheral blood lymphocytes from some of the aged individuals were also tested for levels of Ig-secreting cells in a reverse haemolytic plaque assay; there was a six- to eight-fold increase in the number of plaque-forming cells (PFC) from those individuals whose sera contained appreciable amounts of anti-T cell antibody, as compared with those whose sera contained little or no anti-T cell antibody. Isolated Ig- cells from these individuals were also examined for the presence of regulatory T cell subsets, using sera from juvenile rheumatoid arthritis (JRA) patients. The Ig- cells from the subjects who had no detectable anti-T cell antibodies in their sera and near normal PFC levels were reactive with the JRA sera, whereas the Ig- cells from individuals with increased numbers of PFC and with serum anti-T cell antibodies were only slightly reactive with the JRA sera. These data suggest that a majority of the regulatory JRA+ subset of T cells had been lost in the latter group. When sera from aged individuals containing anti-T cell autoantibodies were reacted with JRA-, Ig- cells isolated from a normal human donor, little positive reactivity was seen, indicating that the autoantibodies in sera from aged humans and from some JRA patients are directed against similar T cell subsets.     

Identification of Tissue-Specific Antigens and Concanavalin A Receptors on Rat Epidermal Cells Using a Radio-Immunoprecipitation Method

Iodination with lactoperoxidaae - ¹²⁵I- - H₂O₂ was used to label surface components of rat epidermal cells. Lysis of the cells in non-idet P₄₀ resulted in the solubilization of tissue-specific antigens and of concanavalin A receptors. These specificities were demonstrated using a radio-immunoprecipitation method. The tissue-specific antigens were recognized using absorbed rabbit anti-rat epidermal cell sera (Lloyd & Darnule 1974); they reacted with two low molecular weight components in the lysate (9,000 and 12,000 daltons). Conca-navalin A reacted with three major components. Two had high molecular weights (75,000 and 95,000 daltons). The possibility that one of these components was radioiodinated lacto-operoxidase, which would have reacted with concanavalin A, was disproved. Another component (which occasionally appeared as two peaks) was similar in size to the species detected by the tissue-specific antisera. Their non-identity was, however, demonstrated by the finding that thr two specificities could be precipitated independently of each other.     

Identification of the B-cell epitopes of the early endosome antigen 1 (EEA1)

Early endosome antigen 1 (EEA1) is a target autoantigen in patients diagnosed with neurological and other autoimmune conditions. Eighteen of 65 sera (28%) that displayed a vesicular cytoplasmic staining pattern also immunoprecipitated the recombinant EEA1. These 18 sera were selected for further clinical, serological and epitope mapping studies. Thirty-six percent of the 18 patients had neurological diseases. Seventeen sera (94%) reacted with the partial length EEA1 constructs that included the C-terminal zinc finger (+FYVE) and the methyl accepting domain (LeuMA: amino acids 82-1411) in an addressable laser bead assay suggesting that the assay may be used for rapid laboratory detection of anti-EEA1 antibodies. Three of seven sera selected for epitope mapping studies bound to EEA1 peptides represented by amino acids 1096-1125, and two reacted with peptides represented by amino acids 1296-1320. One serum reacted only with the C-terminal peptide 1096-1125. The remaining serum reacted with other EEA1 epitopes. This data was supported by the observations that all the sera immunoprecipitated the C-terminal +FYVE (EEA1 1064-1411) construct, a peptide that also contained the linear epitopes 1096-1140. The limited epitope mapping studies suggest that the sera from patients with non-neurological diseases recognized epitopes in the central and C-terminal EEA1 domains, whereas the patients with neurological disease recognized a more restricted set of epitopes in the C-terminal.     

In vivo and in vitro responses to tumor-associated antigens: apparent absence of T cell participation

The tumor-associated antigens of the murine tumors B16, SAD2 and C3HBA readily induced antibody production in vivo, but produced little transplantation immunity. These tumors grew better in normal syngeneic hosts than they did in lethally irradiated or thymectomized lethally irradiated, marrow reconstituted hosts, where they grew equally well. When blood or spleen cells were reacted with anti-H-2 sera directed against the responder cells or with anti-Θ C3H sera and complement and then cultured with irradiated syngeneic tumor cells or extracts of these tumors, the responses to the tumor-associated antigens and to two thymus-independent antigens were not impaired; however, the same treatment greatly reduced the responses to allogeneic tumor or spleen cells and to two thymus-dependent antigens. The observations suggested that the tumor-associated antigens under study primarily stimulated B rather than T cells.     

Demonstration of unique and common antigenic sites located on the SV40 large T and small t antigens

Two distinct antigenic sites were demonstrated on SV40 small t antigen using sera from hamsters bearing tumors induced by SV40-transformed cells. One of the antigenic sites on small t antigen cross-reacts with antigenic sites on large T antigen as rabbit antisera prepared against denatured large T antigen immunoprecipitated both proteins and its activity could be absorbed with extracts prepared from cells infected with the $\text{0.54}\text{0.59}$ deletion mutant dl2005 which synthesized large T antigen but not small t antigen. The second antigenic site was found to be unique for small t antigen and was identified using serum from a hamster bearing an SV40 lymphoma which preferentially immunoprecipitated small t antigen. Its activity to small t antigen could not be removed by absorption with dl 2005-infected cell extracts. Activity against unique antigenic sites on small t antigen was also found in sera from animals bearing another SV40-induced tumor (TSV5). The unique antigenic sites on small t antigen were located on the C-terminal half of the protein since shortened small t polypeptide induced by d1891 (which lacks C-terminal sequences) was not immunoprecipitated by the lymphoma serum. Unique antigenic sites on large T antigen were also demonstrated as certain sera from tumor-bearing hamsters immunoprecipitated only the large T antigen. These results indicate that animals bearing SV40 tumors immunologically distinguish the large T and small t antigens and produce antibodies specific for the common and unique antigenic sites on the two proteins.     

Identification of allergens and antigens of Bermuda grass (Cynodon dactylon) pollen by immunoblot analysis

Allergens and antigens of Bermuda grass pollen fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes were identified using twenty-one sera of Bermuda grass pollen-allergic patients. The IgE- and IgG-binding pollen components transferred to nitrocellulose were detected by reaction with enzyme-labelled anti-human IgE and anti-human IgG, respectively. There was heterogeneity in both IgE- and IgG-binding patterns of the allergic sera tested. Fourteen pollen components, ranging in molecular weight from 16000 to 88000 daltons, bound to IgE antibodies. Only two of the fourteen allergens identified reacted with IgE antibodies of more than 50% of the twenty-one allergic sera. The pollen component with a molecular weight of 32000 daltons showed by far the highest frequency of IgE binding, being recognized by sixteen (76%) of the twenty-one sera examined. Fifteen (71 %) of the twenty-one sera tested had IgE antibodies that reacted with more than one of the fourteen allergenic components identified. Pollen components recognized by IgE antibodies also reacted with IgG antibodies, and there were components only recognized by IgG antibodies. Results obtained from this study should be useful both clinically and in research.     

Development of a recombinant enzyme-linked immunosorbent assay for detection of antibodies against Epstein-Barr virus nuclear antigens 2A and 2B.

The baculovirus expression system was used to produce full-length Epstein-Barr virus nuclear antigens (EBNAs) 2A and 2B. Recombinant baculoviruses that contained the EBNA-2A- and EBNA-2B-encoding sequences were constructed. The proteins were expressed in Spodoptera frugiperda SF-9 cells infected with the recombinant viruses and were characterized by using monoclonal and human polyclonal antibodies by immunoblotting and immunofluorescence techniques. Partially purified extracts of the EBNA-2A- and EBNA-2B-infected insect cells were used to establish a new enzyme-linked immunosorbent assay for the detection of antibodies against EBNA-2A and EBNA-2B. Preferential reactivity toward the type A or type B EBNA-2 protein was observed in 36% of serum specimens from Swiss patients with acute infectious mononucleosis and in 81% of Swiss patients with latent Epstein-Barr virus infection. Of the patients in the latter group, sera from 76% reacted preferentially with EBNA-2A, sera from 5% reacted preferentially with EBNA-2B, sera from 12% showed similar reactivities against both antigens, and sera from 7% were nonreactive.     

Characterization of the Endothelial Surface Proteins Recognized by Anti-Endothelial Antibodies in Primary and Secondary Autoimmune Vasculitis

The antigenic structures recognized by anti-endothelial cell antibodies (AECA) in sera from 10 Wegener's granulomatosis (WG) and 12 systemic lupus erythematosus (SLE) patients with signs of vasculitis were characterized by immunoprecipitation of selectively radiolabeled surface membrane proteins from human umbilical vein endothelial cells. Electrophoretic analysis of the immunoprecipitated proteins revealed reactivities against endothelial antigens ranging in size from 200 to 25 kDa. AECA antigens were not cell specific, since the same sera also reacted, at least in part, with radiolabeled human fibroblast surface proteins. The majority of WG patients displayed a constant precipitation pattern of five proteins (180, 155, 125, 68, and 25 kDa). On the contrary, AECA from SLE sera reacted with a more heterogeneous series of endothelial proteins. A group of four proteins, however, was also found in the majority of SLE sera: 200, 180, 155, and 25 kDa. In addition, some endothelial antigens were immunoprecipitated only by WG (125 kDa) or by SLE sera (200 kDa), suggesting a different endothelial reactivity in different vasculitic processes. The reaction did not involve intracellular proteins as demonstrated by the lack of reactivity of SLE sera negative for AECA but positive for anti-cytoplasmic or anti-nuclear antibodies. These data confirming that AECA recognize surface endothelial determinants further support a potential pathogenetic role for these antibodies in autoimmune vasculitis.     

The heterogeneity ofLeishmania cell-surface antigens

A comparative study of the radioiodinated promastigote cell-surface antigens ofLeishmania mexicana andL. major was carried out under reduced and nonreduced conditions by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Under reduced conditions, the cell surface ofL. mexicana promastigotes showed three iodinated polypeptides with molecular weights of 65 000, 50 000 and 27 000 daltons, whereasL. major promastigotes displayed a single polypeptide of 63 000 daltons. Under nonreduced conditions, the radioiodinated cell-surface component ofL. major shifted to a mol.wt. of 51 000 daltons, whereas only one of the three components ofL. mexicana (mol.wt., 65 000 daltons) underwent a large shift (to 59 000 daltons). The different immunochemical nature of theL. mexicana cell-surface antigens was demonstrated by using different anti-Leishmania sera. The rabbit anti-promastigote serum immunoprecipitated mainly the 50 000- and 27 000-daltonL. mexicana cell-surface polypeptides, whereas the rabbit anti-amastigote serum as well as a serum from a patient with cutaneous leishmaniasis immunoprecipitated almost exclusively the 65 000-dalton polypeptide. Immunoblot studies using a rabbit antibody against theL. major deglycosylated major surface antigen gp63 confirmed the differences in nature of the 65 000- and 50 000-dalton cell-surface antigens ofL. mexicana. The results obtained are discussed in the light of the differences in antigenic cell-surface expression amongLeishmania isolates and their consequences in the development of a differential diagnosis of leishmaniasis.     

Alloantibodies that react with subsets of human T cells

Using the two color fluorescence (TCF) method, alloantibodies against subsets of T cells could be detected in sera from pregnant women with strong HLA antibodies. To preclude interference of these HLA antibodies with the recognition of the T cell antibodies, serum donors were selected which were HLA-Al, -B8, -DRw3. Their sera were tested on a panel of individuals homozygous for HLA-Al, -B8, -DRw3. By enriching peripheral mononuclear blood lymphocytes for Tgamma cells it could be shown that some of the sera reacted mainly with Tgamma and others with Tmu lymphocytes, while some sera reacted with both.