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The Burkitt lymphoma (BL)-derived, HLA-DR antigen positive B cell line, EB1, is a consistently low stimulator in MLC. A rabbit antiserum raised against the strongly stimulating BL line DAUDI, after appropriate absorption with EB1, inhibits MLC stimulation by both B cell lines and allogeneic lymphocytes, whilst lectin-induced proliferation is not significantly affected. Indirect immunofluorescence and 125I-staphylococcal protein A binding to cells pre-incubated with this antiserum suggest that the antigen is present on both peripheral B and T cells, as well as on B lymphoblastoid and myeloma lines. We suggest that this antiserum is directed against lymphocyte activating determinant(s) (LADs) and that these are distinct from the serologically defined DR antigens.

Dinucleotide repeat sequences (‘microsatellites’) have been used as polymorphic genetic markers following amplification in the polymerase chain reaction (PCR). We have compared several methods of analysing the PCR products. The most reliable and unambiguous results were obtained when the PCR products were probed with a specific dinucleotide repeat olignucleotide, so that only the microsatellite-containing products were detectable.

A human monoclonal antibody which reacts preferentially with HLA-DR4 and -DRw10 B-cell targets has been produced. A human B-cell line, secreting antibody which reacted preferentially with DR4 and DR1 targets, was derived from a highly sensitized kidney recipient who had rejected two grafts. This line was fused with the mouse myeloma P3X63Ag8.653 and a selected hybridoma cloned. The clones secrete IgM(λ), which reacts strongly with HLA-DR4 and -DRw10 and more weakly with -DRw14 and a proportion of -DR1 B cells in cytotoxicity assays. Using B-cell lines as targets in cytotoxicity and enzyme-linked immunosorbent assays, the antibody gives a broader pattern of reaction, reacting with HLA-DR1, -DR4, -DR9, -DRw10, -DRw14, and some -DR2 targets. The antibody (NI) is currently in use as a reagent for tissue typing.

The DNA of paired tumour and blood leucocyte samples from a large series of breast cancer patients was analysed to map regions of loss of heterozygosity on chromosome 17. The high frequency of loss of heterozygosity on 17p was confirmed, and a third of informative tumours had also lost an allele at the long arm locus THH59. On the short arm two distinct regions of loss of heterozygosity were identified, in bands p13-3 and p13-1. The latter probably involves the structural gene p53, which has been implicated as an oncogene or as a tumour suppressor in various human cancers. 17p 13-3, however, showed a significantly higher frequency of loss of heterozygosity, and there was no correlation between allele losses at the two sites. Nevertheless, loss of heterozygosity at 17p 13-3 is associated with overexpression of p53 mRNA, suggesting the existence of a gene some 20 megabases telomeric of p53 that regulates its expression. Lesions of this regulatory gene seem to be involved in the majority of breast cancers.

Human B cell lines were screened for production of interleukin 6 (IL 6) by the B9 hybridoma cell bioassay. Some long-established lines such as RPMI 1788, CESS and recently established early-passage Epstein-Barr virus (EBV)-transformed lymphoblastoid lines were constitutive IL 6 producers. Other long-established lymphoblastoid lines such as RPMI 8866 and SKW6.4 and all Burkitt lymphoma lines tested were nonproducers of IL 6. Constitutive production of IL 6 in early passage lines could be enhanced by the phorbol ester phorbol 12-myristate 13-acetate (PMA) and recombinant (r)IL 4 but not by rIL 1 alpha or rIL 1 beta. Nonproducing EBV-transformed lymphoblastoid cell lines could be induced to IL 6 production by PMA, rIL 1 alpha, IL 1 beta and IL 4. Among the nonproducing lines SKW6.4 was induced to IL 6 production by PMA, rIL 1 alpha, rIL 1 beta and rIL 4, whereas RPMI 8866 was induced only by PMA, to a limited extent by rIL 1 alpha and rIL 1 beta but not at all by rIL 4. There was no induction of IL 6 by any recombinant cytokine in the two Burkitt lymphoma lines but measurable production of IL 6 was induced by PMA in one of them (EB4). Cytokines which were neither enhancers nor inducers of cell lines on their own included rIL 2, rIL 5, interferon (rIFN)-gamma, native purified IFN-alpha, tumor necrosis factor (rTNF)-alpha, rTNF-beta and purified platelet-derived transforming growth factor-beta. rIL 4 synergized with either rIL 1 alpha or rIL 1 beta in the induction of IL 6 in the nonproducing line SKW6.4. Similar effects were also seen in this line with combinations of (a) rIFN-gamma and rIL 4 and (b) IFN-alpha and both rIL 1 alpha and rIL 1 beta. rIL 4, with or without rIL 1, was more effective than rIL 6 in the induction of IgM synthesis in SKW6.4 and the effect was only partially inhibited by anti-IL 6 antiserum at a dose which totally inhibited IL 6-induced IgM production. Normal peripheral blood lymphocyte populations pre-activated by anti-immunoglobulin rosetting exhibited enhanced production of IL 6 in the presence of rIL 4 and PMA but not in the presence of rIL 1, in contrast to the behavior of adherent mononuclear blood cells which showed IL 4-induced down-regulation of IL 6 production.(ABSTRACT TRUNCATED AT 400 WORDS)

We have examined the expression on a panel of 22 human kidneys of polymorphic B-cell determinants recognized by mouse monoclonal antibodies. Monoclonal antibodies from a mouse immunized with an antigenic preparation from a DR4 positive B-cell line reacted preferentially with kidneys from DR4 positive donors (p less than 0.005), and the pattern of reactivity with kidney tissues was similar to that of antibodies to monomorphic determinants of class II. However, these antibodies did not show clear specificity for DR4 on lymphocytes in standard serological analyses. These results provide evidence for the expression of polymorphic class II determinants on human kidneys. Reasons for the differences in the apparent specificities of the monoclonal antibodies when tested on kidney sections and lymphocytes are discussed.

Conventional cleavage of linked polypeptide chains by heating in SDS can so alter molecular structure as to interfere with antibody binding, on which both immunoprecipitation and 'western blotting' depend. As an alternative, gentle treatment with acid at room temperature or at 0 degrees C was effective in separating the alpha and beta chains of human MHC Class II glycoprotein dimers and proved superior in terms of preservation of at least one labile epitope on the beta chain.

We have examined the MHC class II β chains in lymphoblastoid cell lines from over 200 individuals and describe one line which possesses, in addition to normal β chains, a species of β chain of unusually high M r and abnormal pI which appears to be a product of the DR locus. This abnormality in M r , detected by SDS-gel electrophoresis, was apparent only in the presence of mercaptoethanol and was shown to be due to difference in polypeptide chain length rather than to extra glycosylation.

Five human lymphoblastoid cell lines which have become tumorigenic, as judged by transplantability into immunosuppressed mice, have been compared with earlier, non-tumorigenic stocks of the same cultures, recovered from liquid nitrogen. Analysis of the cell surface glycoproteins by SDS-PAGE, electro-transfer to cellulose nitrate and "blotting" with radioiodinated lectins, reveals an increase in the molecular weight of one band from around 190 kd to 220 kd, in each case coinciding with the acquisition of the tumorigenic phenotype. Probing the electrotransfers with a monoclonal antibody demonstrates that the altered glycoprotein is a member of the "leukocyte common antigen" family. The increase in molecular weight is due to the addition of carbohydrate residues which are removed by treatment with neuraminidase and endoglycosidase. Screening a larger number of lymphoid cell lines, including those derived from Burkitt's lymphoma, shows an excellent correlation between tumorigenicity and altered molecular weight of a leukocyte common antigen.

Studies with two monoclonal antibodies (DA6.147 and DA6.231) which react, respectively, with isolated human Ia alpha and beta chains are reported. Both antibodies detect epitopes expressed on all DR-heterozygous and DR-homozygous cell lines tested (n = 17) and bind to the Epstein-Barr virus-negative cell line Ramos. Ia subunit specificity of the antibodies was determined by an adaptation of an electroblot technique which transfers separated Ia chains from polyacrylamide gels to nitrocellulose paper. In radioimmunobinding assays, peripheral blood B cell-enriched fractions, phytohemagglutinin-activated T cells and pokeweed mitogen-activated cells gave strong reactions with DA6.231 (anti-Ia beta). In contrast, DA6.147 (anti-Ia alpha) reacted only weakly, if at all, with peripheral B cells, pokeweed mitogen blasts and activated T cells. However, both antibodies bound to isolated Ia from activated T cells and peripheral B cells after Nonidet-P40 solubilization of the cells and DA6.147+ antigens could be found in the cytoplasm of activated T cells by indirect immunofluorescence techniques. Results of serological inhibition procedures following fractionation of lymphoblastoid cell lysates on monoclonal antibody affinity columns showed that the DA6.147 alpha chain epitope is carried on only a minor subpopulation of human Ia.

The biosynthetically radiolabelled secreted proteins of human lymphoblastoid cell lines have been analysed by sucrose gradient velocity sedimentation. The addition of detergent to the gradient buffer dramatically improves the recovery of immunoglobulins from cell line supernatants, allowing the gradient fractions to be analysed further both serologically and biochemically. The results of these analyses show that IgM is synthesised in the 19S form and can be clearly separated from other components which include free light chains. Immunoglobulin appears to form a much larger portion of the proteins secreted by human lymphoblastoid lines than has formerly been recognised, a finding which may be of practical importance for the development and exploitation of human monoclonal antibodies.

Unfixed metaphase and non-metaphase cells were tested for their template activity with RNA polymerase. A device was used which disrupts the cell membrane by centrifugation, and which also ensures that the cells do not continue with their mitotic cycle during the transcription process. The template activity of acid/methanol fixed cells was also tested. None of the unfixed metaphase cells transcribed RNA whereas most of the non-metaphase cells did. In contrast, using fixed cells both classes of cells were transcribed equally well and to a much greater extent. It was concluded that metaphase chromosomes in vivo cannot act as templates for RNA synthesis.

Ia antigens were shown to be present in the cell almost exclusively as mature alpha beta dimers which split into separate alpha and beta chains after boiling in SDS. In contrast metabolically labelling the cells with [35S]methionine resulted in only free alpha and beta chains being labelled. It is concluded that this widely used type of labelling, although useful for studying intermediate synthesis, should not be used for labelling mature cell surface molecules.

Monoclonal antibodies directed against human Ia alpha- and beta-subunit chains have been used as probes to detect polypeptides carrying recognized antigenic determinants or epitopes following two-dimensional PAGE separation. Approximately 4 sets of components differing in isoelectric point but not in molecular weight are recognized by each antibody. The anti-alpha-chain, McAb, reacts weakly with spots designated as epsilon but neither antibody recognizes Im, Ii or delta determinants under the conditions tested.

The solubility and ensuing transfer problems associated with IEF separation and blotting of membrane proteins have been eliminated by confining NP40 to the sample region of the gel and replacing it with octylglycoside in the separating gel. This permitted excellent separation of both the α and β chains of the human MHC antigens and their subsequent detection by Western blotting.

Three monoclonal antibodies which recognise determinants on the surface of human B cells have been studied for their effect on mixed lymphocyte reactions (MLR). MLR inhibition was observed under a variety of conditions. While it was not possible to ascribe lymphocyte activation to a single determinant recognised by any of the antibodies, further analysis of the mechanisms involved in lymphocyte activation should be possible through the use of monoclonal reagents.

“Western blotting” is the name given to a technique whereby antigenic proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (1), then transferred electrophoretically from the gel to a sheet of cellulose nitrate. The positions of the antigens, and hence their approximate molecular weights, can be determined by “probing” the sheet with the corresponding antibody which is either labeled itself (with a radioisotope, for example, or an enzyme) or which can be detected indirectly by a second (labeled) probe (2).

Publisher Summary This chapter describes three monoclonal antibodies—DA6 231, 164, and 147— that precipitate glycoprotein dimers consisting of 34 K and 29 K subunits. 231 and 147 recognize DR-common epitopes, whereas 164 binds to all except DR7 homozygous cell lines. Protein transfer from sodium dodecyl sulfate (SDS) polyacrylamide gels to nitrocellulose paper, followed by probing with the monoclonal antibodies, has permitted assignment of the 231 and 147 epitopes to 29 K and 34 K subunits, respectively. Mutual binding inhibition between 231 and 164 is incomplete in one direction, suggesting that there is a major proportion of 231 + 164 + and a minority of 231 + 164 – 29K molecules expressed on all DR positive cells tested, except DR7 homozygous cells that have lost the 164 epitope. The serological expression of the 147 epitope at the cell surface is complex. The 231/147 ratio on peripheral and activated β lymphocytes is much higher than on β lymphoblastoid lines or on chronic lymphocytic leukemia cells. No significant binding of 147 can be detected on the strongly 231 + 164 + -activated cultured τ cells. Preliminary evidence suggests that molecules that can bind 147 are released on cell lysis even from 147 – activated τ cells.

Twenty-one of the Workshop “L” series antibodies (L5 was missing) were received in April, 1984. They were thawed and stored at 8°C throughout the study (April-August, 1984). Aliquots were diluted 1:20 for use in both serological and biochemical assays, the diluent being RPMI 1640 medium containing 5% fetal calf serum (FCS) and 5% horse serum (HS) plus 0.02% sodium azide. All the antibodies were tested for the ability to bind to a selected panel of cells comprising established human leukocyte lines and fresh tissue from cases of leukemia or lymphoma. Those which gave positive results were then examined by “Western Blotting” on solubilized membrane glycoproteins, derived from human leukemic cells, which had been run in polyacrylamide gel electrophoresis (PAGE) and electrotransferred to cellulose nitrate sheets.

A series of monoclonal antibodies, prepared against the Burkitt's lymphoma line Daudi, define antigens predominantly expressed on cells of the B lymphoid series. Four antibodies which react with HLA-DR or DR-like antigens have enabled the serological definition of at least two and possibly three distinct subsets of molecules. Of these four antibodies, one (DA6.164) defines a DR allotype associated epitope common to all DR types tested except DR 7 homozygotes. A second (DA6.147) recognizes a DR-like antigen which is expressed in abundance on B lymphoblastoid cells, weakly on peripheral blood B cells and not at all on activated T cells. Two further antibodies (DA6.231 and DA6.121), with indistinguishable specificity, detect an epitope apparently common to more than one set of DR molecules.

The requirements for the translation of globin mRNA by ascites ribosomes were studied. It was found that globin was synthesised by a system derived entirely from ascites cells, but the level of synthesis could be increased by the addition of the KCl wash of reticulocyte ribosomes.