Keith Guy

Human MHC class II molecules as differentiation markers

DA6.231 and DA6.164 are mouse monoclonal antibodies that immunoprecipitate HLA-DR-like p34,29 glycoprotein dimers from surface- and metabolically-labeled cells. On lymphoblastoid cell lines the distribution of the 231 epitope is completely nonpolymorphic, while the 164 epitope is present on all cells except on those that are DR7 homozygous. Binding-inhibition studies show that the 231 and 164 epitopes are spatially close to each other when present on the same molecule. The mutual inhibition pattern and the absence of the 164 epitope from the 231+ cells of a few leukemia patients suggest, however, that 231 and 164 epitopes are not invariably present together. Most DR-positive cells possess 231− 164+ and 231+ 164− class 11 molecules in approximately a 2:1 ratio. This has been confirmed by immune depletion studies. Thus DA6.231 appears to define a supralocus epitope. The 164 epitope may be a marker for a subset of class 11 molecules exhibiting differential expression on various cell types immortalized by malignant transformation.

An ordered sequence of expression of human MHC class-II antigens during B-cell maturation?

Studies with monoclonal antibodies confirm that human MHC class-II antigens are encoded by at least three pairs of loci. Here Keith Guy and Veronica van Heyningen suggest that as B cells mature theproducts of these loci are expressed in the sequence SB → DR → DC antigens - a sequence which parallels the order of the genes on chromosome 6.

Subsets of Human D Locus Products Identified by a Series of Monoclonal Antibodies

Abstract A series of monoclonal antibodies, prepared against the Burkitts 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.

Burkitt lymphoma cell lines are prone to recombination in the switch region of the Igμ heavy chain locus

Different recombinations have been found at the Ig heavy chain gene loci in a number of sublines of the Burkitt lymphoma (BL) cell line Namalwa, following prolonged in vitro culture. The Namalwa sublines examined are DNA fingerprint-identical and derived from a monoclonal source. Recombinant DNA clones were used to map the Ig heavy chain gene mutations to a region between the VDJ and C mu segment of the locus. This region is associated with Ig heavy chain class switching in normal B cells. Of 24 clones established from one subline, three were found to have additional VDJ-C mu region mutations, indicating a high frequency of mutation at this locus.

Biochemical Analysis of Antigens Recognized by Workshop B Series Antibodies, Using “Western Blotting”

“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).

Screening of Workshop “B” Series Antibodies by Radioimmunobinding to Human Leukocyte Cell Lines and to Cells from Human Lymphoid Tumors

The fifty-two monoclonal antibodies of the Workshop B series were received in April, 1984. They were allowed to thaw and were held at 8°C for the period of study (April–August, 1984). Aliquots of the original samples were diluted 1:20 with RPM1 1640 culture medium containing 5% fetal calf serum (FCS), 5% horse serum (HS), and 0.02% sodium azide. The diluted antibodies were then tested for binding to a panel of cells from human lymphoid lines or from lymphoid tumors. The phenotypes of the cells forming the panel had already been determined but additional monoclonal antibodies of known specificities were included to act as positive or negative controls in the binding assays.

Monoclonal Anti-B Cell Antibodies: their Effects on Human Mixed Lymphocyte Reactions

Abstract 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.

Analysis of Workshop “L” Series Antibodies: Radioimmunobinding and Biochemical Studies

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.

TOWARD ANALYSIS OF THE HLA-DR SYSTEM WITH MONOCLONAL ANTIBODIES

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.