David S. Secher

Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells

A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.

Intracellular immunoglobulin chain synthesis in non-secreting variants of a mouse myeloma: Detection of inactive light-chain messenger RNA

The intracellular immunoglobulin chain content of a number of non-secreting variants of MOPC 21 mouse myeloma cells has been examined. Pulse-labelling with [14C]lysine, followed by cell lysis, reaction with antibody and analysis of the precipitates on sodium dodecyl sulphate-polyacrylamide gels, reveals three different types of variant. Only one type (NS I) contains light-chains. An abnormal heavy-chain was present in all non-secreting lines and was prominent in one cell line (NS II/1). Pulse-chase experiments indicated a rate of decay of intracellular immunoprecipitable material comparable to its rate of synthesis suggesting intracellular destruction of non-secreted chains. The presence of polyribosomes engaged in immunoglobulin chain synthesis was tested by their addition to a reticulocyte cell-free system. It was confirmed that in the parental cell line immunoglobulin chain synthesis is almost exclusively confined to membrane-derived polyribosomes. Failure of some of the variants (NS II/1 and NS III/1) to synthesize light-chain was not due to misallocation of light-chain mRNA to the free polyribosomes. Isolated 13 S mRNA from the same variants also failed to direct the synthesis of detectable light-chain in the reticulocyte cell-free system. However, fingerprint analysis of the corresponding 32P-labelled 13 S mRNA showed the presence of several oligonucleotides characteristic of the normal light-chain mRNA, indicating the presence of an inactive form of this molecule.

Identification of a monoclonal antibody to abscission tissue that recognises xylose/fucose-containing N-linked oligosaccharides from higher plants.

Monoclonal antibodies raised against extracts of the rachis abscission zone of Sambucus nigra L. were selected for high reactivity towards abscission-zone proteins. One antibody (YZ1/2.23) has been shown to cross-react, by both indirect and competition enzyme-linked immunosorbent assay and by Western blotting, with a number of plant enzymes including horseradish peroxidase, rice α-glucosidase, almond β-glucosidase and the lectins from Phaseolus vulgaris and Erythrina cristagalli.The major N-linked oligosaccharide isolated from horseradish peroxidase has the sequence Manα 3(Manα6)(Xylβ2)Manβ4GlcNAcβ4(Fucα3) GlcNAc. This oligosaccharide was found to be a potent inhibitor of the binding of YZ1/2.23 to the intact glycoprotein. The common determinant is therefore contained within this structure.

Monoclonal antibodies and cell surface antigens

Antibody chains are encoded in three gene clusters containing genes for the variable and constant regions. V and C genes are separated in germ line and during differentiation a rearrangement takes place. But even after this rearrangement the V and C coding sequences are not contiguous. A final splicing must take place in committed cells between the transcription of a discontinuous V-and C-region DNA and the expression of a continuous mRNA coding for an antibody chain. Analysis by cell fusion indicates that the splicing is cis. When two antibody-producing cell lines are fused, the resulting hybrids express the two antibodies that characterize the parental lines. Permanent cell lines producing antibody of predefined specificity have now been derived in this way. Spleen cells from hyperimmunized donors are fused with myeloma cells and a proportion of the hybrids that are established synthesize and secrete antibodies directed against the immunogen. The heterogeneous cell population can be cloned and propagated. This is a potent way of producing monospecific antibodies to complex antigens such as cell membranes and transplantation antigens. Monoclonal xenogeneic antibodies to rat cell-surface membranes have proved very valuable for characterizing and separating rat lymphocyte subpopulations. In more recent experiments, monoclonal xenogeneic antibodies to mouse and human cell-surface antigens have also been produced which permit the characterization of the hitherto undescribed differentiation antigens.

Spontaneous mutation in tissue culture — chemical nature of variant immunoglobulin from mutant clones of MOPC 21

Studies of spontaneous mutants occuring in cell cultures are of considerable interest for the understanding of the nature of somatic mutation in higher organisms. An ideal system for studies of this type would be one in which mutations result in a product which has no effect on the growth of the cell. This condition is approached in a myeloma cell culture, where the secreted immunoglobulin (Ig) confers no apparent benefit on the cell that produced it. Thus many mutations affecting the Ig should be tolerated, and indeed a number of variants with defects in Ig synthesis have been described [ 1 ]. However, none of these, nor any other somatic mutation in higher animal ceils, has been shown to be the result of a mutation in the structural gene coding for the variant protein. We have described a novel technique for the screening of large numbers of clones from a myeloma cell culture for electrophoretic variants of the secreted Ig [2]. The method allowed detection of clones deficient in Ig production and also of a clone that secretes an Ig of altered isoelectric, point (IF-l). A second clone producing a variant IgG (IF-2) has now been isolated. We describe here experiments that indicate that these mutants arise as a result of mutations in Ig structural genes.

Ch3 domain of igg as binding site to fc receptor on mouse lymphocytes.

MEMBRANE receptors recognising the Fc portion of immunoglobulin molecules (Fc receptors) are found in many cells of the immune system1–4. Fc receptors on lymphocytes are readily detected by a rosette test3,5,6 and this reaction is inhibited by pretreatment of the lymphocytes with IgG (ref. 3). IgG proteins lacking almost the entire CH1 and CH3 homology regions have been obtained from mutant cell lines of MOPC 21, a plasmacytoma secreting IgG1 (refs 7 and 8) and the extent of the deletions determined (Fig. 1). To identify that part of the IgG molecule which interacts with the Fc receptor, we have tested the ability of these IgG proteins to inhibit Fc rosette formation on murine lymph node cells. We show (Table 1) that an intact CH3 region is essential for the binding of IgG to Fc receptors on lymph node cells.

Monoclonal Xenogeneic Antibodies to Mouse Leukocyte Antigens: Identification of Macrophage-Specific and Other Differentiation Antigens

The identification and study of cell surface molecules which have specific immune functions and are markers of differentiated white blood cell subpopulations is of great interest in immunology. Antibodies are versatile probes with which to study these molecules. However, it is difficult to obtain highly specific antibodies recognizing individual cell surface molecules, because cell surfaces are complex mosaics of many different immunogenic glycoproteins and glycolipids. A general approach to this problem stems from the experiments of Kohler and Milstein (1). They fused myeloma cells and spleen cells from mice immunized with sheep red blood cells to derive continuous hybrid cell lines secreting antibodies to sheep red blood cells. Such lines can be manipulated in culture so that the multispecific response to a complex immunogen can be resolved into a set of monospecific responses by cloning. Hybrids have been obtained which secrete antibodies to rat major histocompatibility antigens(2), rat cell surface xenoantigens (3), mouse IgD allotypes (4), and mouse H-2K antigens (5).

Kinetics of internalisation and degradation of surface-bound interferon in human lymphoblastoid cells.

The binding of iodinated human interferon‐alpha 2 (IFN‐alpha 2) was studied on the human T cell line, Molt 4. After its initial binding to cells, the IFN is transferred to a trypsin‐resistant compartment before appearing in the medium as TCA‐soluble material, while the total cell‐associated IFN declines to one‐third of its maximum value after 3 h incubation. The Na+/H+ ionophore monensin did not prevent intracellular accumulation of IFN but did completely inhibit its breakdown. We interpret our results as evidence for receptor‐mediated internalisation of IFN followed by intracellular breakdown.

Molecular heterogeneity of alkaline phosphatase

Alkaline phosphatase (EC 3. I .3.1) from E. coli is a dimeric enzyme whose subunits are coded by the same gene [ 11. Molecular heterogeneity of the subunits has been ascribed to differential modification of the N-terminus of each monomer after translation [2, 31. In an attempt to verify this hypothesis we have looked for heterogeneity in the N-terminal sequence of the enzyme using an automatic sequenator [4].

The mechanism of action of interferon-α (IFN-α) in hairy-cell leukaemia; Hu-IFN-α2 receptor expression by hairy cells and other normal and leukaemic cell types

To investigate the possible direct effects of interferon-α (IFN-α) in hairy-cell leukaemia, IFN-α receptor expression by hairy cells (HCs) (11 cases) was measured by a radiolabelling technique and compared with that of MOLT-4, chronic lymphocytic leukaemia (CLL; 14 cases) and various other leukaemic and normal cell types. Purified peripheral blood (PB) and splenic HCs showed higher levels of receptor expression (approx. 1000 ± 200 binding sites/cell; 11 cases tested) than other normal and leukaemic cells types. Purified normal PB and tonsil B cells showed low levels of receptors (approx. 120 ± 100 binding sites/cell), while a range of B-cell leukaemias displayed intermediate levels of expression (approx. 100–500 sites/cell). In the 15 cases of CLL tested, 530 ± 330 binding sites/cell were demonstrated, the high standard deviation reflecting the fact that approximately one third of cases had receptor levels comparable with those in HCL. Normal and HCL T cells, red cells and platelets had no demonstrable IFN receptors. It is suggested that these findings may be relevant to the efficacy of IFN in hairy-cell leukaemia.