Pluripotential cells are present in a mouse embryo until at least an early post-implantation stage, as shown by their ability to take part hi the formation of chimaeric animals1 and to form teratocarcinomas2. Until now it has not been possible to establish progressively growing cultures of these cells in vitro, and cell lines have only been obtained after teratocarcinoma formation in vivo. We report here the establishment in tissue culture of pluripotent cell lines which have been isolated directly from in vitro cultures of mouse blastocysts. These cells are able to differentiate either in vitro or after innoculation into a mouse as a tumour in vivo. They have a normal karyotype.

Establishment in culture of pluripotential cells from mouse embryos

Leukocytes respond to lipopolysaccharide (LPS) at nanogram per milliliter concentrations with secretion of cytokines such as tumor necrosis factor-alpha (TNF-alpha). Excess secretion of TNF-alpha causes endotoxic shock, an often fatal complication of infection. LPS in the bloodstream rapidly binds to the serum protein, lipopolysaccharide binding protein (LBP), and cellular responses to physiological levels of LPS are dependent on LBP. CD14, a differentiation antigen of monocytes, was found to bind complexes of LPS and LBP, and blockade of CD14 with monoclonal antibodies prevented synthesis of TNF-alpha by whole blood incubated with LPS. Thus, LPS may induce responses by interacting with a soluble binding protein in serum that then binds the cell surface protein CD14.

https://science.sciencemag.org/content/sci/249/4975/1431.full.pdf

CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.

Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product.

Xenogeneic immunizations have the advantage of detecting a wide range of antigenic determinants because many commonly occurring proteins have diverged significantly during the course of evolution and are thus antigenic in other species. The broadness of xenogeneic responses, however, means that the antisera they produce are usually complex and require extensive absorptions to make them specific for a single antigen. This problem has now been overcome by generating hybridomas producing monoclonal antibodies (Kohler & Milstein 1975). These permit dissection ofthe xenogeneic response so that large amounts of individual antibodies can be obtained, each of which recognizes only one of the determinants recognized by a broadly reactive conventional antiserum. Williams et al. (1977) used hybridoma monoclonal antibodies obtained after immunizations of mice with rat cells to study rat cell-surface antigens present on subpopulations of rat lymphocytes, i.e., differentiation antigens. Springer et al. (1978a) and Stern et al. (1978) used a similar approach to study mouse lymphocyte antigens. They prepared monoclonal antibodies by immunizing rats with mouse lymphocytes and showed that these monoclonals recognized previously undetected mouse cell surface determinants including a glycoprotein antigen that appears to be specific for macrophages (Springer et al. 1978b). Trowbridge (1978) also used rat anti-mouse immunizations to generate a monoclonal antibody against the non-polymorphic lymphocyte surface antigen T200.

Xenogeneic monoclonal antibodies to mouse lymphoid differentiation antigens.

Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.

We have previously described the derivation of M1/70, a hybrid myeloma line secreting monoclonal rat anti-mouse cell surface antibody (Springer, T., Galfre, G., Secher, D. S. and Milstein, C, Eur. J. Immunol. 1978. 8: 539). We have now investigated the cellular distribution of this antigen using a 125I-labeled anti-rat IgG indirect binding assay, the fluorescence-activated cell sorter, autoradiography and precipitation of cell surface molecules. Screening with a tumor cell panel showed strong reactivity with a macrophage-like line but no reactivity with B or T lymphoma lines. In normal tissues, M1/70 antigen was found to be present in small amounts on spleen and exudate granulocytes and a subpopulation of bone marrow cells, in moderate amounts on spleen and blood monocytes and expressed in much larger amounts on spleen histiocytes and peritoneal exudate macrophages. In contrast, M1/70 antigen was found to be absent from erythroid and lymphoid cells. M1/70 antibody precipitated two polypeptides of 190 000 and 105 000 mol. wt. which were present in much greater amounts on peritoneal exudate macrophages than on spleen cells. The expression on phagocytes of two other antigens identified by monoclonal antibodies M1/69 and M1/9.3 was also examined. Monocytes and granulocytes expressed large amounts of M1/69 and low amounts of M1/70 antigen, while in peritoneal exudate macrophages this pattern was dramatically reversed. M1/70 thus defines a differentiation antigen on mononuclear phagocytes and granulocytes, the expression of which is specifically increased during monocyte maturation. This antibody is the first to be described which recognizes a discrete molecule specific to phagocytes.

Mac‐1: a macrophage differentiation antigen identified by monoclonal antibody

https://www.cell.com/cell/pdf/0092-8674(77)90266-5.pdf

Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes.

Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies.



It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.

Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens

MONOCLONAL antibodies can be produced by cultures of permanent cell lines derived by fusion of suitable myelomas with spleen cells from immunised animals1. So far, mainly two mouse myelomas, X63-Ag8 and NSI/1-Ag4.1, have been used for this purpose1,2. When cells from immunised mice are used in the fusion, the mouse × mouse hybrid myelomas can be grown as tumours in appropriate mouse strains. We describe here the preparation and use of a new clone of a rat myeloma, which is suitable for the derivation of rat × rat hybrid myelomas producing specific rat antibodies. The spent medium of hybrid myeloma cultures usually contains 1–20 μg ml−1 antibody. The serum and ascites of the tumour-bearing mice, with few exceptions, yield 1–20 mg ml−1 of antibody, which is 1,000 times more concentrated and very convenient for larger preparations.

G. Galfre

Papers

Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis.

Mac‐1: a macrophage differentiation antigen identified by monoclonal antibody

Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes.

Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens

Rat × rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG