Lionel A. Manson

A simplified 51Cr-release assay for killer cells☆

Abstract A simplified chronium-51 release assay for lymphoid cell-associated cytotoxic (killer) activity is described that uses a microtest plate for incubation instead of tubes. There is no centrifugation necessary for the assay itself, and it does not require custom equipment. It compares favorably with both the Brunner et al. (1968) assay of which it is a derivative and the assay described by Canty and Wunderlich (1970). The assay was found to be intensitive to changes in incubation media, sera, buffers and vessel shape. Two modifications are described that hasten cell-to-cell contact and greatly reduce the number of target cells required.

Mycoplasma (PPLO) strains with lytic activity for murine lymphoma cells in vitro.

Summary Preliminary characterization of a group of mycoplasma that are cytocidal for L5178Y and P388D1 cells, but not for many other tissue culture cells, is presented. Lysis of these murine malignant lymphoblasts is a property not shared by many other mycoplasma including such agents as Campo, T-5, and Eaton mycoplasma.

The major histocompatibility complex of the rat,RT 1 : II. biochemical evidence for a complex genetic organization.

Recombinational analysis has shown that the rat MHC,RT1 is divided into two regions:RT1.A, which codes for class I (transplantation) antigens, andRT1.B, which controls the humoral immune response and proliferative response to allogeneic cells as well as the expression of class II (Ia) antigens. Serological and sequence studies suggest that there might be more than one antigen-coding locus within theRT1.A region. Results obtained by sequential immunoprecipitation reveal that both regions code for at least two gene products. By implication, theRT1 complex must therefore harbor at least four loci;RT1.A andD coding for class I glycoproteins (45,000 daltons); andRT1.B andE coding for class II (Ia) glycoproteins (35,000 and 28,000 daltons).

A radioimmunoassay for antibodies against surface membrane antigens using adhering cells

A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit 125I-anti-mouse-Fab. The bound radioactive material was eluted with glycine-HCI buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolytic or noncyctolytic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface.

Isolation of transplantation antigens from a leukemic cell grown in vitro.

Attempts to characterize the mouse transplantation antigens have been greatly hampered by the problem of obtaining large amounts of a homogeneous starting material and the difficulties of quantitation in the testing methods. An ideal starting material would be a mouse cell line that could be grown in tissue culture in large quantity relatively easily. One such cell line is the murine lymphoblast, L-5178Y, that was successfully cultured in vitro by Fischer (1957). This line originated many years ago in a DBA/2 mouse after 20 paintings with methylcholanthrene (Law, 1961). It was derived from a generalized leukemia and subsequently converted to an ascitic form. Unlike other cell lines grown in tissue culture, L-5178Y does not adhere to glass, and the cells can therefore be kept in suspension by gentle agitation of the culture flasks. A number of tests should be used for the characterization of a transplantation antigen, since there is no clear agreement by workers in the field on a single test. (1) Ability of a sensitized animal to reject a body skin homograft at an accelerated rate. (2) Ability of a sensitized animal to reject a tail skin homograft on the tail at an accelerated rate. (3) Ability of a sensitized animal to reject a tumor homograft. A preliminary report (Manson et al., 1960) from this laboratory indicated that extracts of L-5178Y cells could be obtained that would, upon injection into allogenic strains of mice, elicit an immunity to subsequent transplants of skin or of L-5178Y tumor tissue, or of both. This paper amplifies and extends our original report of the effectiveness of various cell fractions of the L-5178Y cell line for the immunization of mice.

the lymphocytosis stimulating factor (L.S.F.).

Summary Attempts have been made to confirm the findings of Metcalf with respect to the lymphocytosis stimulating factor. Extracts of a lymphoblast grown in vitro behaved in a similar manner to thymic extracts in the tests used. The test method is subject to fluctuations which yield conflicting data.

Effect of Deuterium Oxide on Growth of HeLa, L and L-5178Y Cells.∗†

Summary The effect of D2O on growth of 3 stable mammalian cell lines (HeLa, L and L-5178Y) has been investigated. As D2O concentration was increased all cells showed increased water content and dry weight and a slower growth rate. Cytological investigations showed an increase in number of multinucleated cells and a moderate increase in sudanophilic material. No significant changes in nucleic acid content were demonstrable with the staining technics used.

Metabolism of Arginine by Mouse Fibroblast, Strain L.

Summary 1. For sustained growth of mouse fibroblast cells (strain L) in suspended culture in Eagles medium, periodic addition of arginine is essential. This requirement can be provided by arginine alone or by equimolar amounts of citrulline and aspartic acid. 2. These cells contain enzymes which catalyze the breakdown of arginine to ornithine and 2 equivalents of ammonia. 3. The requirement for periodic addition of arginine or its precursors is explained by continuous rapid breakdown of arginine observed during growth.

INDUCTION OF THE IMMUNE RESPONSE TO CELL SURFACE ANTIGENS IN VITRO

SummaryTwo tissue culture incubation systems are described in which immune responses to cell surface antigens have been demonstrated In the one-way “mixed lymphocyte interaction” system, a specific stimulation of thymidine uptake was induced by a particulate membrane antigen fraction, the microsomal lipoproteins (MLP)when low levels (0.01 to 0.001 μg per ml) were incubated with spleen or lymph node cells from nonsensitized mice. No stimulation was seen when allogeneic MLP was used at high levels, 10 μg per ml, nor at any level with syngeneic MLP. Specific effectors were demonstrated after 72-hr incubation with stimulatory levels of allogeneic MLP in three separate in vitro assays, a plaque-forming cell reduction assay, a tumor target assay, and an antigen-binding cell assay. In the latter assay, [125I]MLP was used as the source of antigen. This system has limited potential inasmuch as mouse spleen cells do not survive in it beyond the 4th day of culture. The second tissue culture system, the Marbrook system, has much greater possibilities because at least 25% of the inoculum is recovered 7days later. In this culture system a cell-free sheep erythrocyte membrane preparation can induce, plaque-forming cells in the absence of macrophages. Using a sensitive radioimmunoassay, frees specific antibody was detected in culture supernatant fluids. With the same culture system, allogeneic lymphocytotoxic cells (killer) have been induced with spleen cells from unprimed mice in strains differing at the major histocompatibility locus (H-2). Allogeneic MLP induced very significant “killer” cell activity with spleen cells from primed mice. In a syngeneic tumor systems, significant amounts of killer cell activity were induced with unprimed spleen cell inocula, and much larger amounts induced with spleen cells from immunized mice.

Intracellular Localization and Immunogenic Capacities of Phenotypic Products of Mouse Histocompatibility Genes

The transplantation antigens are the phenotypic products of genes which control histocompatibility in vertebrate species. The products of major histocompatibility locus of the mouse, H-2, have been studied as a model. The H-2 transplantation antigens are expressed on cellular membranes in all tissues examined. These gene products have been isolated from cells associated with subcellular membranes. These membranes have been assayed both for their antigen content (antigenicity) and for their capacities to induce a primary humoral and a cell-mediated response (immunogenicity). In all tissues examined, the H-2 antigens (products of the K and D regions of H-2) were found expressed in high concentration on cell surface membrane. However, immunogenic activity was observed only with spleen and thymus preparations, consisting mainly of intracellular membranes (MLP). Immunogenic MLP was also isolated from lymphoblast and fibroblast cells, and again was derived mainly from endoplasmic reticulum. In other tissues, such as liver, kidney, and erythrocytes, H-2 antigens were found only on surface membrane and in an antigenic but nonimmunogenic form. A novel method for tagging surface membrane of mammalian cells is presented. It consists of binding, to whole cells in a covalent linkage, purified preparations of the beta-galactosidase of E. coli. The bound enzyme has proved to be an unambiguous marker for surface membrane. With this marker, the stability of surface membrane to shear forces during homogenization could be assessed. A number of considerations suggest that immunogenicity of transplantation antigens may be due to factor(s) present on the membranes in addition to the H-2 antigenic determinants. There are indications that these factors may be controlled by the I region of the H-2 complex. It is interesting to note that normal tissues which have Ia antigens on their surface membranes yield immunogenic MLP (spleen and thymus), whereas those without Ia surface antigens yield an antigenic MLP that has no immunogenic capacity (liver, kidney, and erythrocytes).