Studies were performed to characterize the effector cells responsible for natural cytotoxicity of mouse lymphoid cells against a variety of syngeneic and allogeneic tumor lines. Since spleen cells from normal nude mice were found to be highly cytotoxic, they were used for most of these experiments. Only a small proportion of the reactivity was affected by treatment with anti-theta serum plus complement. Macrophages dis not appear to be responsible for the reactivity, since treatment with carbonyl iron/magnet or carrageenan did not affect the levels of cytotoxicity. The effector cells were non-adherent, since passage over nylon columns resulted in a considerable increase in activity. The active cells did not have receptors for immunoglobulin or complement, since removal of cells with these receptors by columns or monolayers containing sheep erythrocyte-antibody (EA) complexes or EA-complement complexes did not remove activity. Antibody-dependent cell-mediated cytotoxicity appeared to be ruled out as the mechanism for natural cytotoxicity, since aggregated gamma globulin and a potent anti-immunoglobulin reagent did not inhibit reactivity, and since no role for humoral factors could be demonstrated. The natural effector cell was found to be quite labile at 37 degrees C, losing much of its activity after 4 h. Since no surface markers could be detected on the effector cells, and the mechanism for cytotoxicity appeared distince from others previously described, it is proposed that the natural cytotoxicity against mouse tumor cells is mediated by a unique subpopulation of lymphoid cells, which are tentatively designated N-cells.

Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells

Recently there has been increasing recognition of natural cell-mediated cytotoxicity as a potentially important antitumor effector mechanism in addition to that of specifically immune T cells and of activated macrophages. Although natural cellular cytotoxicity was first recognized only a few years ago (Herberman et al. 1973,1974, McCoy et al. 1973b, Oldham et al. 1973, Rosenberg et al. 1972,1974), there has already been extensive research in many laboratories on the nature of the effector cells, the possible mechanisms of cytotoxicity, the factors regulating the levels of reactivity, and the relevance of natural immunity to in vivo resistance against tumor growth and immune surveillance. A principal component of natural cell-mediated cytotoxicity in rodents and man has been found to be a particular subpopulation of lymphocytes which have been termed natural killer (NK) cells. We have recently reviewed in detail much of the available information on NK cells (Herberman & Holden 1978). In this paper we will only summarize our views on the characteristics of NK cells and focus on a few issues of current interest in our laboratory.

Natural Killer Cells: Characteristics and Regulation of Activity

Glomerulopathies of neoplasia.

Publisher Summary This chapter discusses lymphocyte receptors for immunoglobulin (Ig) The methods used to detect lymphocyte receptors for Ig vary widely both as the type of complex utilized and the technique for measuring binding The chapter describes various techniques available The nature of the lymphocytes that bind complexed Ig are considered with reference to the sensitivity and/or specificity of the various methods A detailed consideration of lymphoid cell subpopulations, including the criteria for defining B lymphocytes, T lymphocytes, and undefined lymphocyte-like (UL) cells, is presented in the chapter Any useful Fc receptor assay system should meet certain basic criteria: (1) the percentage of lymphocytes binding Ig should be titratable to a plateau value This assumes a discontinuous distribution of receptors on different subpopulations (2) The Fc dependence of binding should be demonstrated Because the binding of Ig is dependent on the Fc portion of the molecule, this will help ensure that different assays are detecting similar receptors (3) Cells, other than lymphocytes, that bear Fc receptors should be either identified and excluded from counts or removed from the population being tested Such cells include monocytes, macrophages, neutrophils, and basophils

Lymphocyte Receptors for Immunoglobulin

To investigate the nature of non-endemic Burkitt's lymphoma, we examined neoplastic cells from eight American patients for receptors for sheep erythrocytes (E), complement (EAC), and Fc fragment of lgG (igGEA), and for surface immunoglobulins (Slg) and hydrolytic enzymes. In addition, we reviewed 47 biopsies and 17 autopsies from American patients to ascertain patterns of involvement by tumor in lymph nodes, spleens and Peyer's patches. Neoplastic cells in all cases studies bore monoclonal surface immunoglobulins of the igM class. Receptors for EAC and igGEA were identified on a minority of the cells. Little or no hydrolytic enzyme activity was demonstrable. These results indicate that, like Burkitt's lymphoma in Africans, this histologically identical tumor in American patients consists of B lymphocytes. In 10 biopsies and two autopsies, germinal centers were selectively involved by tumor, suggesting that these neoplastic cells may be related to some B lymphocytes of normal germinal centers.

Non-endemic Burkitts's lymphoma. A B-cell tumor related to germinal centers.

A simplified and efficient technique for separating T lymphoid cells from non-T cells of mouse, rat and man has been developed. The fractionation procedure is based on the capacity of non-T lymphoid cells to bind antigen-antibody complexes through an Fc receptor. Lymphoid cells are adsorbed on antibody-coated sheep erythrocyte (EA) monolayers prepared in polystyrene Petri dishes previously treated with poly-l-lysine. Adsorption of spleen cells on these monolayers, assisted by a 5-min centrifugation, resulted in 90 to 98% depletion of the Fc receptor-bearing cells (mostly B lymphocytes, but also macrophages and polymorphonuclear leukocytes) in the non-adherent cell population. Eighty to 90% of the adherent cells were capable of forming EA rosettes and could be recovered after lysis of the erythrocytes. The non-adherent fraction comprised at least 90% T-cells. This was determined for mouse and rat spleen cells by lysis with anti-thymocyte serum and complement, and by the absence of immunoglobulin-bearing cells. The specificity of the adsorption was further substantiated by the following observations: 1) lymphoid cells lacking an Fc receptor were not adsorbed; 2) the adsorption to EA monolayers was inhibited by antigen-antibody complexes or by aggregated γ-globulin; and 3) only 10 to 15% of the Fc receptor-bearing cells were attached to non-sensitized erythrocyte (E) monolayers. Fractionation of spleen cells obtained from rats immunized against a syngeneic tumor yielded two separate fractions of cytotoxic lymphoid cells: one (non-adherent, T cells) mediating direct target cell destruction, and the other (adherent, non-T cells) mediating lysis of antibody-coated target cells.

Cellular Immunoadsorbents: a Simplified Technique for Separation of Lymphoid Cell Populations

The capability of rat lymphoid cells to destroy antibody-coated target cells (antibody-dependent cell cytotoxicity, ADCC) and to form rosettes with immunoglobulin-coated erythrocytes (EA) was compared. Because both processes require binding of a lymphoid cell to antibody associated with cellular antigen, we undertook to determine if both assays detect the same cell population. Peripheral blood lymphocytes, spleen and lymph node cells had both activities, but there was no correlation between the number of rosettes and the ADCC activity. Blood lymphocytes had the highest cytotoxic potential, while spleen cells produced the highest percentage of rosettes. Bone marrow cells were devoid of cytotoxic activity though many of the cells were able to form rosettes. Thymus cells were neither cytotoxic nor able to form rosettes. Following treatment of spleen cells with 1) anti-rat Ig serum, 2) high concentrations (τ1 mg/ml) of trypsin and Pronase (but not papain), or phospholipase-C, the number of rosettes was strongly diminished, whereas the cytotoxic activity was unimpaired. The ADCC activity was not altered after removal of 60% of the rosetteforming cells by nylon column, but was greatly reduced when 85% of the rosette-forming cells were removed by anti-SRBC serum. These observations indicate that: 1) there is no correlation between the percentage of EA rosette-forming cells and the ADCC activity in various lymphoid tissues, 2) these activities were not equally susceptible to enzymes9 action, 3) the cytotoxic activity is mediated by a subpopulation of the EA rosette-forming cells, and 4) probably fewer immunoglobulin receptor sites per unit surface membrane are required for ADCC cytotoxicity than for rosette formation.

Kedar E

Papers

Cellular Immunoadsorbents: a Simplified Technique for Separation of Lymphoid Cell Populations

Comparative Studies of Immunoglobulin Receptors and Antibody-Dependent Cell Cytotoxicity (ADCC) in Rat Lymphoid Organs

Antibody-dependent cellular cytotoxicity to a syngeneic Gross virus-induced lymphoma.