Anders Örn

A rapid method for the separation of functional lymphoid cell populations of human and animal origin on PVP-silica (Percoll) density gradients.

Subclasses of lymphocytes can be separated on gradients of non‐toxic polyvinylpyrrolidone‐coated colloidal silica (Percoll) by virtue of differential densities. Such gradients can yield functionally active lymphocyte populations after brief centrifugation. Gradients can he generated in a discontinuous step fashion and centrifuged in standard table‐top laboratory centrifuges or as self‐generating gradients duriny ultracentrifugation. The density medium has low viscosity and can be made isotonic for virtually any use. Gradients have proved useful in both human and experimental animal studies, and high percentage yields allow for separations from small cell numbers. Methods are described for separation of whole blood and lymphotd subpopulations. including blasts stimulated with mitogens or in mixed lymphocyte reactions. The cytotoxic capability of various density fractions was evaluated for mixed lymphocyte culture‐induced allogeneic killing and spontaneous, so‐called ‘natural’ killer cell activity. The lower density associated with blast transformation allows for significant enrichments of stimulated cells from in vitro cultures. Higher thymidine incorporation, restimulation in mixed lymphocyte reactions, and greater cytotoxic capacity are associated with these‘blast’fractions.

Enhancement by interferon of natural killer cell activity in mice.

Abstract Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for 51 chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10 3 to 10 4 units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic ( nu/nu ) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro . Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.

Study of the Mechanism for in Vitro Activation of Mouse NK Cells by Interferon

The enhancement of the lytic capacity of mouse splenic ‘natural killer’ (NK) cells by interferon has been studied in vitro as a model for NK cell differentiation from inactive immediate precursors. We show that the increased cytotoxicity is a function of interferon concentration, and that two stages in the NK cell differentiation pathway can be distinguished. The first, very brief, can he performed at 0°C and without protein synthesis and probably corresponds to the fixation of interferon on its cell surface receptors. The second, resulting from the inductive signal given by interferon, proceeds for several hours and requires both RNA and protein synthesis. Our results also indicate that target cells for interferon‐induced cytotoxicity are cells for interferon‐induced cytotoxicity arc cells with ‘null’ characteristics, similar to the mature NK cells. Finally, they suggest that no soluble Intermediary factor other than interferon is involved in the enhancing of NK cell cytotoxicity.

Pigment mutations in the mouse which also affect lysosomal functions lead to suppressed natural killer cell activity.

The impact of five pigment mutations in the mouse on natural killer (NK) activity was examined in inbred strains congenic for the respective mutation. Whereas the nature of pigmentation disorder was similar in the five mutant strains (beige, pallid, reduced pigmentation, pale ear, and sepia), all mutations except sepia also led to a significant change in lysosomal enzyme activities in the kidney. A significant reduction in NK activity was observed in the four strains with lysosomal impact, whereas homozygous sepia mice displayed normal NK activity. The pigment mutations analysed are located on different chromosomes and fail to cross‐interact negatively with each other in the heterozygous mice. This would indicate that pigment mutations with a parallel impact on lysosomal enzyme activities probably always result in a reduction in natural killer cell activity.

Severe Suppression of the B‐cell System has No Impact on the Maturation of Natural Killer Cells in Mice

Mice were treated with a heterologous anti‐IgM serum to obtain B‐cell‐deprived mice. Spleen cells from normal and B‐cell‐deprived mice were tested in three different cytolytic systems: natural killer cells (NK); antibody‐dependent cell‐mediated cytolysis (ADCC) against an NIC‐sensitive tumour, P815; and ADCC against chicken erythrocytes. The impact of administration of an interferon‐inducing NK enhancing agent, Tilorone, was also investigated. Whereas the cell population from B‐cell‐deprived mice was significantly suppressed in antibody‐producing cells, the capacity to function in NK or ADCC was largely unimpaired both before and after administration of Tilorone. Our results would imply that mature B cells play no significant role in either the maturation of the NK cells or the expression of their cytolytic ability. Furthermore, effector cells for both NK and ADCC against antibody‐coated tumour target cells were found to be distinct from those functioning in ADCC against chicken erythrocytes.