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This report describes the in vitro inhibition of several classes of human lymphocyte-mediated lysis by antisera from mice and rats immunized with concanavalin A-stimulated human T-enriched lymphocytes. The inhibitory antibodies in these antisera recognized molecules on the surface of cytotoxic lymphocytes, and did not bind to B-cell lines. Experiments were performed to demonstrate that these antisera were not inhibiting cytolysis by trivial mechanisms such as (i) masking of target cell antigens by anti-target cell antibodies, (ii) agglutinating effector cells, thereby preventing effector-target cell contact, or (iii) toxicity to the effector cells. Dextran dispersion experiments revealed that these antisera inhibit the post-conjugate formation phase of cytolysis mediated by cytotoxic T lymphocytes. These antisera inhibited natural killer-like cytotoxicity and lectin-dependent cell-mediated cytotoxicity as well as lysis mediated by both mixed cell culture-generated cytotoxic T lymphocytes and cytotoxic T-lymphocyte cell lines. These findings suggest that these in vitro systems of cell-mediated lysis may share a common step; however the different classes of cytolysis may have been inhibited by separate antibodies in the antisera. This rapid reproducible method of generating inhibitory antisera will facilitate the study of the molecules involved in the post-conjugate formation phase of human cytotoxic T-lymphocyte-mediated lysis. © 1982.

High levels of lymphotoxin-like activity (LT) were found in supernatants from secondarily stimulated immune mouse splenocytes activated with concanavalin A (Con A) in vitro. Splenocytes obtained from C57Bl/6 mice immune to the P815 mastocytoma were restimulated in vitro with mitomycin C-treated P815 cells, and then stimulated with Con A. High levels of unstable LT activity are rapidly (2-4 hr) released by these lectin-stimulated splenocytes. The introduction of a crosslinking agent, glutaraldehyde, was found to stabilize this LT activity and allowed us to perform more defined biochemical studies and to examine the functional activities of the LT classes. The lytic activity in these supernatants resided in the high-molecular-weight classes, termed Complex (Cx > 200,000 daltons) and alpha-heavy (αH 130,000-160,000 daltons). It was found that the Cx and αH LT classes from the secondarily stimulated immune splenocytes cause lysis of allogeneic target cells, P815 and EL-4, in a 16-hr 75Semethionine release assay, and in some cases, this lysis was specific for the sensitizing target cell. © 1981.

The high-molecular-weight (MW) complex class (MW = greater than or equal to 200,000) of lymphotoxin (LT) may be involved in the process of lymphocyte-mediated cytolysis. This LT class was produced by concanavalin A-stimulated human adenoid and tonsil lymphocytes in vitro and purified approximately 1000 fold by a scheme employing lectin affinity chromatography on concanavalin A-Sepharose, negative hydrophobic affinity chromatography on phenyl-Sepharose, and molecular sieving on Ultrogel AcA 44. A cell-lytic subunit, termed C-alpha toxin, was dissociated from this partially purified complex LT (Cx) preparation. The identification of C-alpha toxin as an alpha 2 LT form was established by its comigration with the alpha 2 LT form during native polyacrylamide gel electrophoresis (PAGE), molecular sieving, and isoelectricfocusing. In addition, C-alpha toxin was neutralized by antiserum raised against purified alpha 2 LT, and the latter was neutralized by antiserum raised against C-alpha toxin. Furthermore, preliminary evidence indicates that both alpha 2 LT and C-alpha toxin are composed of peptides with apparent MW of approximately 69,000. Additional data indicate the Cx LT form is composed of alpha 2 LT and other subunits.