Immune Cytolysis of Mouse Thymic Lymphocytes

Abstract

Immune cytolysis was carried out by mixing a suspension of mouse thymic lymphocytes containing 97 to 99% viable cells with complement and various dilutions of rabbit antiserum or isoantiserum. Cellular integrity was determined after incubation for 2 hr at 37° C, by use of vital staining. An end point of 50% cell deformation was employed. As source of complement, hamster serum was almost nontoxic to mouse thymic lymphocytes. Two absorptions with mouse red cells and three absorptions with mouse liver removed the slight toxicity and any tendency for clumping. Guinea pig serum similarly absorbed was still slightly toxic. The count of deformed cells in the assay tubes containing antiserum changed relatively little between 1 and 3 hr after the start of incubation. This could be attributed to a balance between the rate of increase in the number of stained cells and their rate of disappearance due to complete lysis. Optimal conditions for assay of complement were determined. Complement titers determined with cells sensitized with varying concentrations of isoantiserum were relatively constant; in contrast, the complement requirement decreased with increasing concentrations of rabbit antiserum. This complement-sparing action of rabbit antiserum was only partially correlated with its complement-independent cytotoxicity. Optimal conditions for assay of cytolysin were determined. Direct proportionality between cytolytic titer and cell concentration was obtained over a range from approximately 5 to 20 million cells/ml. There was progressive loss of proportionality at lower cell concentrations. An unabsorbed rabbit antiserum to AKR thymic lymphocytes gave no evidence of specificity in its cytolysis of thymic lymphocytes taken from four different strains of inbred mice. The two isoantisera that were tested showed potent cytolysis of AKR thymic lymphocytes.