Potentiation of Cultured Mouse Thymocyte Responses by Factors Released by Peripheral Leucocytes

Abstract

Lymphoid cell interactions in the immune response, both in vivo and in vitro, have been the subject of much interest recently (1, 2). In this regard, substances have been found in supernatants of cell cultures which can affect the response of other lymphoid cells (3, 4). The following report describes a new effect wherein mouse thymocytes are highly stimulated by a combination of nonspecific stimulants and substances released by human leucocytes.Five × 106 thymus or spleen cells from male CBA/H or CBA/J mice, aged 6 to 12 weeks, were cultured in duplicate in plastic tubes (Falcon #2001) in 1.0 ml MEM-S (Microbiological Associates, Bethesda, Md., with added antibiotics and l-glutamine), supplemented with 8% normal human serum. These cultures were stimulated by the following, added singly or in combination: a) 1.0 µl phytohemagglutinin-P (PHA, Difco Laboratories, Detroit, Mich.); b) different concentrations of human white blood cells (obtained by gravitational sedimentation of the erythrocytes); c) supernatants (SUP) from cultures of human leucocytes. cultures of human leucocytes. The latter were prepared by incubating 1.5 × 106 human leucocytes in 2.0 ml of the above medium with or without stimulants for 24 hr, then removing the cells centrifugally and storing at — 20°C. These human cultures were stimulated by 1.0 µl PHA or 100 µg endotoxin (LPS, Difco, lipopolysaccharide B, Escherichia coli 0111 :B4). The supernatants at various dilutions were added in a volume of 0.5 ml to the mouse cells in the same volume. Treatment with mitomycin C (MIT, Nutritional Biochemicals Corp., Cleveland, Ohio) was carried out by incubating mouse thymocytes (40 × 106/ml) or human leucocytes (15 × 106/ml) with the drug (final concentration 100 µg/ml) for 30 min at 37°C. The cells were then spun down and washed twice with the medium. All cultures were incubated for 72 hr at 37°C in 5% CO2 in air. A pulse of tritiated thymidine (3HT, Schwarz BioResearch, Orangeburg, N.Y. 0.5 or 1.0 µCi) was given during the last 24 hr of incubation. Incorporation of 3HT was determined by washing the cells twice with saline, followed by precipitation with trichloroacetic acid. The precipitates were dissolved in 0.4 ml formic acid and 3.0 ml ethanol and counted with 10 ml scintillation fluid (PPO, 0.5% and POPOP, 0.025% in toluene) in a Nuclear Chicago (Des Plaines, Ill.) Mark I scintillation counter. Counts in duplicate cultures regularly differed from the mean value by less than 15%, except with counts below 1000. The data recorded below are from representative experiments; the results were found to be reproducible in repeated tests.