Bovine costimulator. I. Production kinetics, partial purification, and quantification in serum-free Iscove's medium

Bovine peripheral blood leukocytes were examined for blast transformation in response to T-cell lectins in serum-containing RPMI 1640 medium and serum-free Iscove's medium. Phytohemagglutinin-induced blastogenesis was significantly greater in Iscove's medium than in RPMI containing ten percent fetal calf serum. Concanavalin A-induced blast transformation was equivalent in both media. However, the kinetics of lectin response and the quantity of lectin required for optimum blastogenesis was considerably different in the two culture media. Concanavalin A-induced blast transformation of bovine thymocytes in Iscove's medium revealed that a concentration of 10(6) cells/ml, inconsequential blastogenesis ensued; but at 10(7) cells/ml blast transformation was significant and dose-dependent. Therefore, conditioned media from concanavalin A-stimulated bovine peripheral blood leukocytes, prepared in serum-free Iscove's medium, were assayed for costimulator activity using bovine thymocytes at 10(6) cells/ml in Iscove's medium as indicator cells. Both optimum lectin requirements and cell concentrations for production of costimulator activity were found. Conditioned medium, operated with the total exclusion of serum and with optimal costimulator activity, was fractionated via gel exclusion chromatography. A quantitative assay was described, and results indicated that bovine costimulator had an approximate molecular weight of 20,000 daltons.