DNA Synthesis in Mixed Cultures of Dog Leukocytes: Differential Effect of X-Radiation and Freeze-Thawing on Cellular Isoantigenicity

Summary A marked induction of DNA synthesis occurred in 50:50 mixed cultures of peripheral blood leukocytes derived from sets of allogeneic (Beagle and mongrel) normal dog donors. DNA-synthetic activity at selected times was determined by liquid scintillation counting following pulse labeling of cell populations with 3 H-thymidine. In a typical experiment, DNA synthesis in mixed cultures attained a maximum value at the 6th day (61,000 cpm) followed by a steady decline thereafter (to 7500 cpm by day 9), as contrasted with control values (65 to 86 cpm). In leukocyte cultures of cells isolated and mixed on the same day, considerable DNA labeling (1900 to 3000 cpm) occurred at time 0. These values declined, (600 cpm at 18 hr; 230 cpm at day 2), and then rose later (2400 cpm at day 4). Incubation of individual cell suspensions for 18 hr prior to mixing eliminated the elevated zero time label. X-irradiation (2500 rads) of one (or the other) of the cell components of the mixture changed the reaction from a “two way” to a “one way” immunologic reaction. When one (or the other) of a pair was frozen (-196°C) and thawed (37°C) three times and momentarily homogenized before mixing with intact allogeneic leukocytes, no induction of DNA synthesis occurred. These results suggest that leukocyte isoantigenic factors are not inactivated or destroyed by x-irradiation and can induce DNA synthesis in allogeneic leukocytes; whereas, they are labile to freeze-thawing. Our results are compatible with the known chemical and immunologic properties of transplantation antigens.