PREPARATION AND USE OF BLASTOGENIC MATERIAL FOR UNIDIRECTIONAL TISSUE MATCHING.

摘要

Sucrose density gradient centrifugation of cell-free medium (CFM) from mixed human leukocytes cultures resulted in the isolation of a 19S blastogenic fraction (I) containing some 4% of the CFM dry weight. Chromatography of fraction I on DEAE-Sephadex A-50showed 3well-resolved peaks. Factors affecting the strength of the unidirectional stimulation were investigated in order to improve the reliability of techniques for unidirectional quantitation in the mixed leukocyte reactions. Also,two methods of unidirectional stimulation,using 'X-irradiated cells'and 'Mitomycin C treated cells'were compared. Two normal,unrelated female donors' (W. and C.) leukocytes regularly produced only minimal blastogenesis when mixed together in vitro. This was demonstrated both with one-way stimulation (using X-irradiated leukocytes) and with two-way stimulation. Both of these donors'leukocytes responded normally to X-irradiated leukocytes from three other normal subjects. The role of various cell types in the mixed leukocyte reaction was investigated. The results indicated that cultures containing a high percentage of lymphocytes (77-93%) produced only very weak blastogenesis in mixed cultures compared with whole,untreated leukocyte suspensions: (39-52% lymphocytes). Macrophages,obtained by culturing whole leukocyte suspensions in vitro in flat-sided flint glass bottles,regularly showed blastogenic activity when added to cultures of allogeneic lymphocytes. The reproducibility of results of mixed leukocyte reactions with given donor pairs was also investigated. No consistent pattern was observed. There are in vitro and/or in vivo variables which are still not understood. (Author)