Shinpei Kasakura

A factor stimulating DNA synthesis derived from the medium of leukocyte cultures.

BAIN et al.1,2 have shown that when human peripheral blood leucocytes from two individuals are mixed and cultured, large immature cells appear and the incorporation of tritiated thymidine into DNA is stimulated. It has been suggested that variations in this response may reflect differences in the number of histocompatibility antigens shared by the donors of the cells1–3. It has also been shown that transplantation antigens can be detected in the medium from tissue cultures of rabbit4 and dog5 spleen cells. We, therefore, decided to determine whether factors released into the medium from cultured peripheral blood leucocytes might stimulate tritiated thymidine uptake (radioactive content) in cultures of leucocytes from another individual.

THE EFFECT OF UREMIC BLOOD ON MIXED LEUKOCYTE REACTIONS AND ON CULTURES OF LEUKOCYTES WITH PHYTOHEMAGGLUTININ

The effect of uremia on mixed leukocyte reactions and on leukocyte cultures with phytohemagglutinin (PHA) was investigated. Generally, the reactivity of lymphocytes from uremic patients to X-irradiated allogoneic leukocytes in mixed cultures was significantly lower than that of normal lymphocytes. Uremic plasma usually depressed the mixed leukocyte reaction of normal lymphocytes. Uremic lymphocytes reacted normally to PHA. Uremic plasma did not depress the reactivity of normal lymphocytes to PHA. Possible explanations for the depressed reactivity of uremic lymphocytes to allogeneic antigens are discussed. When the mixed leukocyte reaction test is applied to kidney homotransplantation, one should consider the effect of uremia on this test.

A blastogenic factor in unidirectional mixed cultures with x-irradiated cells.

SUMMARY Cell-free medium (CFM) from cultures of unmixed or mixed irradiated human leukocytes stimulated 3H-thymidine uptake by fresh allogeneic leukocytes. At least some component of the blastogenic factor was antigenically specific. The blastogenic factor from cultures of irradiated leukocyte mixtures was much more active than that from cultures of unmixed irradiated leukocytes. The activity of the blastogenic factor was affected by the dose of irradiation and the time interval between the end of irradiation and the preparation of cultures. The blastogenic factor from mixed cultures of irradiated and intact cells was consistently less stimulatory to fresh leukocytes from the donor of the irradiated cells in the original mixture (source of CFM) than it was to fresh leukocytes from the original donor of the intact cells. These data raise the possibility that production of a blastogenic factor may be involved in the stimulation of responding lymphocytes in mixed cultures.

Irradiated and Preserved Leukocytes in Mixed Leukocyte Cultures.

Summary and conclusions When leukocytes received 1,5-00 r or more in vitro, they were rendered incapable of DNA synthesis and mitosis in mixed leukocyte cultures, but did not lose their ability to stimulate the blastogenesis of allogeneic intact leukocytes. The sum of the “one-way” (Ax + B) and (A -f- Bx) reactions was approximately equal to the “two-way” (A + B) reaction. Therefore, irradiated leukocytes can be used for unidirectional quantitation of the mixed leukocyte reaction. When leukocytes were irradiated with less than 6,000 r and cultured with PHA, some transformation to blasts occurred. Leukocytes stored at —70°C for 2 weeks retained their capacity for blast transformation when cultured in the presence of X-irradiated allogeneic cells. Frozen storage of irradiated leukocytes reduced their blas-togenic effect variably up to 30% of that of fresh controls.

Nature of the blastogenic material in the medium from peripheral leukocyte cultures.

Some of the physical and chemical properties of blastogenic material contained in cell-free culture medium of 5-day mixed leukocyte cultures have been described. These properties seem similar in many respects to those of dog and mouse transplantation antigens which have been described by other workers. Characterization and purification of the blastogenesis factor may lead to increased understanding of transplantation immunity. In view of the relatively stable qualities of this factor, it may be possible to concentrate it in sufficient quantity to have practical application in tissue matching.