Lymphocyte populations primed in mixed lymphocyte cultures have been used to determine if the allogeneic effect is mediated by alloreactive T lymphocytes. A microculture system with excess B cells, sheep erythrocytes, and limiting numbers of allogeneic T cells was used to compare the frequencies of allogeneic “helper” cells in primed and unprimed populations. A positive allogeneic effect, i.e., the production of anti-sheep erythrocyte antibodies, could be detected when low numbers of primed T cells were added to the cultures. After priming to the H-2 antigens of the B cells used in the cultures, the frequency of allogeneic helper cells was increased at least 10-fold to approximately one in every 30 cells. Priming to different H-2 antigens did not produce this effect. Therefore, the allogeneic effect can clearly be mediated by cells that specifically recognized and respond to alloantigens.

As the number of primed T cells increased, the number of responding microcultures decreased. Lymphocytes mediating the suppressive effect were clearly different from those mediating positive effects with respect to their susceptibility to irradiation and mitomycin-C treatment. The frequency of the set of cells mediating the negative effect was estimated, but varied from experiment to experiment. That the helper activity could be detected only under conditions of limiting T cells stresses the importance of limiting dilution analysis in the functional assessment of mixed populations of cells.

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This work was supported in part by United States Public Health Service Grants CA 14049 and GM10356.

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