Activated T cells in human peripheral blood: quantitation with a reverse hemolytic plaque assay

Rabbit antiserum to culture supernatant of concanavalin A-stimulated peripheral blood mononuclear cells was used in a reverse hemolytic plaque assay to detect activated T lymphocytes in human peripheral blood. The number of plaque-forming cells in fresh, unstimulated lymphocyte preparations was approximately 400/10(6) cells, and increased 7- to 14-fold after stimulation with a variety of mitogens and antigens. The kinetics of the increase paralleled 3H-thymidine incorporation, with a maximum on day 3 of culture. Plaques were eliminated by treatment of cells with anti-Leu-I + complement or by depletion of E-rosettes. The activated T cells were not restricted to a given inducer, suppressor, or Ia+ T cell subset, however. Both mitogen-stimulated culture supernatants and mitogen-activated lymphocytes, but not resting lymphocytes, were effective in absorbing the capacity of the rabbit antiserum to develop plaques. This suggested that the predominant specificity of this antiserum was directed toward surface membrane determinant(s) of T cells actively shed into the medium. This process was shown to require ongoing protein synthesis, and, in mitogen-stimulated lymphocyte preparations, DNA synthesis as well. This approach has enabled detection of a surprisingly large number of endogenously activated T cells in normal human blood, and should be useful for the analysis of immunoregulatory events at the single-cell level.