Behaviour of human immunoregulatory cells in culture. I. Variables requiring consideration for clinical studies

The suppressor function of lymphocytes stimulated with concanavalin A (Con A) provides a potential method for examining disorders of immunoregulation. Clinical application, however, requires definition of the culture conditions that influence the expression of normal suppressor cell activity. In the present studies culture conditions were modified until a sensitive assay for non-specific suppressor cell function was reproducible utilizing the response to varying doses of phytohaemagglutinin (PHA) as an indicator system. Practical conclusions included (1) that sensitivity was not lost if the suppressor cells and responder cells were allogenic; (2) that fresh responder cells were as sensitive as precultured responder cells; (3) that a wide range of Con A concentrations could induce suppressor activity; and (4) that the sensitivity of the assay was much enhanced by using suboptimal mitogen doses of PHA. Twelve percent of normal subjects gave false negative results but these could be avoided by studying cells at more than one time point after stimulation with Con A. Cells resting in culture for 7 days could be induced to suppress after stimulation with Con A and these suppressor cells were very sensitive to pharmacological doses of dexamethasone. Studies utilizing different times of cell pre-incubation before Con A stimulation and different periods of exposure to Con A revealed fluctuation in the induction of suppression that may represent alternating periods of suppression and amplifying activity among stimulated cells in vitro. Such variations will need to be taken into account in the application of this type of assay to clinical studies seeking disordered immunoregulation.