Lymphocyte activation. V. Quantitation of the proliferative responses to mitogens using defined T and B cell populations

Abstract

Although mitogens are frequently used for polyclonal lymphocyte stimulation in both experimental and clinical studies, biological tests of this kind encounter several technical difficulties. The relative importance of some of these were studied by using `calibration curves' for mitogenic stimulation. Thymocytes from cortisone-treated mice (T cells) and spleen cells from T cell-deprived mice (B spleen cells) were mixed in different known proportions and stimulated by Concanavalin A (Con-A), which is a T cell mitogen, or lipopolysaccharide (LPS), which is a mouse B cell mitogen.

We have confirmed earlier observations showing that the amount and specific activity of [³]thymidine have to be adjusted to the culture method used. A linear relationship between the responding cell number and incorporated thymidine was promoted when the extracellular thymidine concentration was constant during the labelling period (16 hr). In our system this could be achieved by adding sufficient amount of thymidine in the form of isotope of relatively low specific activity (50 mCi/mmol). In contrast, when isotope of very high specific activity was used, a low number of responsive cells gave almost `normal' incorporation values.

The validity of the use of mitogens was also tested with the T6 chromosome marker method and cell surface immunoglobulin analysis. It was found that the selective responses of T and B cells to T or B cell specific mitogens were maintained in cultures containing varying numbers of T and B cells. The only indication that B cell activating or `potentiating' factors are released by Con-A-stimulated T lymphocytes was observed in cultures with a high proportion (75%) of `T' cells.

Radio-autographic analysis revealed that under the culture conditions in which the majority of T cells from the spleen were stimulated by Con-A, only 20–30% of B cell population responded to LPS and pokeweed mitogen (PWM).