GENETIC-REGULATION OF LYMPHOCYTE-T RESPONSIVENESS TO PHA IS INDEPENDENT OF CULTURE CONDITIONS

Abstract

A maximal interline separation was obtained after 10 consecutive generations of selective breeding for the character quantitative in vitro response of lymph node lymphocytes to the mitogenic effect of phytohemagglutinin [PHA]. At the selection limit the difference between high and low responder lines was about 20-fold. A similar interline separation was demonstrated for the T[thymus-derived]-mitogen effect of concanavalin A. The identical response to PPD (purified protein derivative of tuberculin), a B [bone marrow-derived] mitogen, proved that the genetic selection has only modified the potentialities of T lymphocytes. During selective breeding, responsiveness to PHA stimulation was always measured under identical culture conditions. To demonstrate that the interline difference in responsiveness was due essentially to genetic factors independent of environmental effects, a systematic study of various culture conditions was undertaken. The optimal stimulation was found after 2 days of culture for high line cells and after 3 days for low line cells. The difference between maximal responses was only slightly lower than that obtained after a 2-day culture as used for the selection test. Increase in cell concentrations produced higher thymidine incorporation. In the 2 lines, a linear correlation was established between the cell concentration and the response produced. The maximal response given by the highest number of low line lymphocytes was equivalent to that given by a number, 11-fold smaller, of high line cells. Within certain limits, changes in the amount of tritiated thymidine added to the culture did not affect the interline separation. With a thymidine of high specific activity, a sub-evaluation of uptake by high line cells decreased the interline difference. Results in mixed culture of lymph node cells from high and low lines indicated that the low response was not due to the release of inhibiting factors or to presence of suppressive cells in low responder mice. Separation of these 2 lines was due to genetic factors acting independently of the cell culture conditions.