LIMITING DILUTION ANALYSIS OF ALLOANTIGEN-REACTIVE LYMPHOCYTES-T .3. EFFECT OF PRIMING ON PRECURSOR FREQUENCIES

Abstract

The effect of specific priming with alloantigens on the frequency of cytolytic T [thymus-derived] lymphocyte precursors (CTL-P) was investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14 day unidirectional mixed leukocyte cultures [MLC] (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3- to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25- to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5-20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood or thymus cells were cultured for 14 days in MLC. Direct evidence that the pool of specific CTL-P can be expanded after alloimmunization was provided. The very high frequencies observed after in vitro priming indicated that this system should be particularly useful for future studies of the progeny of individual CTL-P.