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Limiting Dilution Analysis of Alloantigen-Reactive T Lymphocytes

III. Effect of Priming on Precursor Frequencies¹

From the Department of Immunology, Swiss Institute for Experimental Cancer Research and Unit of Human Cancer Immunology, Ludwig Institute for Cancer Research, Epalinges s/ Lausanne, Switzerland

Abstract

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2^b) mice primed with DBA/2 (H-2^d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2^d 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 to 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 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.

Footnotes

¹ This work was supported in part by a grant from the Swiss National Foundation for Scientific Research.

² Present address: Department of Pathology, University of Geneva, School of Medicine, Geneva, Switzerland.

³ Correspondence: Dr H. R. MacDonald, Unit of Human Cancer Immunology, Ludwig Institute for Cancer Research, 1066 Epalinges s/Lausanne, Switzerland.

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