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Increased Cytolytic T Lymphocyte Activity Induced by 2-Mercaptoethanol in Mixed Leukocyte Cultures: Kinetics and Possible Mechanisms of Action¹

From the Department of Immunology, Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland

Abstract

The 20- to 50-fold increase in cytolytic T lymphocyte (CTL) activity caused by the addition of 50 µM 2-mercaptoethanol (2-ME) at the onset of a one-way murine mixed leukocyte culture (MLC) between C57BL/6 and DBA/2 splenic lymphocytes appears to be unrelated to early events in the culture: if 2-ME was present for the first 24 hr of culture only, there was no increase on day 4, but if addition of 2-ME was delayed until the last 24 hr of culture, the CTL activity was almost as high as that of cultures that were exposed to 2-ME for the entire 4-day culture period.

The increase of CTL activity caused by delayed addition of 2-ME ("2-ME rescue") was used to investigate the mechanism by which the thiol induces differentiation of CTL from precursor cells. 2-ME rescue was mimicked by two other thiols, dithiothreitol and cysteamine phosphate, but at higher concentrations. Because the latter compound has no free sulfhydryl group until it diffuses into cells and is enzymatically dephosphorylated, we conclude that thiols may increase the differentiation of CTL from precursor cells by an intracellular process involving free sulfhydryl groups rather than by interaction with membrane sulfhydryls or destruction of inhibitor cells or their products.

Cell separation experiments indicated that 2-ME rescue was independent of the presence of B lymphocytes and of adherent cells (macrophages) and was restricted to a subpopulation of T lymphocytes that developed into large lymphoid precursor cells during the first 3 days in culture even without 2-ME. The development of this subpopulation required DNA synthesis between 24 and 72 hr after the onset of MLC. When 2-ME was added to day -3 MLC, CTL activity increased slightly as early as 4 hr later, but the major increase occurred during the second half of the 24 hr "rescue" period. Because this increase was inhibited by cytosine arabinoside (ARA-C), it seems likely that DNA synthesis is associated with and may be required for the differentiation of large precursor lymphoid cells into CTL after the addition of 2-ME.

Footnotes

¹ This work was supported by grants from the Swiss National Foundation and by the United States Energy Research and Development Administration (JWH).

² Present address: Laboratory of Radiobiology, University of California, San Francisco, California 94143.

³ Present address: Ontario Cancer Foundation, London, Ontario, Canada N6A 4G5.

⁴ Present address: Department of Pathology, University of Chicago, Chicago, Illinois 60637. FWF is a fellow of the John Simon Guggenheim Memorial Foundation.