Cytolytic T lymphocytes (CTL) and specific CTL precursor cells can be generated during the immune response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse. MSV-immune spleen cells yield high numbers of specific CTL after incubation in vitro with irradiated tumor cells in syngeneic secondary mixed leukocyte-tumor cell cultures (sec-MLTC). Peak cytolytic activity generated in sec-MLTC is detectable on day 7 after culture initiation and decreases slowly thereafter. This report presents data showing that highly active CTL could be regenerated upon tertiary stimulation of long-term sec-MLTC with irradiated tumor cells bearing MSV-associated antigens. CTL activity recovered from tertiary MLTC could be detected in a short-term (3 hr) 51Cr release assay, and increased levels of cytolysis were already evident 24 hr after culture re-initiation. It was also shown that cells recovered from MLTC 12 days after tertiary restimulation could be further stimulated by specific tumor antigen in quaternary MLTC to yield increased CTL numbers. These results thus indicated that T lymphocytes could be repeatedly stimulated by specific tumor antigen in syngeneic MLTC to yield increasing numbers of cytolytic cells. Furthermore, the tumor antigen used for the initial stimulation of MSV-immune spleen cells in sec-MLTC appeared to have induced a specific selection of CTL precursor cells, since only syngeneic tumor cells bearing MSV-associated antigens were capable of inducing high levels of CTL regeneration in tertiary MLTC.

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This work was supported by contracts and grants from the Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France, and the Swiss National Foundation for Scientific Research, Lausanne, Switzerland.

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