GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO: I. RESPONSE OF NORMAL AND IMMUNE MOUSE SPLEEN CELLS IN MIXED LEUKOCYTE CULTURES

doi:10.1084/jem.140.3.703

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GENERATION OF CYTOTOXIC T LYMPHOCYTES IN VITRO : I. RESPONSE OF NORMAL AND IMMUNE MOUSE SPLEEN CELLS IN MIXED LEUKOCYTE CULTURES

Jean-Charles Cerottini ¹, Howard D. Engers ¹, H. Robson MacDonald ¹, and K. Theodor Brunner ¹

¹ From the Department of Immunology Swiss Institute for Experimental Cancer Research, 1011 Lausanne, Switzerland

Mouse cytotoxic T lymphocytes (CTL) were generated in mixedleukocyte cultures (MLC) using spleen cells as responding cellsand irradiated allogeneic spleen cells as stimulating cells.Cytotoxicity was assessed by a quantitative ⁵¹Cr assay systemand the relative frequency of CTL in individual cell populationswas estimated from dose-response curves. Inclusion of 2-mercaptoethanolin the MLC medium resulted in a 20–40-fold increase inthe relative number of CTL generated at the peak of the response.Under these culture conditions, cell-mediated cytotoxic activitywas detectable in MLC populations as early as 48 h after theonset of the cultures.

When spleen cells from mice immunizedwith allogeneic tumor cells 2–4 mo previously were culturedwith irradiated spleen cells of the same alloantigenic specificity(MLC-Imm), it was found that the cell-mediated cytotoxic responsewas detectable earlier and reached higher levels than that observedin a primary MLC. At the peak of the response, MLC-Imm populationswere observed to lyse up to 50% of the target cells within 3h at a lymphocyte: target cell ratio of 0.3:1. Immunologicaland physical characterization of the effector cells generatedin MLC-Imm indicated that they were medium to large-sized Tlymphocytes. Altogether, these studies suggested the existenceof an anamnestic cell-mediated cytotoxic response in MLC-Imm.

Submitted on May 1, 1974

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