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Lymphocytes from a healthy HLA-identical bone marrow transplant donor were tested for their ability to destroy her brother's acute myelogenous leukemia blasts in vitro. Primary mixed lymphocyte culture (MLC) and cell-mediated lysis (CML) responses between the patient's remission (pretransplant) and donor's lymphocytes were negative. Stimulation of donor lymphocytes for 7 d in vitro with irradiated leukemia cells, leukemia cells plus allogeneic irradiated lymphocytes, or a pool of irradiated lymphocytes from 10 donors, did not activate any cytotoxic cells able to destroy the HLA identical leukemic blasts. Further culturing for 7 additional d in T cell growth factor (TCGF) generated lymphocytes that induced effective cytotoxicity against the leukemic blasts, but not against autologous lymphocytes. Effective killing against the leukemia was observed only in cultures initially stimulated with the irradiated leukemia cells. These cytotoxic cells were maintained in TCGF and mediated persistent killing against the leukemic target cells. They were also able to destroy lymphocytes from the patient's mother and father, but not from an unrelated cell donor. This suggested specific recognition of non-HLA antigens inherited by the patient, that were foreign to the HLA identical bone marrow donor. These lymphocytes were cloned by a limiting dilution technique and one clone maintained cytotoxicity to the AML blasts and the father's lymphocytes, but not lymphocytes from the mother or an HLA-identical donor. This cytotoxicity was inhibited by a monoclonal anti-HLA antibody. Thus, in vitro sensitization of this sibling's lymphocytes with AML blasts followed by TCGF expansion, and cloning, enabled the detection of HLA-restricted cytotoxic cells that recognize minor locus histocompatibility antigens. This immune recognition may be relevant to the "graft vs. leukemia" effect that has been observed in leukemic animals and patients following histocompatible hematopoietic transplants.

HLA identical leukemia cells and T cell growth factor activate cytotoxic T cell recognition of minor locus histocompatibility antigens in vitro.