Generation of leukemia-reactive cytotoxic T lymphocytes from HLA-identical donors of patients with chronic myeloid leukemia using modifications of a limiting dilution assay

Donor leukocyte transfusions (DLT) have an anti-leukemic effect in most patients with a relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. However, DLT are often complicated by graft-versus-host disease, Selection of donor lymphocytes with a relative specificity for leukemic cells is desirable, The generation of leukemia-reactive cytotoxic T lymphocyte (CTL) responses between HLA-identical donors and patients in bulk cultures showed major variations, and false negative results were observed, In a modification of a limiting dilution analysis (LDA) two-fold serial dilutions of HLA-identical donor mononuclear cells (MNC) were cultured in the presence of CML cells, The anti-leukemic CTL precursor frequencies in these donors varied between <1 and 9 per 10(6) MNC, KLA-restricted CD4(+) or CD8(+) lymphocytes as well as MHC non-restricted gamma delta T cells were responsible for the anti-leukemic responses, A positive correlation between cytotoxicity in the various wells after 3, 4 and 5 weeks of culture could be found, The LDA may be superior to bulk cultures in selecting stable immune responses and in separating multiple different antileukemic T cell responses in each donor-patient combination.