Effect of CTL specific for WT235 and WT126 on progenitor/stem cells.

(A) Analysis of CTL-mediated inhibition of colony formation (CFU-GM) of CD34⁺ cells purified from CML patients and healthy donors. Purified CD34⁺ cells were cocultured with WT235-specific CTLs for 4 hours or cultured for 4 hours in the absence of CTLs, followed by triplicate plating of 3000 CD34⁺ cells in methylcellulose medium supplemented with stem cell factor, interleukin 3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF). After 14 days the CFU-GMs were determined as means of triplicate cultures. The graph shows the means and SD of 15 independent experiments with CD34⁺ from 6 HLA-A2⁺ CML patients, 5 A2⁺ healthy donors, and 4 HLA-A2⁻ CML patients. (B,C) Analysis of CTL-mediated inhibition of LTC-ICs of CD34⁺ cells purified from CML patients (B) and normal cord blood (CB) or mobilized peripheral blood (PB; C). CD34⁺cells were mock treated or treated with WT126-specific CTLs (CML nos. 2 and 3; CB no.1) or WT235-specific CTLs (CML nos. 1 and 2; CB nos. 2 and 3; PB nos. 1-4) for 4 hours, followed by culture for 5 weeks on irradiated M2-10B4 feeder cells. The assay was carried out in quadruplicate cultures to reduce culture variation. Cells from adherent and nonadherent fraction of the cultures were pooled at week 5, and 20% of the cells were plated in a CFU-GM assay in triplicate. The results are expressed as CFU-GM output (means ± SD).