Cytokine-maturated LRH-1⁺ CD34⁻ CML cells are not lysed by LRH-1–specific CTLs. (A,B) Purified CD34⁺ CML cells were cultured for 1 week with growth factors before incubation with LRH-1–specific CTL RP1 (▴), HLA-B7-specific CTL KOR18 (▾; ie, positive control), or medium (♦) only. Survival of CFSE-labeled cytokine-maturated LRH-1⁺ CD34⁻ CML cells from 2 LRH-1⁺ CML patients was determined in the absence or presence of CTLs at an E/T ratio of 0.5:1. Data are depicted as mean plus or minus SD of triplicate wells. (C) P2X5 mRNA expression was determined by real-time quantitative RT-PCR in sorted CD34⁺ CML cells (d0; ■) from 2 LRH-1⁺ CML patients and in the cytokine-matured CD34⁻ progeny of these cells (d7; ). Cytokine-maturation of CD34⁺ CML cells results in a down-regulation of P2X5 expression, confirming the marginal susceptibility for LRH-1 CTL-mediated lysis. The arbitrary threshold of 0.4 is indicated with a dashed horizontal line. (D) Cytospins of CD34⁺ progenitor cells (top panel) and cytokine-maturated CD34⁻ cells (bottom panel) from LRH-1⁺ CML Pt8. Target cells were cocultured with CTLs at an E/T ratio of 0.5:1 for 2 days, after which the cells were analyzed. Morphologically, the CD34⁺ progenitor cells consisted of myeloblasts, whereas the cytokine-matured CD34⁻ progeny of these cells consisted of promyelocytes, myelocytes, and granulocytes. The progenitor cells were clearly lysed by LRH-1–specific CTL RP1, whereas the more differentiated cells were no longer attacked. In contrast, both cell populations were susceptible to the allo-HLA-B7 control CTL KOR18. Imaging was performed with a Zeiss Axioskop microscope (Zeiss, Sliedrecht, The Netherlands) equipped with a Sony 3CCD color video camera system. Pictures were taken using a 40×/0.75 objective.