Proliferative responses to interleukin-3 and granulocyte colony-stimulating factor distinguish a minor subpopulation of CD34-positive marrow progenitors that do not express CD33 and a novel antigen, 7B9

Human hematopoietic colony-forming cells (CFC) express the CD34 antigen (CD34⁺) as well as differentiation antigens such as CD33 and HLA-DR. CD34⁺ cells that do not express these latter differentiation antigens have been shown to contain few CFC in direct culture, but generate increasing numbers of CFC when cultured over a marrow stromal cell layer in the long-term culture system. In this study we determined if CD34⁺ cells with low or absent expression of CD33 and a novel antigen, 7B9 (CD34⁺CD33^-7B9^-), could be distinguished from CD34⁺ cells expressing these antigens (CD34⁺CD33⁺7B9⁺) based on their proliferative responses to interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) in a short-term liquid culture system. These two populations were separated by fluorescence-activated cell sorting, cultured with IL-3 (10 ng/mL), G-CSF (100 ng/mL), or IL-3 and G-CSF, and ³H-thymidine uptake was measured. CD34⁺CD33^-7B9^- cells proliferated in the presence of IL-3, but not G-CSF. However, a synergistic response to the combination of IL-3 and G-CSF was seen in most experiments. In contrast, CD34⁺CD33⁺7B9⁺ cells proliferated in the presence of either IL-3 or G-CSF but did not display an additive or synergistic response to the combination of IL-3 and G-CSF. In colony-forming assays performed before and after liquid culture, the CD34⁺CD33^-7B9^- cells in two experiments contained 0.3% and 2.2% of all sorted marrow CFC before liquid culture and generated 40-fold and ninefold increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), respectively, after liquid culture with IL-3 and G-CSF. In contrast, the CD34⁺CD33⁺7B9⁺ cells contained 99.7% and 97.8% of all sorted marrow CFC before liquid culture and had no change or a threefold increase in the number of CFU-GM, respectively, after liquid culture with IL-3 and G-CSF. Single-cell liquid cultures containing IL-3 and G-CSF with cells that were either CD34⁺CD33^-7B9^- and depleted of mature lymphoid cells (CD34⁺lin^-) or were CD34⁺lin⁺ showed that a higher proportion of wells containing a CD34⁺lin^- cell gave rise to one or more CFC (8.7%) than did wells containing a CD34⁺lin⁺ cell (2.9%), with the responding cells in the former population giving rise to an average of 2.9 ± 0.6 CFC and in the latter population, 2.0 ± 1.0 CFC. Thus, we have identified a presumably immature precursor population that is CD34⁺CD33^-7B9^- and is distinguishable from a population containing the vast majority of CFC that is CD34⁺CD33⁺7B9⁺ based on its proliferative responses to IL-3 and G-CSF and its ability to generate increased numbers of CFC upon culture in the presence of these factors.