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.(ABSTRACT TRUNCATED AT 400 WORDS)
Litzow
M R
MR
Program in Pediatric Oncology, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Brashem-Stein
C
C
Andrews
R G
RG
Bernstein
I D
ID
D000911:Antibodies, Monoclonal; D000941:Antigens / Q000032:analysis*; D015703:Antigens, CD / Q000032:analysis*; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis*; D001854:Bone Marrow Cells; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells; D003114:Colony-Forming Units Assay; D004261:DNA Replication / Q000187:drug effects*; D004347:Drug Interactions; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D007700:Kinetics; D011994:Recombinant Proteins / Q000494:pharmacology; D063268:Sialic Acid Binding Ig-like Lectin 3; D013936:Thymidine / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000941:Antigens; D015703:Antigens, CD; D018952:Antigens, CD34; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D007377:Interleukin-3; D011994:Recombinant Proteins; D063268:Sialic Acid Binding Ig-like Lectin 3; D016179:Granulocyte Colony-Stimulating Factor; D013936:Thymidine