Characterization of adult human marrow hematopoietic progenitors highly enriched by two-color cell sorting with My10 and major histocompatibility class II monoclonal antibodies

Monoclonal antibodies, My10 (HPCA-1) and major histocompatibility class II (HLA-DR), were used to enrich and phenotype normal human marrow colony-forming unit: granulocyte-macrophage (CFU-GM), burst-forming unit: erythroid (BFU-E), and multipotential colony-forming unit: granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) progenitor cells. Nonadherent low density T lymphocyte-depleted marrow cells were sorted on a Coulter Epics 753 dye laser flow cytometry system with the use of Texas Red-labeled anti-My10 and phycoerythrin conjugated anti-HLA-DR. Cells were separated into populations with nondetectable expression of antigens (DR^-My10^-) or with constant expression of one antigen and increasing densities of the other antigen. More than 98% of the CFU-GM, BFU-E, and CFU-GEMM were found in fractions containing cells expressing both HLA-DR and My10 antigens. The cloning efficiency (CE) of cells in the DR^-My10^- cell fraction was 0.01%. In the antigen-positive sorted fractions, the CE was highest (up to 47%) in the fractions of cells expressing high My10 and low DR (My10⁺⁺⁺DR⁺) antigens and was lowest (2.5%) in the fraction of cells expressing low My10 and low DR (My10⁺DR⁺) antigens. Populations of cells varying in the density of HLA-DR, but not My10, antigens varied in the proportion and types of progenitor cells present. When My10-positive cells were sorted for HLA-DR density expression, the CE for CFU-GM was similar in the DR⁺ and DR⁺⁺ fractions, but most of the BFU-E and CFU-GEMM were found in the DR⁺ fraction. Within the CFU-GM compartment, most of the eosinophil progenitors were found in the DR⁺ fraction, whereas a greater proportion of macrophage progenitors were detected in the DR⁺⁺ fraction. CFU-GM and BFU-E in the fractions of cells positive for DR and My10 were assessed for responsiveness to the effects of recombinant human tumor necrosis factor-α, recombinant human interferon-γ, and prostaglandin E₁. Colony formation from CFU-GM was suppressed by the three molecules, and colony formation by BFU-E was suppressed by recombinant human tumor necrosis factor-α and interferon-γ and enhanced, in the presence of T lymphocyte-conditioned medium, by prostaglandin E₁ in all antigen-positive fractions. These effects were at least as great in the highly enriched progenitor cell population, containing up to 47% colony-forming cells, as they were in the presorted population of nonadherent low density T-lymphocyte-depleted cells with a cloning efficiency of approximately 0.4% and strongly suggest that these factors can act directly on the progenitor cells themselves.