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The efficacy of immunomagnetic beads to purge human myeloma cells from bone marrow ex vivo was evaluated. The optimal conditions for purging were studied first by using three myeloma cell lines: RPMI-8226, SKO- 007, and SKMM-2. Myeloma cells labeled with the vital fluorescent dye Hoechst 33342 were admixed with normal bone marrow cells, and two monoclonal antibodies reactive with the myeloma cells (PCA-1 and BL-3) were added alone or in combination with the cells. Magnetic beads coated with goat antimouse immunoglobulin G were then added, and the tumor cells to which beads were attached were separated from the mixture with a magnet. The efficacy of tumor cell removal was dependent on the bead-to-tumor ratio; a ratio of more than 500 was optimal in the presence of excess normal marrow cells. The combination of monoclonal antibodies PCA-1 and BL-3 increased the tumor cell removal as compared with either antibody alone. Two cycles of treatment were more effective than one cycle was. Under optimal conditions, 2.3 to 4 logs of tumor cells could be removed from the mixture containing 10% myeloma cells without a significant loss of normal hematopoietic progenitors as measured by CFU-GM, CFU-GEM, and BFU-E. When the efficacy of this procedure was tested on fresh bone marrow from patients with multiple myeloma (MM) by using the combination of PCA-1, BL-3, and J-5, 1.6 to 2.5 logs of tumor cells could be removed by one cycle of treatment, even from marrows containing less than 10% myeloma cells. These observations support the use of monoclonal antibody combinations and immunobeads as a reliable and nontoxic method to eliminate contaminating myeloma cells ex vivo in preparation for autologous bone marrow transplantation in patients with MM.

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony- forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.

To reexamine the influence that T lymphocytes have on the regulation of human peripheral blood burst-forming unit (BFU-E) proliferation in the absence of a statistically significant number of monocytes, very low numbers (3 to 10 X 10(3)/mL) of a null cell fraction highly enriched for BFU-E were cultured alone and in the presence of 5 X 10(5) sheep erythrocyte-purified, autologous T lymphocytes in a methylcellulose culture system containing erythropoietin. T lymphocytes consistently enhanced the growth of BFU-E from the null cell fraction, as reflected in both their number and size. Irradiation of T lymphocytes prior to coculture with null cells markedly reduced this enhancement, strongly suggesting that T lymphocytes synthesize erythroid burst-promoting factors (BPA). To determine whether there were functional differences between the two major T lymphocyte populations as defined by OKT4 (T helper/inducer) and OKT8 (T suppressor/cytotoxic) murine monoclonal antibodies to stimulate the growth of BFU-E, both T cell subpopulations were isolated by negative (panning) or positive (fluorescence-activated cell sorting) selection and cocultured with null cells. No statistically significant differences emerged between unseparated, OKT4+ and OKT8+ T lymphocytes in their ability to stimulate the growth of BFU-E. Thus, these studies provide further evidence that T lymphocytes are a major population of BPA-producing cells and further that OKT4+ and OKT8+ T lymphocytes equally elaborate these factors.

The activities of four purified human growth factors: biosynthetic (recombinant) granulocyte-macrophage colony-stimulating factor (GM- CSF); recombinant erythroid-potentiating activity (EPA); natural and recombinant pluripoietin (Ppo); and natural pluripoietin alpha (Ppo alpha), were compared on the growth of hematopoietic colonies from enriched populations of human marrow and blood progenitor cells. Conditioned medium from the Mo T cell line (MoCM) was used as a standard positive control. We found that activities of GM-CSF and Ppo alpha on the growth of hematopoietic colonies were indistinguishable; Ppo alpha is now believed to be identical to GM-CSF. Both factors were able to promote the growth of colonies derived from subpopulations of CFU-GM, BFU-E, and CFU-GEM. Colonies derived from CFU-GM and CFU-GEM in cultures stimulated by GM-CSF and Ppo alpha were much smaller than in cultures stimulated by MoCM. In contrast to previous reports in which less highly enriched progenitors were used as target cells, Ppo had no detectable activity on the growth of colonies derived from BFU-E or CFU- GEM but promoted the growth of a subpopulation of CFU-GM derived colonies. Ppo is now recognized to be identical to G-CSF. The GM colonies in cultures stimulated by G-CSF (Ppo) were much smaller than in cultures stimulated by MoCM. EPA had no detectable activity on either the size or number of colonies derived from CFU-GM, BFU-E, or CFU-GEM. Results from experiments using target cell populations of marrow fractions separated by velocity sedimentation and marrow populations following freezing suggested that GM-CSF (Ppo alpha) and G- CSF (Ppo) primarily affect the growth of relatively mature subpopulations of progenitor cells. It is clear from these results that additional factor(s) are present in MoCM that are necessary to stimulate CFU-GM, BFU-E, and CFU-GEM maximally in vitro.

The studies described compare the subpopulations of granulocyte- macrophage progenitor cells present in normal marrow with those derived from the marrow of patients with Ph1-positive chronic myelogenous leukemia (CML). The subpopulations were separated on the basis of size by velocity sedimentation and measured for their proliferative capacity by the colony formation technique. A pattern of development of colonies in the individual fractions was obtained by assaying the absolute number of colonies present at time intervals from 3 to 21 days. The number of colonies present at 3 days was taken as 100%, and the percentage of increase or decrease from this value was determined on subsequent days. In the fractions containing the most rapidly sedimenting large cells, the pattern of development of colonies derived from normal and CML marrow was similar. The CML colony-forming units in culture (CFU-C) began to show a deviation from the normal CFU-C pattern of development in the fractions containing CFU-C intermediate in size, and this deviation became progressively more pronounced in the slowest sedimenting small cell fractions. In these latter fractions, the CFU-C derived from CML marrow decreased in number at a rate similar to those arising from the more rapidly sedimenting fractions. This is in contrast to CFU-C derived from normal marrow, which increased in number in the more slowly sedimenting fractions and in the intermediate fractions, remained constant in number, or decreased at a rate slower than those arising from the more rapidly sedimenting fractions. The most likely explanation for these findings is accelerated maturation of the early small granulocyte-macrophage progenitor cells in CML so that these cells show the same limited proliferative capacity as do the later larger progenitor cells.