Radiolabeled antibody therapy of lymphoma.
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Antibodies, Monoclonal↗
Biomedical subjects
I D Bernstein
Publications and source records attributed to I D Bernstein.
The ligand for c-kit, stem cell factor, stimulates the circulation of cells that engraft lethally irradiated baboons.
Recombinant human stem cell factor (SCF), the ligand for c-kit, has been shown to stimulate increased numbers of hematopoietic progenitor cells of multiple types to circulate in the blood of baboons, but it was not known if the cells stimulated to circulate by SCF contained cells capable of engrafting and rescuing lethally irradiated baboons. Peripheral blood mononuclear cells (PBMNC) were collected by leukapheresis from four untreated control baboons and from three baboons on the 10th or 11th day of treatment with SCF (200 micrograms/kg/d). All animals were transplanted with 1.00 to 1.04 x 10(8)/kg of cryopreserved autologous PBMNC after treatment with a single dose of 1,020 cGy total body irradiation (TBI). Three animals were transplanted with PBMNC that had been collected during SCF treatment, 24 to 38 days after the last dose of SCF. Rapid trilineage engraftment was documented by bone marrow biopsy in all three. The mean time to a total white blood cell count (WBC) > or = 500/microL, WBC > or = 1,000/microL, and an absolute neutrophil count (ANC) > or = 500/microL was 15 +/- 3 (mean +/- SD), 19 +/- 1, and 19 +/- 2 days, respectively. Two animals remain alive with stable engraftment more than 180 and 245 days posttransplant. The third died of sepsis 32 days posttransplant with a hypercellular marrow showing trilineage engraftment. The surviving animals were transfusion independent by 10 and 59 days posttransplant. Four control animals were transplanted with PBMNC collected in the absence of SCF stimulation. One was treated for 11 days with SCF (200 micrograms/kg/d) after PBMNC were collected. This animal was transplanted 25 days after the last dose of SCF. None of the four control animals engrafted and they died 13, 16, 28, and 38 days posttransplant with marrow aplasia. Treatment with SCF stimulates the circulation of cells that engraft and rescue lethally irradiated baboons. The characteristics of the transplantable cells present in the circulation are now amenable to direct study.
Animals↗
CD34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons.
CD34+ cells devoid of detectable mature and immature T and B lymphocytes, expressing the CD2, CD10, and CD20 antigens, were isolated from marrows of three pairs of sex-mismatched, mixed lymphocyte culture (MLC) nonreactive, sibling baboons. Reciprocal transplants were performed between members of each pair, using the sex chromosomes, identified by standard cytogenetic techniques, as markers of the transplanted cells. Five animals from these three pairs were transplanted with 0.6 to 2.1 x 10(6)/kg of isolated cryopreserved and/or fresh isolated cells that were greater than 95% to 97% CD34+. Before transplantation, animals were treated with either single (920 or 1,020 cGy) or split (700 cGy x 2) dose total body irradiation. All animals engrafted with donor cells, as demonstrated by cytogenetic analysis of bone marrow metaphase cells 4 weeks after transplantation, with days to white blood cell count (WBC) greater than 500 being 19 +/- 2, to WBC greater than 1,000 23 +/- 2, to absolute neutrophil count greater than 500 24 +/- 3, and to platelets greater than 20,000 30 +/- 7. Three animals died of infectious-related complications at 34, 42, and 109 days after transplantation with evidence of host and donor cells (mixed chimerism) in marrow. Two animals remain alive and healthy more than 545 and 455 days after transplantation with stable mixed chimerism in marrow and blood. For these two animals, cytogenetic analysis of granulocyte/macrophage and erythroid colonies derived from marrow precursors between weeks 25 and 42 posttransplant showed evidence of mixed chimerism. Cytogenetic studies of CD2+ T cells and CD20+ B cells isolated from blood of these two animals between weeks 21 and 51 posttransplant showed the presence of mixed chimerism in both lymphocyte populations. Thus, isolated allogeneic CD34+ marrow cells devoid of detectable mature and immature T and B lymphocytes can engraft and reconstitute stable long-term myelopoiesis and lymphopoiesis in lethally irradiated baboons. These results are consistent with the hypothesis that CD34+ marrow cells contain pluripotent hematopoietic stem cells capable of fully reconstituting lymphohematopoiesis in the transplanted host.
Animals↗
A c-kit ligand, recombinant human stem cell factor, mediates reversible expansion of multiple CD34+ colony-forming cell types in blood and marrow of baboons.
The ligand for the human c-kit, recombinant human stem cell factor (SCF), was administered to baboons at doses of 200, 100, 50, 25, and 10 micrograms/kg/d. SCF induced a dose-dependent expansion of hematopoietic colony-forming cells (CFC) of multiple types in both blood and marrow, including colony-forming unit (CFU) granulocyte-monocyte, burst-forming unit-erythroid, CFU-MIX, and high proliferative potential-CFC. These changes were associated with a dose-dependent leukocytosis, involving all leukocyte lineages, a reticulocytosis, and increases in marrow cellularity. At 200 micrograms/kg/d of SCF, CFC in blood were increased 10-fold to greater than 100-fold. This correlated with an increased frequency of CD34+ cells in blood. The frequency of CFC in blood approached that of marrow in some animals. These changes were reversed within 7 to 14 days of stopping SCF. The results of these studies suggest a role for the c-kit ligand in stimulating the expansion of multiple CFC types in blood and marrow for potential therapeutic purposes.
Animals↗
Monoclonal antibody-based therapies of leukemia and lymphoma.
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Animals↗
Blasts from patients with acute myelogenous leukemia express functional receptors for stem cell factor.
Stem cell factor (SCF) acts in concert with lineage-specific growth factors to stimulate the growth of hematopoietic colonies. To determine if neoplastic human hematopoietic cells would also respond to SCF, we cultured marrow mononuclear cells from 20 patients with newly diagnosed acute myelogenous leukemia (AML) and two normal donors with SCF, interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or combinations of growth factors in semisolid medium, and assessed colony growth. SCF receptors (c-kit receptors) were quantitated by equilibrium binding studies with 125I-SCF, and binding parameters were estimated using the ligand program. The cellular distribution of c-kit receptors was determined by autoradiography. Our results show that SCF alone or in combination with IL-3 or GM-CSF increased both the number and size of colonies in 10 of the patients. Receptors for SCF were identified on the blasts from all 20 AML patients. The number of receptors ranged from 600 to 29,000 per cell. In the majority of patients, both high- and low-affinity binding sites were identified. Neither the number of receptors per cell nor the finding of one or two classes of receptors correlated with growth response to SCF. Autoradiographic analysis of 125I-SCF binding to normal marrow mononuclear cells revealed grains associated with blasts and megakaryocytes. Grain counts on blasts from 10 AML patients and on normal marrow blasts suggested that high-affinity c-kit receptor expression on AML blasts is lower than or similar to that of normal blasts. These results identify c-kit receptors on human AML blasts, and indicate that SCF acts synergistically with IL-3 or GM-CSF to stimulate colony growth from the marrow cells of a portion of patients with AML.
Autoradiography↗
Expression of lymphoid-associated cell surface antigens by childhood acute myeloid leukemia cells lacks prognostic significance.
The prognostic significance of cell surface antigens associated with lymphoid and myeloid lineage differentiation on the blasts of children with acute myeloid leukemia (AML) was evaluated. Leukemic blasts from 176 patients enrolled on Childrens Cancer Study Group Protocol 213 determined to have AML by standard morphologic and cytochemical criteria were immunophenotyped. Cell surface antigens associated with myeloid differentiation were found on blasts from 88.1% of patients (CD15, 44%; CD33, 65%; CD36, 53%; glycoprotein Ib, 9.3%). However, blasts from 30.7% of patients expressed surface antigens thought to be specific for lymphoid lineage differentiation (CD2, 9.4%; CD5, 2.7%; CD19, 34.5%; CD20, 0.8%). In addition, CD34, a glycoprotein found on immature cells of both myeloid and lymphoid lineages, was expressed on the blast cells of 48.2% of patients. With the exception of the lymphoid lineage nonspecific antigen CD4, no correlation was found between white blood cell count at diagnosis, age, and French-American-British morphology, and the expression of any of the lymphoid- or myeloid-associated cell surface antigens studied. Nor was the expression of these antigens prognostically significant with respect to response to induction therapy, death during induction, survival, event-free survival, or survival/event-free survival following remission induction. Multivariate analysis showed that CD4 expression was not an independent predictor of outcome. Thus, this prospective study suggests that expression of lymphoid-associated cell surface antigens as well as myeloid-associated antigens by childhood AML blasts lacks prognostic significance.
Adolescent↗
Differences in the frequency of normal and clonal precursors of colony-forming cells in chronic myelogenous leukemia and acute myelogenous leukemia.
Acute myelogenous leukemia (AML) is a clonal disease that is heterogeneous with respect to the pattern of differentiative expression of the leukemic progenitors. In some patients, the involved stem cells manifest pluripotent differentiative expression, whereas in others, the involved progenitors manifest differentiative expression mainly restricted to the granulocytic pathway. This is in contrast to chronic myelogenous leukemia (CML) which is a clonal disease known to arise in a pluripotent stem cell. Therefore, we tested whether these leukemias could be distinguished with respect to their involvement of immature precursors by studying colony-forming cells (CFC) and their precursors from four glucose-6-phosphate dehydrogenase (G6PD) heterozygous patients with AML and five patients with CML. CFC were separated from their precursors by FACS for expression of CD33 and CD34 followed by growth in a long-term culture (LTC) system. The vast majority of CFC express both the CD33 and CD34 antigens, but their less mature precursors, detected by their ability to give rise to CFC in LTC, express only CD34. In three of the four patients with AML, the CD33-CD34+ cells produced CFC in LTC that appeared to be predominantly or completely normal (ie, nonclonal) in origin. In the fourth patient, a significant enrichment of nonclonal progenitors was obtained in the CD33-CD34+ population, but these cells may also have included significant numbers of clonal cells. In contrast, in four of five patients with CML, cultures of both the CD33-CD34+ and CD33+CD34+ populations produced CFC in LTC that were almost entirely clonal in origin, whereas in the fifth patient a substantial number originated from nonclonal stem cells. These data indicate that granulocyte/monocyte progenitors are predominantly clonally derived in CML and AML. In CML, their precursors are also predominantly clonal, but in some cases of AML they are not. These findings may have implications for understanding the success or failure of current therapies of AML and CML.
Antigens, CD↗
Selective radiation of hematolymphoid tissue delivered by anti-CD45 antibody.
The ability to deliver radiation selectively to lymphohematopoietic tissues may have utility in conditions treated by myeloablative regimens followed by bone marrow transplantation. Since the CD45 antigen is the most broadly expressed of hematopoietic antigens, we examined the biodistribution of radiolabeled anti-CD45 monoclonal antibodies in normal mice. Trace 125I or 131I-labeled monoclonal antibodies 30G12 (rat IgG2a), 30F11 (rat IgG2b), and F(ab')2 fragments of 30F11 were injected i.v. at doses of 5 to 1000 micrograms. For both intact antibodies, a higher percentage of injected dose/g (% ID/g tissue) in blood was achieved with higher antibody doses. However, as the dose of antibody was increased, the % ID/g in the target organs of spleen, marrow, and lymph nodes decreased. At doses between 5 and 10-micrograms, % ID/g in these tissues exceeded that in lung, the normal organ with the highest concentration of radiolabel. In contrast, thymus was the only hematopoietic organ in which the % ID/g increased with increasing antibody dose, although at high dose the % ID/g was still far below that achieved in the other hematopoietic organs. Antibody 30F11 F(ab')2 fragments were cleared more quickly than intact antibody from blood and from both target and nontarget organs, although the relationship between increasing antibody dose and decreasing % ID/g in spleen, marrow, and lymph nodes was observed. The time-activity curves for each dose of antibody were used to calculate estimates of radiation absorbed dose to each organ. At the 10-micrograms dose of 30G12, the spleen was estimated to receive a radiation dose that was 13 times more than lung, the lymph nodes 3 to 4 times more, and the bone marrow 3 times more than lung. For each antibody fragment dose, the radiation absorbed dose per MBq 131I administered was lower because the residence times of the fragments were shorter than those of the intact antibody. Thus these estimates suggested that the best "therapeutic ratio" of radiation delivered to target organ as compared to lung was achieved with lower doses of intact antibody. We have demonstrated that radiolabeled anti-CD45 monoclonal antibodies can deliver radiation to lymphohematopoietic tissues with relative selectivity and that the relative uptake and retention in different hematolymphoid tissues change with increasing antibody dose.
Animals↗
Effect of interleukin-2 on biodistribution of monoclonal antibody in tumor and normal tissues in mice bearing SL-2 thymoma.
BACKGROUND: Our laboratory demonstrated previously that treatment with a tumor-specific, radiolabeled anti-Thy 1 murine monoclonal antibody (MAb) in mice can eradicate a T-cell lymphoma mass, but the doses required were toxic to normal organs. Approaches to increase MAb concentration in tumor tissue versus normal tissue may overcome this problem. Interleukin-2 (IL-2) has been shown to increase capillary permeability, as indicated by extravasation of albumin. PURPOSE: The purpose of this study was to determine whether increased extravasation of MAb at the tumor site might result in selective binding to tumor antigen, increasing localization of radiolabeled MAb at the tumor site. METHODS: We studied the effect of IL-2 on biodistribution of 1A14 MAb (anti-Thy 1.1) in normal AKR/Cum (Thy 1.2+) mice and in AKR/Cum mice bearing SL-2, a spontaneous T-cell lymphoma (Thy 1.1+), compared with biodistribution of albumin in normal mice and biodistribution of the nonreactive G3G6 MAb in tumor-bearing mice. IL-2 was given intravenously in the tail vein in doses of 0, 25,000, 50,000, 100,000, or 200,000 U twice a day for a total of seven doses over 3.5 days. Mice received injections of a mixture of 1A14 MAb (250 muCi/100 micrograms) and albumin or G3G6 MAb (145 muCi/100 micrograms) in a total volume of 200 microL at 12 hours after the last IL-2 dose. RESULTS: In normal mice, IL-2 caused a dose-dependent increase of both radiolabeled MAb and albumin in the spleen, liver, lung, and lymph node, but it spared the brain. In tumor-bearing mice, IL-2 resulted in higher levels of MAb in the tumors 72 hours after receiving injections, with 17.5% and 24.3% of the injected dose per gram of tumor present in the mice pretreated with 100,000 or 200,000 U of IL-2 twice a day for 3.5 days, compared with 13.4% in the controls. In IL-2-treated mice, levels of MAb were greater in the tumors than in critical normal organs; the differences were statistically significant for tumors versus lungs at 24 hours after injection and for tumors versus livers at 48 hours and 72 hours after injection. CONCLUSIONS: These results suggest that pretreatment with IL-2 may lead to enhanced distribution of tumor-specific MAb to the tumor site, compared with normal tissues, thus increasing therapeutic efficacy of radiolabeled MAb.
Albumins↗
Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor.
Stem cell factor (SCF) stimulates the growth of burst-forming unit-erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU-GM) by binding to a specific cell surface receptor. The receptor for SCF is encoded by the protooncogene c-kit. After immunizing mice with the human erythroleukemia cell line OCIM1, we obtained a monoclonal antibody (MoAb) that recognizes the human c-kit receptor. This MoAb, designated SR-1, blocks binding of 125I-human SCF to the c-kit receptor, and neutralizes the biologic effects of SCF in hematopoietic colony assays. With few exceptions, c-kit expression was identified on all hematopoietic and lymphoid cell lines tested by indirect immunofluorescent analysis using SR-1 and by binding studies with 125I-SCF. SR-1 recognizes a small fraction of normal bone marrow mononuclear cells, and these cells have the morphologic appearance of blasts. Colony assays show that BFU-E and CFU-GM display the c-kit receptor. SR-1 does not cross-react with murine c-kit protein, indicating that the binding epitopes of the human and murine c-kit receptors are antigenically distinct. This MoAb may be useful to characterize the spectrum of cells that display the c-kit receptor and to further define the role of SCF in hematopoiesis.
Animals↗
Localization of radiolabeled antimyeloid antibodies in a human acute leukemia xenograft tumor model.
Acute myeloid leukemia is an attractive disease to treat with radiolabeled antibodies because it is radiosensitive and antibody has ready access to the marrow cavity. In order to evaluate potentially useful radiolabeled antibodies against human acute myeloid leukemia, we have developed a nude mouse xenograft model using the human acute leukemia cell line, HEL. Mice with s.c. xenografts of HEL cells received infusions of radioiodinated anti-CD33 antibody. Examination of the biodistribution of the antibody showed that uptake in the s.c. tumor was maximal [16.9% injected dose (ID)/g at 1 h after infusion] following infusion of 1-10 micrograms of antibody and decreased following infusion of 100 micrograms (6.5% ID/g at 1 h) presumably as a result of saturation of antigen sites. The radiolabel was poorly retained in tumor (4.5-8.2% ID/g at 24 h after infusion). These results were consistent with in vitro studies demonstrating rapid internalization and catabolism of the anti-CD33 antibody. Uptake in tumor could be improved by using either a radiolabel that is retained intracellularly, 111In-DTPA (18.5% ID/g at 24 h), or by targeting a surface antigen that does not internalize upon antibody binding, CD45 (20.5% ID/g at 24 h). These results indicate that this model system will be useful in evaluating the interaction of radiolabeled antibodies with human acute myeloid leukemia cells in an in vivo setting.
Acute Disease↗
Expression of myeloid differentiation antigens in acute nonlymphocytic leukemia: increased concentration of CD33 antigen predicts poor outcome--a report from the Childrens Cancer Study Group.
Ninety-eight cryopreserved specimens of acute nonlymphocytic leukemia (ANLL) cells obtained at initial diagnosis of children enrolled on the Childrens Cancer Study Group 251 protocol (CCG 251) were examined by indirect immunofluorescence using four monoclonal antibodies to myeloid differentiation antigens. The relationship between the level of differentiation of ANLL cells as determined by their antigen phenotype and the clinical outcome of treatment, including complete remission (CR) rate, survival, and event-free survival, was evaluated. Most leukemic specimens were determined to express the CD33 antigen (L4F3), a 67-kD protein. Because the level of differentiation of normal myeloid cells is reflected by the concentration of the CD33 antigen expressed, samples were categorized as CD33-bright (immature) versus CD33-dull (mature). Patients with CD33-bright leukemic blasts had a marginally inferior CR rate to those with CD33-dull blasts (P = 0.08). With respect to survival and event-free survival, there was a significantly inferior outcome in the CD33-bright patients (P = 0.04 and P = 0.06, respectively). Reactions of ANLL with anti-CD15 antibody (1G10), anti-CD36 antibody (5F1), or anti-CD17 antibody (T5A7) did not predict clinical outcome. This study indicates that patients whose ANLL blasts displayed the CD33 antigen in an amount associated with immature myeloid cells experienced a worse outcome than patients with ANLL blasts that expressed a phenotype associated with more mature cells.
Antibodies, Monoclonal↗
Monoclonal antibodies in the study and therapy of hematopoietic cancers.
Recent advances utilizing monoclonal antibodies to phenotype acute leukemias have revealed no prognostic significance of the expression of lymphoid-associated antigens by acute myeloid leukemia blasts and conflicting results regarding 'biphenotypic' acute myeloid leukemia. Several studies treating patients with refractory lymphoma with immunotoxins reported encouraging results but significant production of anti-mouse or anti-toxin antibody. Radiolabeled antibodies that react with panhematopoietic antigens have delivered selective radiation to marrow, spleen and lymph nodes in animal models and are being used in Phase I studies of marrow transplantation for acute leukemia.
Antibodies, Monoclonal↗
Is there a better way to deliver total body irradiation?
The available data suggest that the antileukemic, immunosuppressive and toxic effects of TBI are highly dose dependent and if TBI is given as single daily fractions at 5-10 cGy/minute, an optimal dose for the treatment of most leukemias is somewhere between 12 and 16 Gy. Giving radiation as a single dose rather than fractionating it increases both its immunosuppressive as well as its toxic effects. The relative anti-leukemic effects of single vs. fractionated irradiation are less clear, but available data support the view that fractionation does not substantially reduce the effects of TBI on myeloid tissue. Increasing the dose rate increases toxicities and, although data are not yet complete, likely increases both immunosuppressive and anti-tumor effects. The impact of total dose, dose fractionation and dose rate are highly interdependent and how best to manipulate these 3 factors to lead to an optimal combination is as yet unknown. Directing radiotherapy to sites of leukemia using monoclonal antibodies or other carriers such as growth factors is feasible and, although there are many aspects of this approach which have yet to be worked out, targeted radiotherapy may prove to be the best way to achieve the therapeutic goals of increased tumor ablation and immunosuppression without increased toxicities.
Bone Marrow Transplantation↗
Recombinant human stem cell factor, a c-kit ligand, stimulates hematopoiesis in primates.
Recombinant human stem cell factor (SCF) is homologous with recombinant rat SCF (rrSCF) and is a ligand for c-kit. We determined the influence of SCF on hematopoiesis in vitro and in vivo in baboons. In vitro, SCF alone stimulated little growth of hematopoietic colony-forming cells from baboon marrow, but did increase the number of colonies formed in response to erythropoietin (Epo), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In vivo, SCF caused an increase in the peripheral blood of the number of erythrocytes, neutrophils, lymphocytes, monocytes, eosinophils, and basophils. In marrow, it caused an increase in marrow cellularity and in the absolute number of colony-forming unit-granulocyte-monocyte (CFU-GM) and burst-forming unit-erythroid (BFU-E) in marrow following infusion of SCF. The in vivo stimulation of multiple lymphohematopoietic lineages corroborates previous in vitro studies and suggests a potentially important clinical role for SCF.
Animals↗
Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+lin- cells, and the generation of colony-forming cell progeny from CD34+lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor.
We tested the ability of recombinant human stem cell factor (SCF) to stimulate isolated marrow precursor cells to form colonies in semisolid media and to generate colony-forming cells (CFC) in liquid culture. SCF, in combination with interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) caused CD34+ cells to form increased numbers of granulocyte-macrophage colonies (CFU-GM), and to form macroscopic erythroid burst-forming units (BFU-E) in the presence of IL-3, erythropoietin (Epo), and SCF. We tested isolated CD34+lin- cells, a minor subset of CD34+ cells that did not display antigens associated with lymphoid or myeloid lineages, and CD34+lin+ cells, which contain the vast majority of CFC, and found that the enhanced colony growth was most dramatic within the CD34+lin- population. CD34+lin- cells cultured in liquid medium containing SCF combined with IL-3, GM-CSF, or G-CSF gave rise to increased numbers of CFC. Maximal numbers of CFU-GM were generated from CD34+lin- cells after 7 to 21 days of culture, and required the presence of SCF from the initiation of liquid culture. The addition of SCF to IL-3 and/or G-CSF in cultures of single CD34+lin- cells resulted in increased numbers of CFC due to the proliferation of otherwise quiescent precursors and an increase in the numbers of CFC generated from individual precursors. These studies demonstrate the potent synergistic interaction between SCF and other hematopoietic growth factors on a highly immature population of CD34+lin- precursor cells.
Antigens, CD↗
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)
Antibodies, Monoclonal↗