CFU-C Colony Formation Research Articles

Colony growth characteristics in chronic myelomonocytic leukemia

Using cell culture studies specific in-vitro characteristics have been reported for Philadelphia chromosome positive myelogenous leukemia (Ph+ CML) and for juvenile chronic myelogenous leukemia (JCML) previously. We performed cell culture studies in four patients with chronic myelomonocytic leukemia (CMML) and demonstrated the following in-vitro features: excessively increased circulating CFU-C, while BFU-E and CFU-mix were either moderately increased or not detectable; CFU-C colony formation from CMML mononuclear cells (MNC) without addition of exogenous colony stimulating activity (CSA), even after depletion from adherent cells; failing inhibition of CMML MNC on normal BFU-E colony formation. These in-vitro characteristics point to CMML as a distinct entity. In two CMML-patients investigated CFU-C proliferation appeared to some extent inhibited by the addition of IFN-α, IFN-γ and TNF-α to cell cultures.

Hematopoetic precursors respond to a unique B lymphocyte-derived factor in vivo

In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi- CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)- containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.

Hematopoetic Precursors Respond to a Unique B Lymphocyte-Derived Factor In Vivo

In this study we detected a factor that stimulates the proliferation of bone marrow-derived hematopoietic precursors in diffusion chambers implanted in mice. This factor, called diffusible colony-stimulating factor (D-CSF), was found in medium conditioned in the presence of spleen and peripheral blood cells from mice with B cell leukemia (BCL1). After the administration of D-CSF, the number of colonies formed in the plasma clot inside the chamber (CFU-DG) was increased, as were the number of hematopoietic precursors (CFU-MIX, CFU-S, CFU-C, and BFU-E) as judged by a subculture of diffusion chamber contents. Depletion of macrophages and T cells from the spleen cell suspension did not decrease the production of D-CSF, thereby indicating that it was derived from B cells. Neoplastic BCL1 cells appear to be the source because D-CSF could not be detected in medium conditioned with normal B cells. BCL1-conditioned medium (CM) did not enhance CFU-MIX, BFU-E, and CFU-C colony formation in vitro, which suggested that D-CSF is different from multi-CSF, EPA, or CSF. The addition of BCL1 CM to multi-CSF-, erythroid potentiating activity (EPA), and CSF (EL-4CM)-containing cultures had no effect on CFU-MIX, BFU-E, and CFU-C colony formation, thus indicating the absence of a synergistic or inhibitory activity. On the other hand, EL-4 CM, which stimulates CFU-MIX, BFU-E, and CFU-C in vitro, had no effect on CFU-DG in vivo. Biochemical characterization of BCL1 CM revealed that D-CSF is relatively heat stable and loses its bioactivity with protease treatments. It binds to lentil-lectin, according to gel-filtration chromatography has a relative molecular weight of approximately 43,000, and on reverse-phase high-performance liquid chromatography elutes with acetonitrile. These data also indicate that transformed B cells may serve as a source for hematopoietic regulators that act on hematopoietic precursors in vivo.

The Regulatory Role of Interleukin 2-Responsive T Lymphocytes on Human Marrow Granulopoiesis

The effect of recombinant interleukin 2 (IL2) on marrow CFU-C colony formation was evaluated to define the role for T lymphocytes in human marrow granulopoiesis. The colony-stimulating factor (CSA) used in our experiments was found to contain IL2. IL2 depletion from CSA resulted in a reduction in CFU-C colony proliferation. Addition of exogenous IL2 caused an increase in CFU-C colony numbers in a dose-dependent manner. This increase could be prevented by anti-Tac, a monoclonal antibody (MoAb) to the IL2 receptor. Moreover, anti-Tac in the absence of exogenous IL2 resulted in an overall decrease in colony numbers. Depletion of either adherent cells or T lymphocytes abolished the effect of IL2 and anti-Tac on colony growth. In the presence of IL2, re-addition of T lymphocytes to the T-depleted marrow or adherent cells to adherent cell-depleted marrow resulted in a significant increase in CFU-C colony numbers, whereas no significant effect was found when IL2-depleted CSA was used. Although T lymphocytes were not themselves essential for CFU-C colony growth, our studies indicate that IL2 and IL2-responsive T cells can regulate in vitro granulopoiesis.

The regulatory role of interleukin 2-responsive T lymphocytes on human marrow granulopoiesis

The effect of recombinant interleukin 2 (IL2) on marrow CFU-C colony formation was evaluated to define the role for T lymphocytes in human marrow granulopoiesis. The colony-stimulating factor (CSA) used in our experiments was found to contain IL2. IL2 depletion from CSA resulted in a reduction in CFU-C colony proliferation. Addition of exogenous IL2 caused an increase in CFU-C colony numbers in a dose-dependent manner. This increase could be prevented by anti-Tac, a monoclonal antibody (MoAb) to the IL2 receptor. Moreover, anti-Tac in the absence of exogenous IL2 resulted in an overall decrease in colony numbers. Depletion of either adherent cells or T lymphocytes abolished the effect of IL2 and anti-Tac on colony growth. In the presence of IL2, re- addition of T lymphocytes to the T-depleted marrow or adherent cells to adherent cell-depleted marrow resulted in a significant increase in CFU- C colony numbers, whereas no significant effect was found when IL2- depleted CSA was used. Although T lymphocytes were not themselves essential for CFU-C colony growth, our studies indicate that IL2 and IL2-responsive T cells can regulate in vitro granulopoiesis.

Radiosensitization of hematopoietic precursor cells (CFUc) in glioblastoma patients receiving intermittent intravenous infusions of bromodeoxyuridine (BUdR)

The potential use of bromodeoxyuridine (BUdR) as a radiosensitizer given by an intermittent intravenous route is being studied in a Phase I/II trial at the National Cancer Institute. In order to assess the extent of radiosensitization, we have studied the radiation response of human bone marrow cells CFUc taken from 6 patients prior to and after a 14-day infusion of BUdR. Varying concentrations (1000–1500 mg) of BUdR were infused for 12 hours every 24 hours for up to 14 consecutive days. Cell survival was determined by colony formation of CFUc in soft agar suspensions. X ray survival curves were generated over a dose range of 0–300 rad and the slopes of the survival curves (D 0) before and after BUdR infusion were compared. Radiation enhancement ratios (ER) (D 0 pre-BUdR/D 0 post-BUdR) ranged from 1.0–2.2 and appeared to be BUdR dose dependent. Above 650 mg/m 2 the radiation ER was ≥ 1.5. Dose dependent systemic toxicity to bone marrow and skin was also observed with intermittent intravenous infusions of BUdR. From our study, it appears that an intravenous dose of <700 mg/m 2/12 hours is well tolerated and may result in radiosensitization of CFUc in man.

Quantitative and qualitative analysis of stem cells of patients with aplastic anaemia.

3 different methods, (1) assays of CFU-Cs and CFU-Es, (2) responsiveness of CFU-Cs and CFU-Es to low doses of CSF and ESF, respectively and (3) effects of ALG on CFU-C colony formation in vitro, were used to evaluate the quantitative and qualitative defects of stem cells in 28 patients with aplastic anaemia. Some patients with aplastic anaemia who had attained complete remission several years previously, exhibited severely depressed in vitro CFU-C colony formation. This suggests that the defect persists for a long time after clinical complete remission. Residual marrow CFU-Cs and CFU-Es did not have defective responses to the humoral stimulating factors, CSF and ESF. No patient showed a rise of colony number after treatment with ALG in vitro.

Generation of CFU-C suppressor T cells in vitro. III. Failure of mitogen-primed T cells from patients with chronic granulocytic leukemia to inhibit the growth of normal CFU-C

T lymphocytes were derived by E rosetting from the peripheral blood (PB) and bone marrow (BM) of 15 patients with chronic granulocytic leukemia (CGL) in the chronic phase of their disease. T cells were also obtained from 12 healthy individuals. T cells were incubated overnight either in culture medium (RPMI) or RPMI plus pokeweed mitogen (PWM). The supernatants were then recovered and the cells washed in fresh RPMI. T cells from normal donors and from CGL patients were then cocultured with normal allogeneic marrow cells grown in soft agar for CFU-C colony formation. Target marrow cells were also grown in agar in the presence of T-derived supernatants. The results of this study can be summarized as follows. (1) Normal PB and BM T cells efficiently suppressed autologous and allogeneic CFU-C growth after PWM stimulation. (2) T cells derived from peripheral blood or marrow of CGL patients failed to inhibit CFU-C growth, whether pretreated with PWM or not. (3) The supernatants of PWM-treated normal T cells strongly inhibited CFU-C colony formation, whereas the supernatants of PWM-treated CGL T cells had no CFU-C/suppressor activity. These data indicate that T cells from CGL patients cannot be primed to become CFU-C suppressor cells after PWM: stimulation in vitro and cannot release a soluble inhibitor of granulopoiesis produced by PWM-primed normal T cells.

Generation of CFU-C suppressor T cells in vitro. III. Failure of mitogen-primed T cells from patients with chronic granulocytic leukemia to inhibit the growth of normal CFU-C

AbstractT lymphocytes were derived by E rosetting from the peripheral blood (PB) and bone marrow (BM) of 15 patients with chronic granulocytic leukemia (CGL) in the chronic phase of their disease. T cells were also obtained from 12 healthy individuals. T cells were incubated overnight either in culture medium (RPMI) or RPMI plus pokeweed mitogen (PWM). The supernatants were then recovered and the cells washed in fresh RPMI. T cells from normal donors and from CGL patients were then cocultured with normal allogeneic marrow cells grown in soft agar for CFU-C colony formation. Target marrow cells were also grown in agar in the presence of T-derived supernatants. The results of this study can be summarized as follows. (1) Normal PB and BM T cells efficiently suppressed autologous and allogeneic CFU-C growth after PWM stimulation. (2) T cells derived from peripheral blood or marrow of CGL patients failed to inhibit CFU-C growth, whether pretreated with PWM or not. (3) The supernatants of PWM-treated normal T cells strongly inhibited CFU-C colony formation, whereas the supernatants of PWM- treated CGL T cells had no CFU-C/suppressor activity. These data indicate that T cells from CGL patients cannot be primed to become CFU- C suppressor cells after PWM: stimulation in vitro and cannot release a soluble inhibitor of granulopoiesis produced by PWM-primed normal T cells.

1α,25-dihydroxyvitamin D 3 suppresses proliferation of murine granulocyte-macrophage progenitor cells (CFU-C)

Summary The effect of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] on cell growth was examined in murine bone marrow granulocyte-macrophage progenitor cells (CFU-C). The colony formation of CFU-C in agar cultures induced by a colony-stimulating factor (CSF) was markedly suppressed by 10−10−10−8 M of 1α,25(OH)2D3 in a dose-dependent manner. Two to three higher orders of concentrations of 25-hydroxyvitamin D3, 1α-hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 than 1α,25(OH)2D3 were required to induce similar suppression. The inhibitory effect of 1α,25(OH)2D3 on cell growth was confirmed by measuring [3H]-thymidine incorporation into DNA of bone marrow cells in liquid cultures. These results suggest that 1α,25(OH)2D3 is involved in the regulation of proliferation of normal bone marrow cells.

Inhibitory effects of T cells on in vitro granulopoiesis, erythropoiesis, and immunoglobulin production in patients with aplastic anaemia.

Coculture with mononuclear cells from aplastic anaemia (AA) patients and normal bone marrow cells produced different responses among 10 patients studied. In 4 cases, CFU-E growth was inhibited while in another 2 cases CFU-C growth was inhibited. To determine the suppressive effects of mononuclear cells from 6 AA patients, mononuclear cells were separated into T cells, B cells, and monocytes. It was found that T cells from 6 AA patients exhibited suppressive effects on normal CFU-C or CFU-E growth, whereas T cells from normal subjects enhanced the colony formation of CFU-C and CFU-E. On the other hand, monocytes fom 6 AA patients and normal subjects strongly suppressed normal CFU-C and CFU-E growth, although no significant difference was observed in both groups. The immune system in 10 AA patients was evaluated, 3 AA patients (Cases No. 1, 6, 8) exhibited an increase in Tr cells, impairment of PHA responsiveness, and low level of immunoglobulin. T cells from these patients showed a significant suppressive effect on PWM-induced differentiation of allogenic normal B cells. Thus, T cells from 3 out of 10 AA patients exhibited suppressor activities on both haematopoietic cell differentiation and B cell differentiation into plasma cells in vitro.

Autoimmune pancytopenia: Lymphocyte inhibition of autologous but not allogenic bone marrow growth in vitro

A patient with autoimmune renal failure, cavitary lung lesions and arthritis experienced pancytopenia while prednisone therapy was being tapered. Utilizing semisolid culture techniques, a population of nonadherent peripheral blood mononuclear cells was demonstrated, which inhibited autologous but not allogeneic bone marrow erythroid colony-forming units (CFU-E) and myeloid colony-forming units (CFU-c) in vitro. No inhibition of CFU-E or CFU-c colony formation was seen when patient's serum or immunoglobulin G (IgG) was added to cultures. Reinstitution of prednisone therapy resulted in normalization of peripheral blood counts, which was accompanied by the loss of the hemopoietic inhibitor cell activity in the patient's peripheral blood. These results demonstrate the need for testing autologous marrow samples when looking for possible immunemediated inhibition of hematopoiesis.

Myelopoietic stem cells (CFUc) in the blood and bone marrow of children with acute lymphoblastic leukaemia and lymphosarcoma, cultivated without an exogenous supply of colony stimulating factor (CSF).

26 children with acute lymphoblastic leukemia (ALL) and 7 additional patients with lymphosarcoma in leukaemic transformation (LSA) have been studied with respect to the content of myelopoietic stem cell (CFUc) in blood and bone marrow. The methylcellulose culture technique (Iscove et al 1974) was employed in the absence of an exogenous source of colony stimulating factor (CSF). During active disease, CFUc colony formation was absent from patients with ALL, but was present in 2 patients with LSA. 2 therapeutic regimens were employed. Colony formation from bone marrow CFUc was highly variable during remission maintained by either regimen, with no clear relation to clinical stage, number of monocytes or circulating neutrophils. Patients with LSA consistently had high numbers of bone marrow CFUc. CFUc were low or absent from the blood. In conclusion, CFUc are absent from the bone marrow in active ALL, but may be present in active LSA. For the purpose of monitoring children with ALL during therapy, determination of blood or bone marrow CFUc was not found in this study to be helpful.

Cytotoxicity of anti-beta 2-microglobulin xenoantisera to human and murine myeloid progenitor cells and lack of cross-reactivity with colony-stimulating activity.

A previous report has suggested an antigenic relationship between beta 2-microglobulin (beta 2 mu) and granulocyte colony-stimulating activity (CSA). Since human myeloid progenitor cells (CFU-C) express HLA antigens and beta 2 mu is a known molecular component of HLA antigens, we wondered whether the reported effect of anti-beta 2 mu heteroantisera on in vitro granulopoiesis might result from cytotoxicity to CFU-C rather than from cross-reactivity with CSA. To test this, we used rabbit antibody reactive with human and murine beta 2 mu (anti-beta 2 mu). Treatment of human and murine bone marrow cells with anti-beta 2 mu and complement resulted in 95+% inhibition of CFU-C colony formation compared to controls. To test for an effect on CSA, anti-beta 2 mu was incubated with human and murine sources of CSA. After addition of goat anti-rabbit Ig antiserum to precipitate immune complexes and unbound anti-beta 2 mu, the supernatant fluid retained CSA but was no longer cytotoxic to CFU-C. These results indicate that human and murine CFU-C express membrane beta 2 mu and that anti-beta 2 mu antibody does not cross-react with human or murine CSA.