Association of cell cycle expression of Ia-like antigenic determinations on normal human multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells with regulation in vitro by acidic isoferritins

An association has been established between human Ia-like antigenic determinants, expression during DNA synthesis on multipotential (CFU- GEMM) and erythroid (BFU-E) progenitor cells, and the regulatory action of acidic isoferritins in vitro. Treatment of human bone marrow cells with monoclonal anti-Ia (NE1-011) plus complement inhibited colony formation of CFU-GEMM) and BFU-E by 50%-70%. Reduction of colonies was similar whether bone marrow cells were exposed to anti-Ia plus complement, high specific tritiated thymidine (3HTdr), or acidic isoferritins. No further decrease was apparent with 3HTdr or acidic isoferritins after Ia-antigen+ CFU-GEMM or BFU-E were removed, or with anti-Ia plus complement or acidic isoferritins after S-phase CFU-GEMM or BFU-E were removed. Anti-Ia, in the absence of complement, had no effect on colony formation but blocked the inhibition of CFU-GEMM and BFU-E by acidic isoferritins. Demonstration of Ia-antigens on BFU-E and inhibition of BFU-E by acidic isoferritins appeared to require the presence of phytohemmagglutinin leukocyte conditioned medium (PHA-LCM) in the culture medium during the 14-day incubation period. these results implicate Ia-antigen+ cells, acidic isoferritins, and PHA-LCM in the regulation of multipotential and erythroid progenitor cells in vitro.     

Modulation of the expression of HLA-DR (Ia) antigens and the proliferation of human erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells by prostaglandin E

The relationship between the presence of Ia-like antigens on human CFU-GEMM and BFU-E, and their responsiveness to the regulatory effects of AIF and PGE have been studied using normal human bone marrow cells. In primary methylcellulose culture the addition of 10(-6)-10(-9) M PGE1 results in the enhancement of the total number of BFU-E detected, with no observed effect on the number of CFU-GEMM. Addition of acidic isoferritins to primary cultures results in an approximately 50% inhibition of both BFU-E and CFU-GEMM proliferation. Removal of Ia+ cells by cytotoxic treatment with monoclonal antihuman HLA-DR (Ia) antibody plus C' resulted in: (a) reduction of total CFU-GEMM and BFU-E by approximately 50%, (b) abrogation of the enhancing effect of PGE on BFU-E, and (c) detection of populations of CFU-GEMM and BFU-E that are no longer sensitive to inhibition by AIF. Culture of marrow cells in suspension culture at 37 degrees C for 24 h prior to methylcellulose culture resulted in the loss of detectable Ia antigen on BFU-E and CFU-GEMM, loss of their responsiveness to AIF, loss of the enhancing effect of PGE on BFU-E, and the inability to detect cycling cells. Exposure of marrow cells to PGE, however, during the suspension phase augmented the total number of BFU-E, and CFU-GEMM detected and resulted in the detection of S-phase cells, expression of Ia antigens of both BFU-E and CFU-GEMM, and restoration of the ability to detect BFU-E and CFU-GEMM sensitivity to inhibition by AIF. After suspension culture with PGE, no further enhancement of BFU-E by PGE was observed. These results indicate that the expression of Ia antigens is important in the regulation of BFU-E and CFU-GEMM proliferation and add further evidence for a role for PGE in controlling progenitor cell Ia-antigen expression, cell cycle and, as a consequence, their proliferative capacity.     

SDF-1-induced actin polymerization and migration in human hematopoietic progenitor cells.

OBJECTIVE: The capacity of hematopoietic progenitor cells (HPCs; CD34(+) cells) to respond to chemotactic stimulation is essential for their homing efficiency, e.g., during stem cell transplantation. Previous studies established that stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 play an important role in the homing of HPCs. The aim of the present study was to analyze SDF-1-induced actin polymerization and migration of HL-60 cells and primary human CD34(+) cells. MATERIALS AND METHODS: SDF-1-induced migration of CD34(+) cells from cord blood (CB) and peripheral blood (PB) across fibronectin-coated filters was measured in a Transwell assay. Actin polymerization was detected using fluorescent phalloidin and analyzed by confocal microscopy and FACS analysis. RESULTS: SDF-1 induced a rapid and transient increase in actin polymerization and in polarization of the actin cytoskeleton in primary CD34(+) cells and HL-60 cells. SDF-1 was found to induce significantly more actin polymerization in CB CD34(+) cells that show fast migration in vitro compared to slow migrating PB CD34(+) cells. Moreover, CB CD34(+) cells that had migrated toward SDF-1 showed an elevated and prolonged rise in F-actin upon second exposure to SDF-1 compared to nonmigrated cells, although both cell types expressed equal levels of the SDF-1 receptor CXCR-4. CONCLUSIONS: The relatively high migratory capacity of CB-derived human HPCs is not related to cellular polarization or high expression of the SDF-1 receptor but is largely determined by their capacity to efficiently polymerize F-actin in response to SDF-1.     

Synergistic antiproliferative effect of recombinant interferon-gamma with recombinant interferon-alpha on chronic myelogenous leukemia hematopoietic progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM)

Recombinant interferons, alpha (rIFN-alpha) and gamma (rIFN-gamma), have been demonstrated to have significant antitumor activity as single agents in the treatment of chronic myelogenous leukemia (CML). Due to their possible synergistic efficacy, a combination rIFN therapy in CML has been proposed. To establish a biologic basis for this, we have studied the suppressive effects of rIFN-alpha and rIFN-gamma on the in vitro growth of CML-derived progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, CFU-GM), the optimal conditions for rIFN synergism, and the possible role of hematopoietic accessory cells (T-lymphocytes and monocytes- macrophages) in mediating rIFN-induced growth inhibition. When added to unseparated bone marrow cells, rIFN-alpha and rIFN-gamma significantly reduced colony formation, with 50% inhibition occurring at 71 and 186 U/mL for CFU-GEMM, 40 and 152 U/mL for CFU-Mk, 222 and 1,458 U/mL for BFU-E, and 119 and 442 U/mL for CFU-GM, respectively. A small amount of rIFN-gamma (5 U/mL) acted synergistically with increasing doses of rIFN- alpha, and the values of 50% inhibitory concentrations fell outside the lower limit (10 U/mL) used in our experiments. This synergy was evident even when rIFN-gamma was added 72 hours after the initiation of cultures; however, it was completely lost when the target cells were depleted of accessory cells. When a low dose of rIFN-alpha (5 U/mL) was added to rIFN-gamma, the 50% inhibitory concentration values were decreased up to tenfold. These studies (1) confirm that CML-derived hematopoietic progenitors are responsive to the suppressive activity of both rIFN-alpha and rIFN-gamma in vitro, (2) demonstrate that different mechanisms are responsible for the suppressive activity of the two rIFNs, and (3) characterize their synergistic interaction, providing a basis for future clinical trials aimed at investigating combination rIFN therapy in CML.     

血管紧张素Ⅱ对定向造血祖细胞BFU-E体外扩增的影响

目的：研究血管紧张素Ⅱ(AngⅡ)参与造血调控的机制及其对造血干／祖细胞BFU-E定向优势分化的作用。方法：将脐血来源的CD34^ 细胞在不同的细胞因子组合中悬浮培养，使它们向红系造血祖细胞优势分化，分别在不同时间对培养细胞进行3种处理：一部分细胞提取：RNA，用RT-PCR方法检测AngⅡ受体：mR-NA的转录；一部分添加AngⅡ和其他细胞因子继续培养3天后转移到半固体培养基中进行集落计数；其余细胞在原来的条件下继续悬浮培养。结果：新分离的CD34^ 细胞表面无AngⅡ受体mRNA的表达，此时添加AngⅡ对造血祖细胞生长无明显作用；在CD34^ 向红系优势分化体系中悬浮培养7天后，细胞中能检测到AngⅡ受体mRNA的表达，此时添加AngⅡ可刺激BFU-E的扩增；在培养第14天，细胞中己无AngⅡ受体表达，此时添加AngⅡ对红系祖细胞的扩增已无明显作用。结论：红系祖细胞在分化过程中有AngⅡ受体mRNA的表达，AngⅡ可刺激早期红系造血细胞的增生，并可提高造血细胞向红系定向优势分化的效率。     

Effects of recombinant alpha and gamma interferons on the in vitro growth of circulating hematopoietic progenitor cells (CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM) from patients with myelofibrosis with myeloid metaplasia

Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder due to clonal expansion of a pluripotent hematopoietic progenitor cell with secondary marrow fibrosis. No definitive treatment has as yet been devised for this condition, which shows a marked variability in clinical course. To evaluate whether excessive hematopoietic progenitor cell proliferation could be controlled by recombinant human interferon alpha (rIFN-alpha) and gamma (rIFN-gamma), we studied the effects of these agents on the in vitro growth of pluripotent and lineage-restricted circulating hematopoietic progenitor cells in 18 patients with MMM. A significant increase in the growth (mean +/- 1 SEM) per milliliter of peripheral blood of CFU-GEMM (594 +/- 253), CFU-Mk (1,033 +/- 410), BFU-E (4,799 +/- 2,020) and CFU- GM (5,438 +/- 2,505) was found in patients as compared with normal controls. Both rIFN-alpha and rIFN-gamma (10 to 10(4) U/mL) produced a significant dose-dependent suppression of CFU-GEMM, CFU-Mk, BFU-E, and CFU-GM growth. Concentrations of rIFN-alpha and rIFN-gamma causing 50% inhibition of colony formation were 37 and 163 U/mL for CFU-GEMM, 16 and 69 U/mL for CFU-Mk, 53 and 146 U/mL for BFU-E, and 36 and 187 U/mL for CFU-GM, respectively. A marked synergistic effect was found between rIFN-alpha and rIFN-gamma: combination of the two agents produced inhibitory effects greater than or equivalent to those of 10- to 100- fold higher concentrations of single agents. These studies (a) confirm that circulating hematopoietic progenitors are markedly increased in MMM, (b) indicate that these presumably abnormal progenitors are normally responsive to rIFNs in vitro, and (c) show that IFNs act in a synergistic manner when used in combination. Because rIFN-gamma can downregulate collagen synthesis in vivo, this lymphokine could be particularly useful in the treatment of patients with MMM.     

Influence of peripheral blood admixture on the number of hematopoietic progenitor cells (CFU-GM and BFU-E) in human bone marrow aspirates

Peripheral blood contamination in human bone marrow aspirates was calculated from the ratios between the erythrocyte and nucleated cell counts in both the bone marrow aspirate and a simultaneously obtained peripheral blood sample. This method is based upon experimental data which showed that the erythrocytes in bone marrow aspirates are mainly derived from the intravascular blood compartment. A strong negative correlation was found between the peripheral nucleated cell fraction (FBl) and the number of myeloid progenitor cells in 65 bone marrow samples (correlation coefficient r = -0.51; p less than 0.001). A similar correlation was found between erythroid progenitor cells and peripheral blood fraction (r = -0.55; p less than 0.01). The culture conditions were continuously monitored, using large batches of frozen marrow samples as controls. The correction for peripheral blood admixture permits a more reliable and reproducible interpretation of the quantitative results obtained from studies on the number of clonogenic cells in human bone marrow aspirates. Moreover, the method allows the interpretation of data obtained from bone marrow samples which are heavily contaminated by peripheral blood.     

The role of interleukin 3 and interleukin 6 in the protection from 4-hydroperoxycyclophosphamide and the proliferation of early human hematopoietic progenitor cells

Previous reports have shown that interleukin 1 (IL-1) has radioprotective effects when given to mice 20 h before a lethal dose of irradiation and enhances granulocyte recovery in mice treated with cyclophosphamide. We have recently reported that IL-1 can provide protection for human bone marrow colony-forming cells including blast colony-forming cells (B1-CFC) treated with high doses of 4-hydroperoxycyclophosphamide (4-HC). In view of the recent reports that IL-1 induces interleukin 6 (IL-6) in fibroblasts and macrophages and that IL-6 and interleukin 3 (IL-3) are the main growth factors for B1-CFC, we have examined the ability of these interleukins to protect early human hematopoietic progenitor cells from the cytotoxic effects of 4-HC. In addition, we have also studied the ability of IL-3 to promote colony formation by 4-HC-treated bone marrow cells with or without IL-1 preincubation. In this study, we report that preincubation of bone marrow mononuclear cells with IL-3 or IL-6 prior to 4-HC results in no protection and, in fact, may be detrimental to early hematopoietic progenitor cells. On the other hand, addition of IL-3 to 5637-conditioned medium and erythropoietin enhanced colony formation by early progenitors following 4-HC treatment. These findings suggest that IL-3 and IL-6 are not responsible for the protection of early progenitor cells from 4-HC seen with IL-1, but that IL-3 does promote colony formation following 4-HC treatment.     

In vitro biology of human hematopoietic stem and progenitor cells

Hematopoietic stem and progenitor cells from human umbilical cord blood have been the focus of both basic and clinical research during the last 20 years. It has been clearly demonstrated that such sells possess higher proliferation and expansion potentials, as compared to their adult counterparts, and their capacity to reconstitute the hematopoietic system of mammals has also been shown. Different In vitro systems have been used to characterize the biology of these hematopoietic cells and some culture methods are being currently used to expand the numbers of such cells for clinical purposes.     

Megakaryocytic differentiation capacity of human pluripotent bone marrow progenitor cells CFU-GEMM in vitro after cryopreservation

The effect of cryopreservation on the pluripotent haemopoietic progenitors CFU-GEMM as well as on the megakaryocytic (CFU-Mk), erythroid (BFU-E) and granulocytic-monocytic (CFU-GM) progenitor cells was analyzed. Progenitor cell recovery after freezing, as determined in 5 experiments, averaged 89% for CFU-GEMM (range: 63% - 194%), 85% for CFU-Mk (range: 62% - 96%), 92% for BFU-E (range: 43% - 174%) and 60% for CFU-GM (range: 31% - 93%). Immunological analysis of individual mixed colonies using a double labelling immunoalkaline phosphatase slide technique and monoclonal antibodies against megakaryocytic and granulocytic cells revealed megakaryocytic cells in more than 79% (range: 73% - 94%) and 84% (range: 75% - 87%) of mixed colonies before and after freezing, respectively. Our results indicate that cryopreservation of human bone marrow cells does not alter the megakaryocytic differentiation capacity of the haemopoietic progenitor cells CFU-GEMM and CFU-Mk in vitro.     

Megakaryocyte differentiation capacity of human pluripotent bone marrow progenitor cells CFU-GEMM in vitro after cryopreservation*

The effect of cryopreservation on the pluripotent haemopoietic progenitors CFU-GEMM as well as on the megakaryocyte (CFU-Mk), erythroid (BFU-E) and granulocytic-monocytic (CFU-GM) progenitor cells was analyzed. Progenitor cell recovery after freezing, as determined in 5 experiments, averaged 89% for CFU-GEMM (range: 63% - 194%), 85% for CFU-Mk (range: 62% - 96%), 92% for BFU-E (range: 43% - 174%) and 60% for CFU-GM (range: 31% - 93%). Immunological analysis of individual mixed colonies using a double labelling immunoalkaline phosphatase slide technique and monoclonal antibodies against megakaryocytic and granulocytic cells revealed megakaryocyte cells in more than 79% (range: 73% - 94%) and 84% (range: 75% - 87%) of mixed colonies before and after freezing, respectively. Our results indicate that cryopreservation of human bone marrow cells does not alter the megakaryocytic differentiation capacity of the haemopoietic progenitor cells CFU-GEMM and CFU-Mk in vitro.     

Regulation of human peripheral blood BFU-E growth in vitro by leukaemic B-lymphocytes

We report a stimulating effect of leukaemic B-lymphocytes from anaemic and non-anaemic patients with CLL on the proliferation of normal peripheral blood BFU-E. Coculture of leukaemic B-cells at various concentrations (2.5 X 10(3)-10(6)) with 2.5 X 10(5) mononuclear cells from normal peripheral blood increased the number of BFU-E derived erythroid colonies. The same effect was observed when the number of target cells was varied in the presence of a fixed number of B-lymphocytes, with a clear linear relationship. B-cell conditioned medium gave a similar increase when added to the culture instead of B-cells. At high concentration of B-cells from anaemic patients, the size of the colonies was increased and a large number of macroscopic colonies was seen. The place of the B-cells in the regulation of erythroid progenitors in relation to monocytes and T-lymphocytes has still to be established.     

Long-term generation and expansion of human primitive hematopoietic progenitor cells in vitro

Although sustained production of committed human hematopoietic progenitor cells in long-term bone marrow cultures (LTBMC) is well documented, evidence for the generation and expansion of human primitive hematopoietic progenitor cells (PHPC) in such cultures is lacking. For that purpose, we attempted to determine if the human high proliferative potential colony-forming cell (HPP-CFC), a primitive hematopoietic marrow progenitor cell, is capable of generation and expansion in vitro. To that effect, stromal cell-free LTBMC were initiated with CD34+ HLA-DR-CD15- rhodamine 123dull bone marrow cells and were maintained with repeated addition of c-kit ligand and a synthetic interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein. By day 21 of LTBMC, a greater than twofold increase in the number of assayable HPP-CFC was detected. Furthermore, the production of HPP-CFC in LTBMC continued for up to 4 weeks, resulting in a 5.5-fold increase in HPP-CFC numbers. Weekly phenotypic analyses of cells harvested from LTBMC showed that the number of CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 CD34+ HLA-DR- cells increased from 10(4) on day 0 to 56 x 10(4) by day 21. To examine further the nature of the in vitro HPP-CFC expansion, individual HPP- CFC colonies were serially cloned. Secondary cloning of individual, day 28 primary HPP-CFC indicated that 46% of these colonies formed an average of nine secondary colony-forming unit--granulocyte-macrophage (CFU-GM)--derived colonies, whereas 43% of primary HPP-CFC gave rise to between one and six secondary HPP-CFC colonies and 6 to 26 CFU-GM. These data show that CD34+ HLA-DR- CD15- rhodamine 123dull cells represent a fraction of human bone marrow highly enriched for HPP-CFC and that based on their regeneration and proliferative capacities, a hierarchy of HPP-CFC exists. Furthermore, these studies indicate that in the presence of appropriate cytokine stimulation, it is possible to expand the number of PHPC in vitro.     

Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium.

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow, such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for > or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described, but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues, we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor, interleukin (IL)-3, and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand, steel factor, IL-3, IL-6, granulocyte colony-stimulating factor, and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days, approximately 40% of which included > or = 1 LTC-IC. In contrast, in similar cultures containing methylcellulose, input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications.     

Prostaglandin E acts at two levels to enhance colony formation in vitro by erythroid (BFU-E) progenitor cells

The prostaglandin E (PGE) enhancement of erythroid colony formation by human bone marrow erythroid progenitor cells (BFU-E) is mediated by a T8+ subset of lymphocytes. Medium was conditioned by bone marrow and blood T-lymphocytes and T-lymphocyte subsets (T8+, T8-, T4+, and T4- cells) in the absence or presence of PGE1 in order to determine if the cells could release a cell-free source of erythroid colony enhancing activity and what the conditions for this release would be. The T-lymphocyte conditioned medium was assayed for its effects on erythroid colony formation by nonadherent low-density T-lymphocyte depleted (NALT-) bone marrow cells plated in the presence of erythropoietin, hemin, phytohemagglutinin-stimulated leukocyte conditioned medium, or medium conditioned by 5637 cells, in the absence or presence of PGE1 and in the presence or absence of serum. PGE1 induced the release of an erythroid colony enhancing activity from the T8+ and T4-, but not from the T8- and T4+ subsets of lymphocytes, but this cell-free source of activity was only apparent if it was tested for colony formation in the presence of added PGE1. The release and action of the PGE1 induced T-lymphocyte erythroid enhancing activity did not require the presence of serum. Erythroid colony formation by NALT- bone marrow cells was not enhanced by PGE1 alone, by medium conditioned by T-lymphocytes in the absence of PGE1, or by PGE1 plus medium conditioned by T-lymphocytes in the absence of PGE1. The results suggest that the PGE1 enhancement of erythroid colony formation occurs by an apparently synergistic action on non-T-lymphocytes by PGE1 itself and by a factor or factors released from T8+ lymphocytes in response to PGE1.     

Growth kinetics of CFU-GEMM, BFU-E, and CFU-C in diffusion-chamber cultures of normal human bone marrow cells

The proliferation and differentiation of human pluripotent hemopoietic precursor cells CFU-GEMM as well as of the committed precursors BFU-E and CFU-C within conventional diffusion chambers (DC) was studied. Low-density human bone marrow cells from eight normal donors were cultured in DC implanted into 750-rad-preirradiated mice. Cells harvested from DC after 1-14 days were assayed for the content of CFU-GEMM, BFU-E, and CFU-C. The mean number of progenitor cells inoculated per DC on day 0 was 59 (range 12-144) for CFU-GEMM, 367 (range 165-878) for BFU-E, and 632 (range 122-1168) for CFU-C. After an initial decrease in the number of all progenitors, an increase of CFU-GEMM was observed in five out of eight experiments and in the number of BFU-E and CFU-C in seven and five experiments, respectively. The assessment of the percentage of precursor cells sensitive to ara-C demonstrated a rise in the proportion of cells in S phase after implantation into DC consistent with reentry into active proliferation. From the observed behavior of the CFU-GEMM it is concluded that the earliest detectable human precursor cells do proliferate within DC.     

Negative regulation of DNA synthesis in early erythropoietic progenitor cells (BFU-E) by a protein purified from the medium of C57BL/6 mouse marrow cells.

During studies on the influence of Fv-2 on the cycle state of the erythroid burst-forming unit (BFU-E), an activity was found in bone marrow supernatants from C57BL/6 (B6) mice that shut down DNA synthesis of the BFU-E in vitro. It acted within minutes, its action was completely reversed by a single wash, and it appeared specific to the BFU-E. The activity-causing substance, being macromolecular, heat stable, and trypsin-sensitive, was evidently a protein and was named negative regulatory protein. We purified a negative regulatory protein from a bone marrow-derived "B6 Pan" cell line with properties thus far indistinguishable from those of the negative regulatory protein obtained directly from B6 marrow. By gel filtration the protein has a Mr of approximately equal to 79,000, by cation- and anion-exchange chromatography it appears to be a neutral molecule at physiological pH, and the molecule does not bind to Con A. After five sequential chromatographic steps, we obtained a preparation active at a concentration of 25 ng/ml. Our findings are compatible with the hypothesis that quiescence of BFU-E with respect to DNA synthesis in vivo in B6 mice is mediated by negative regulatory protein molecules.     

Production of colony-stimulating factor(s) for granulocyte-macrophage and multipotential (granulocyte/erythroid/megakaryocyte/macrophage) hematopoietic progenitor cells (CFU-GEMM) by clonal lines of human IL-2-dependent T-lymphocytes

Human T-lymphocyte lines that were selected for recognition of HLA-DR6 antigen and were dependent for growth in vitro on an added source of interleukin-2 (IL-2) were derived from the peripheral blood of normal individuals. Each was tested for production of a lymphokine(s) with properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) using as target cells nonadherent cells from human long-term bone marrow cultures (LTBMC) or fresh marrow. Each of eight T-lymphocyte lines that were OKT3, OKT4, and HLA-DR positive produced GM-CSF that stimulated colony formation by both LTBMC cells and fresh marrow. Individually examined single-cell-derived bone marrow colonies growing in T-cell GM-CSF contained peroxidase-positive neutrophils, and macrophage-monocytes (GM-CFUc). Supernatant from a single-cell-derived T-cell clonal line designated F1 stimulated formation of granulocyte-macrophage colonies, megakaryocyte colonies, macroscopic erythroid bursts, and multipotential colonies containing erythroid cells, megakaryocytes, neutrophilic and eosinophilic granulocytes, and monocyte-macrophages (CFU-GEMM) in the presence of added erythropoietin. These data indicate that human IL-2-responsive T-lymphocytes produce lymphokine(s) that stimulate proliferation of primitive as well as committed hematopoietic stem cells, and implicate human T-lymphocytes in regulation of human multipotential hematopoietic stem cells in vivo.     

Effects of B cell stimulatory factor-1/interleukin 4 on hematopoietic progenitor cells

B cell stimulatory factor-1 (BSF-1)/Interleukin 4 (IL 4) is a T cell product originally characterized on the basis of its actions on B lymphocytes. Recently it has been reported that BSF-1 activates T cell and mast cell lines. We now provide evidence that BSF-1, purified to homogeneity, also has a broad spectrum of activity on hematopoietic progenitor cells (HPC). However, like its action on B cells, prolierative effects were only observed when BSF-1 was combined with an additional factor. Thus BSF-1, in costimulation with recombinant G-CSF, enhances the proliferation of granulocyte-macrophage progenitor cells (CFU-GM). BSF-1 increases the proliferation of CFU-e in the presence of recombinant erythropoietin (rEPO). Furthermore, BSF-1 induces, together with rEPO, colony formation by primitive erythroid (BFU-e) and multipotent (CFU-mix) progenitor cells comparable to that observed with rEPO and interleukin 3 (IL 3). BSF-1 is also active as a megakaryocyte colony-stimulating factor; in combination with recombinant interleukin 1, rEPO or the supernatant of the T cell hybridoma FS7-20.6.18, BSF-1 induces megakaryocyte colony formation (CFU-Mk). The same factors that synergize with BSF-1 also enhance CFU-Mk proliferation induced by IL 3. Although the precise mechanisms of action of BSF-1 on HPC is not yet known, we propose that BSF-1 represents an activation factor for HPC and prepares the progenitor cells to respond to specific growth or differentiation factors.     

Infection of hematopoietic progenitor cells by human cytomegalovirus.

The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.