Immunophenotypic analysis of myelodysplastic syndromes

BACKGROUND AND OBJECTIVES: In contrast with hematologic malignancies in which the value of immunophenotypic studies is well established, information on the immunophenotypic characteristics of myelodysplastic syndromes (MDS) is scanty. The main goal of the present study was to explore the immunophenotypic differences between patients with MDS and normal individuals, including changes in distribution of cell lineages as well as phenotypic aberrations and blockades in cell maturation pathways. DESIGN AND METHODS: In MDS the proportion of bone marrow CD34+ cells was higher than in normal patients but the most immature progenitors (CD34+CD38-) were less represented. By contrast the proportion of myelomonocytic CD34+ cells was greater than in normal individuals, translating into an increased myeloid/non-myeloid CD34+ hematopoietic progenitor cell ratio. RESULTS: This suggests that in MDS, the majority of CD34+ cells are already committed to the myeloid lineage. Upon analyzing the granulo-monocytic differentiation pathway, MDS patients showed an increased proportion of monocytic cells with a decreased percentage of cells of neutrophil lineage, leading to a lower neutrophil/monocytic cell ratio. Maturational arrests in the monocytic but not in the neutrophil differentiation pathway were observed. In refractory anemia with excess blasts in transformation (RAEB-t) such blockades mainly occurred during the earliest stages of differentiation but in the other MDS subtypes they occurred in later stages. INTERPRETATION AND CONCLUSIONS: Phenotypic aberrations occurred in 90% of patients and a high proportion of cases showed >=2 aberrations. In summary, our results show that, in addition to an abnormal distribution of the bone marrow cell compartment, MDS patients frequently show aberrant phenotypes and maturational arrests. Some of these features may help in cases in which the diagnosis of MDS is questionable.     

Combined immunophenotyping and DNA in situ hybridization to study lineage involvement in patients with myelodysplastic syndromes.

Clonality of myeloid and lymphoid cell fractions obtained from peripheral blood (PB) or bone marrow (BM) of five patients with a myelodysplastic syndrome (MDS), was studied by combined immunophenotypic analysis and DNA in situ hybridization. This novel technique enables quantitative and direct analysis of cytogenetic alterations in nondividing cells of distinct cell lineages. Four patients with a trisomy 8 and one patient with a translocation (1;7) were studied. For cell lineage determination, antibodies specific for progenitor cells (CD34), myeloid cells (CD15), monocytes (63D3), T cells (CD3), and B cells (CD19,20,22) were used. In one patient with a trisomy 8, BM cells were available and the erythroid lineage could be studied. For detection of cytogenetic aberrations, we used chromosome-specific repetitive DNA probes. In three patients, all nonlymphoid cells carried the cytogenetic abnormality; in two patients, mosaicism within these lineages was suggested by the relative low numbers (35% to 55%) of aberrant cells. None of the T or B cells of the five patients carried the chromosomal aberrations. We conclude that combined immunophenotyping and in situ hybridization is a feasible technique to study lineage involvement. Our data suggest that the chromosomal aberrations studied in MDS are restricted to the myeloid lineages.     

Apoptosis in relation to CD34 antigen expression in normal and myelodysplastic bone marrow

An increased bone marrow (BM) apoptosis is one of the mechanisms responsible for the ineffective hematopoiesis of myelodysplastic syndromes (MDS). It is controversial whether the excessive apoptosis in myelodysplasia predominantly involves the subset of progenitor cells or of maturing cells. We investigated the degree of apoptosis in MDS BM and its differences from normal marrow in relation to CD34 antigen expression. A double-labelling technique that combined the Tdt-mediated dUTP nick end labelling (TUNEL) method with immunocytochemistry for CD34 antigen was used on BM slides of 18 MDS patients and 11 controls. The apoptotic rate (AR) appeared significantly higher in CD34-negative than in CD34-positive cell subsets both in myelodysplastic and in normal BM. When MDS and normal CD34-negative cell populations were compared, a greater AR in MDS CD34-negative cells was found, while no statistical difference in AR resulted from the comparison between MDS and normal CD34-positive cell populations. Our results suggest that in myelodysplastic as well as in normal BM the apoptotic phenomenon predominantly involves the maturing cells. The increase in apoptotic levels which can be observed in myelodysplastic compared to normal BM seems to be mainly due to an increase in apoptosis in the differentiated cell population.     

Proliferation and differentiation of myelodysplastic CD34+ cells: phenotypic subpopulations of marrow CD34+ cells

In a search for a mechanism to explain the impaired growth of progenitor cells in patients with myelodysplastic syndromes (MDS), marrow CD34+ cells were purified up to 94.9% +/- 4.2% for normal individuals and 88.1% +/- 17.6% for MDS patients, using monoclonal antibodies and immunomagnetic microspheres (MDS CD34+ cells). Phenotypic subpopulations of these CD34+ cells were analyzed for CD38, HLA-DR, CD33, CD13, CD14, CD41 and CD3 plus CD19, in association with proliferative and differentiative capacities. The 15 studies performed included 12 MDS patients. Coexpression rate of CD13 significantly increased in the MDS CD34+ cell population with a value of 91.4% +/- 11.6% and ranging from 60.3% to 100%, and exceeded 99% in four studies, whereas that of normal CD34+ cells was 49.9% +/- 15.8%, ranging from 28.2% to 70.1% (P < .001). Coexpression rate of CD38, HLA-DR, CD33, CD14, and CD3 plus CD19 in MDS CD34+ cells did not significantly differ from that of normal CD34+ cells. The total number of colonies and clusters grown from 100 normal marrow CD34+ cells was 40.4 +/- 8.6, the range being from 27.2 to 50.3; this varied in MDS marrow CD34+ cells with a value of 34.0 +/- 28.7, the range being 0 to 95.9. The lineage of colonies and clusters promoted by MDS marrow CD34+ cells was predominantly committed to nonerythroid with impaired differentiation in 13 of 15 studies (87%). CD13 is first expressed during hematopoiesis by colony-forming unit granulocyte-macrophage and is absent in erythroid progenitors. Therefore, this study provides direct evidence for the lineage commitment of MDS CD34+ cells to nonerythroid with impaired differentiation and explains the mechanism of nil or low colony expression of MDS progenitor cells to erythroid lineage.     

Cytogenetic clonality analysis: typical patterns in myelodysplastic syndrome and acute myeloid leukaemia

The cell morphology and karyotype of bone marrow samples from 24 patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) were studied simultaneously with a combined technique of May-Grünwald-Giemsa (MGG) staining and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes. This enabled us to investigate cell lineage involvement in three malignant conditions: MDS ( n  = 12), leukaemia-transformed MDS (LT-MDS) ( n  = 5) and de novo AML ( n =7). In MDS we found blasts and often significant proportions of mature granulocytic and erythroid cells to be cytogenetically abnormal. Percentages of granulocytic and erythroid cells with cytogenetic aberrations were generally less than those of blasts. These data support the involvement of a transformed pluripotent stem cell that has retained maturation abilities. In two patients with chronic myelomonocytic leukaemia (CMMoL) the clonal involvement of monocytes was predominant. Results in the five patients with LT-MDS were similar to those in MDS. In the bone marrow of five of the seven de novo AML patients the cytogenetic abnormalities were restricted to the blasts and did not include the more mature granulocytic or erythroid populations. In the other two patients with AML, both with a t(8;21) and a loss of the Y chromosome, high percentages of mature neutrophils were cytogenetically abnormal. These patterns of clonal lineage involvement in MDS, LT-MDS, t(8;21) AML and AML appear typical and may be of clinical use, for example, for distinguishing LT-MDS from de novo AML in newly presenting patients.     

Characteristics of bone marrow cell dysplasia and its effectiveness in diagnosing myelodysplastic syndrome

Background: Although dysplasia plays an important role in the diagnosis of myelodysplasia syndrome (MDS), its morphologic variety and irregularity result in difficulties in its clinical application.

Methods: Bone marrow smears from cases with MDS and non-clonal disease were collected and performed microscopy analysis. We respectively recorded the percentage of specific dysplastic cells (PSDC) and incidence of specific dysplasia (ISD) of each dysplastic type in three hematopoietic cell lineages for the comprehensive analysis of diagnostic efficacy to MDS.

Results: Compared with non-clonal anemia, the PSDCs and ISDs of the four specific dysplastic types as petal nucleus and internuclear bridging in erythroid lineage, pseudo-Pelger-Huet in granulocytic lineage and lymphoid small megakaryocyte in megakaryocytic lineage were significantly higher in MDS; and their area under the curves were all greater than 0.600. If the dysplastic rate in each lineage was higher than 10%, their corresponding false positive rates (FPRs) were below 0.033, 1?×?10^?4 and 1?×?10^?4, respectively. If the dysplastic rates in three cell lineages reached 0.065, 0.045 and 0.040, respectively, their corresponding FPRs were all below 0.050.

Conclusion: Four specific dysplastic types possess higher diagnostic efficacy for the diagnosis of MDS. Though the dysplastic rate over 10% in any hematopoietic cell lineage presents a lower FPR, it is possibly considered to lower the diagnostic threshold of MDS if a specific dysplastic type with higher diagnostic efficacy presents.     

Clonal analysis of myelodysplastic syndrome: monosomy 7 is expressed in the myeloid lineage, but not in the lymphoid lineage as detected by fluorescent in situ hybridization.

Conflicting results have been published on whether or not myelodysplastic syndromes (MDS) affect all cell lineages. Involvement of myeloid and erythroid cell lineages has been regularly observed, but it remains controversial whether the different lymphoid cell lineages are involved. In this study of eight patients with MDS associated with monosomy 7, fluorescent in situ hybridization (FISH) was used to enumerate the chromosomes 7 in interphase cells. With the probe D7Z1, the rate of false-positive detection of monosomy 7 was 3% +/- 2% in normal cells. T- and B-cell lines were established from eight patients with MDS and monosomy 7. As determined by FISH in interphase cells, 1.9% (0% to 3%) of the cells in the B-cell lines showed one fluorescent spot and 1.1% (0% to 2.9%) of the cells in the T-cell lines. These values do not differ from normal values. However, the possibility that normal cells were selected when the T- and B-cell lines were established could not be excluded. Therefore, peripheral blood cells were obtained, separated according to surface markers specific for lymphoid and myeloid cell lineage with a cell sorter, and analyzed for the expression of monosomy 7 by FISH. Antibodies recognizing T cells (CD3), B cells (CD20), natural killer (NK) cells (CD57), monocytes and granulocytes (low and high expression of CD11b antigen), and myeloid progenitors (CD33) were used to separate cells. The expression of monosomy 7 in the T cells, NK cells, and B cells did not differ from control values. These results in the lymphoid subpopulations are in stark contrast with the observations in the myeloid populations; the percentage of cells with monosomy 7 ranged from 9% to 78% (controls: 6% +/- 2%) in cells with low CD11b expression, 20% to 89% in cells with a high expression of the CD11b antigen (controls: 7% +/- 3%), and 23% to 91% in the CD33 positive cells (controls: 5% +/- 3%). The results of this study suggest that monosomy 7 does not usually affect lymphoid subpopulations but is restricted to committed progenitor cells with the capacity to differentiate into mature myeloid cells.     

Proliferation and differentiation of myelodysplastic CD34+ cells in serum-free medium: II. Response to combined colony-stimulating factors

Abstract: To investigate the role of colony stimulating factors (CSFs) in the proliferation and differentiation of progenitor cells from myelodysplastic syndromes (MDS), marrow progenitor cells from 18 MDS patients were highly purified using CD34 monoclonal antibody and immunomagnetic microspheres (MDS CD34+ cells). These cells were cultured in serum-free medium with various combinations of five colony stimulating factors (CSFs): recombinant human interleukin-3 (rIL-3), granulocyte/macrophage-CSF (rGM-CSF), granulocyte-CSF (rG-CSF), macrophage-CSF (rM-CSF), and erythropoietin (rEP). Among the tested CSFs, such as rM-CSF, rG-CSF, rGM-CSF and rIL-3, a combination of the first three CSFs was the most effective stimulus for the proliferation of non-erythroid MDS progenitor cells. An increase of undifferentiated “blast” cell colonies in 5/18 MDS patients occurred and these 5 patients belonged to the high-risk group. In the presence of these three CSFs, rIL-3 had no effect on the proliferation and differentiation of MDS CD34+ cells; however, IL-3 was efficient for the proliferation of MDS CD34+ cells to the erythroid lineage. rGM-CSF or rIL-3 alone did not efficiently support proliferation and differentiation of CD34+ cells. M-CSF is present in normal human serum at a concentration of 550 ±110 U/ml, a concentration exceeding that used in this study (100 U/ml). Therefore, in vivo administration of G-CSF combined with GM-CSF to MDS patients may be one of the most effective CSF combinations for proliferation of MDS progenitor cells to the non-erythroid lineage. However, the effect on the capacity for differentiation was minimal, especially in patients belonging to the high-risk group.     

The significance of myelodysplasia in untreated patients with multiple myeloma]

Microscopic analysis of bone marrow smears from ten untreated patients with multiple myeloma (MM) revealed that seven patients had myelodysplastic changes. Of these, five patients had anemia alone while the other two had anemia and leucopenia. The myelodysplastic changes seen in MM were less extensive than those seen in myelodysplastic syndrome (MDS). Moreover, the dysplastic changes in MM were determined to be limited to two or three lineage cells. Dysplastic changes were observed even after clinical signs of MM had improved due to therapy. We consider that the myelodysplastic changes seen in MM can be attributed to MM itself, rather than to the coexistence of MDS and MM. Such findings suggest that the pathogenesis of MM involves a common stem cell which differentiates into multiple lineage cells.     

Molecular cytogenetics of stem cells: target cells of chromosome aberrations as revealed by the application of fluorescence in situ hybridization to fluorescence-activated cell sorting

The lineage involvement in stem cell disorders, such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), remains unclear. To explore this issue, we used fluorescence in situ hybridization for cells sorted by fluorescence-activated cell sorting (FACS) from 12 patients with chronic-phase CML. Philadelphia chromosome (Ph) was found in pluripotent stem cells (CD34+Thy-1+), B cells (CD34+CD19+), and T/natural killer (NK) progenitor cells (CD34+CD7+) collected by FACS from bone marrow cells. B (CD19+), T (CD3+), and NK (CD3-CD56+) cells showed a marked decrease in Ph+ cells between progenitor cells and mature cells The Ph+ T and NK cells decreased to below background levels. These data suggest that Ph+ lymphocytes either do not differentiate or are eliminated during their maturation process Among 7 MDS patients associated with trisomy 8, sorted lymphocytes from peripheral blood did not have +8. CD34+ subpopulations from bone marrow including B,T/NK progenitors, and pluripotent progenitor cells also did not have +8.Trisomy 8 was identified from the level of multipotent colony-forming units (CD34+CD33+), and the lymphoid lineage was not involved. Thus, MDS with trisomy 8 conceivably arises from nonlymphoid progenitor cells, sparing T, B, or NK cells. Further studies using molecular cytogenetics may clarify the mechanism of leukemia happening at the level of stem cells.     

Establishment and characterization of a B-cell line derived from a patient with a myelodysplastic syndrome which expresses myelomonocytic and lymphoid markers

We describe a novel continuous B-cell line (PV-90) derived from a patient with myelodysplastic syndrome (MDS) and originating from spontaneous infection with the Epstein-Barr virus (EBV). The patient progressed to acute myeloblastic leukaemia (AML) 5 months after clinical onset of MDS. PV-90 is of clonal origin as indicated by the presence of immunoglobulin (Ig) gene rearrangements, monoclonal surface immunoglobulins, and a single DNA restriction fragment corresponding to the EBV genomic termini. PV-90 cells also express a number of myelomonocytic markers, including alpha-naphthyl acetate esterase (ANAE), coagulation factor XIII, and CD68 antigen. Moreover, PV-90 cells constitutively express the c-fms proto-oncogene mRNA as the patient's blast cells did. Whereas a trisomy 11 (+11) was found in the patient's bone marrow cells, PV-90 cells had a normal karyotype initially, but at 4 months showed two different and independent chromosomal abnormalities: 90, XX, -Y, -Y, t(9;16) (q11;p13), and 90, XX, -Y, -Y, t(17;18) (p13;q21), the latter possibly involving the p53 (17,p13) and bcl-2 (18, q21) proto-oncogenes. The early development of these chromosomal aberrations is consistent with a genetic instability of PV-90 cells. Expression of bi-lineage markers and genetic instability may suggest that PV-90 cells originated from transformation of a myelodysplastic progenitor cell capable of both myeloid and B-cell differentiation. The PV-90 cell line might be useful in a number of studies, including the possible role of c-fms in cell differentiation, pathogenetic mechanisms of human preleukaemia and lineage promiscuity in acute leukaemia.     

Expression of p15(ink4b) gene during megakaryocytic differentiation of normal and myelodysplastic hematopoietic progenitors.

In myelodysplastic syndrome (MDS), the expression of the cyclin-dependent kinase inhibitor p15(ink4B) (p15) is frequently decreased because of the aberrant methylation of the gene promoter; p15 is normally up-regulated during megakaryocytic differentiation. It was hypothesized that p15 methylation and deregulation of gene expression contribute to defective megakaryocytopoiesis in patients with MDS. Here it is shown that the increasing autocrine production of TGF-beta1 stimulates megakaryocytic differentiation in normal CD34(+) cells and that p15 mediates, at least in part, this effect. This TGF-beta1-dependent pathway is altered in MDS CD34(+) progenitors because of p15 methylation. The demethylating agent 2-deoxyAZAcytidin can restore the normal demethylated state of the p15 gene and increase its expression. Nevertheless, MDS CD34(+) cells only poorly differentiate to the megakaryocytic lineage. These findings suggest that p15 methylation occurs in a neoplastic clone with a profound defect of cell proliferation, survival, and differentiation that cannot be overcome by using a demethylating drug.     

Knockdown of Hspa9, a del(5q31.2) gene, results in a decrease in hematopoietic progenitors in mice

Heterozygous deletions spanning chromosome 5q31.2 occur frequently in the myelodysplastic syndromes (MDS) and are highly associated with progression to acute myeloid leukemia (AML) when p53 is mutated. Mutagenesis screens in zebrafish and mice identified Hspa9 as a del(5q31.2) candidate gene that may contribute to MDS and AML pathogenesis, respectively. To test whether HSPA9 haploinsufficiency recapitulates the features of ineffective hematopoiesis observed in MDS, we knocked down the expression of HSPA9 in primary human hematopoietic cells and in a murine bone marrow-transplantation model using lentivirally mediated gene silencing. Knockdown of HSPA9 in human cells significantly delayed the maturation of erythroid precursors, but not myeloid or megakaryocytic precursors, and suppressed cell growth by 6-fold secondary to an increase in apoptosis and a decrease in the cycling of cells compared with control cells. Erythroid precursors, B lymphocytes, and the bone marrow progenitors c-kit(+)/lineage(-)/Sca-1(+) (KLS) and megakaryocyte/erythrocyte progenitor (MEP) were significantly reduced in a murine Hspa9-knockdown model. These abnormalities suggest that cooperating gene mutations are necessary for del(5q31.2) MDS cells to gain clonal dominance in the bone marrow. Our results demonstrate that Hspa9 haploinsufficiency alters the hematopoietic progenitor pool in mice and contributes to abnormal hematopoiesis.     

Abnormal megakaryopoiesis in patients with myelodysplastic syndromes: analysis of cellular and humoral defects

In 13 patients with myelodysplastic syndrome (MDS) mature and immature erythropoietic (CFU-E, BFU-E), granulopoietic (CFU-GM) and megakaryopoietic (CFU-Meg) colony formation from human bone marrow mononuclear cells was evaluated in a microagar culture system. All but three patients exhibited abnormal CFU-Meg. The defect of CFU-Meg paralleled the reduction of BFU-E, whereas CFU-GM number declined to a lesser extent. Not only the CFU-Meg number, but also the number of megakaryocytes (Mk) per colony was reduced suggesting an additional functional CFU-Meg defect. Megakaryocytic growth factor (Meg-CSF) abnormalities in MDS patients were detected using normal nonadherent T-lymphocyte depleted bone marrow cells as target cells for serum testing. Even for sera from patients with a reduction of platelets and bone marrow megakaryocytes Meg-CSF levels were not increased. No cellular or humoral inhibition could be detected in an MDS patient with a 5q- karyotype, who had an isolated defect of the megakaryocytic cell lineage at presentation. Some patients revealed a spontaneous formation of mixed erythrocytic, granulocytic and megakaryocytic clusters in the presence of fetal calf serum or autologous patient serum, probably representing autonomous proliferation of the malignant clone. In conclusion, both cellular and humoral factors can cause abnormalities of the megakaryocytic cell lineage in MDS patients.     

骨髓增生异常综合征免疫表型分析

目的：探讨免疫表型测定在骨髓增生异常综合征（MDS）诊断及分型中的价值。方法：采用一组系列相关单克隆抗体和流式细胞术对19例MDS患者免疫表型进行检测，并对其中的10例进行了细胞遗传学检查。结果：MDS患者骨髓单个核细胞（MNC）CD13，CD33抗原表达率平均分别为36．69％和41．86％，而T淋巴系抗原CD3的表达平均仅为14．49％，且随着低危的难治性贫血（RA）向高危的难治笥贫血伴原始细胞增多（RAEB）或难治性贫血伴原始细胞增多－转变型（RAEB-t)的进展，较早期的髓系抗原CD13，CD33及干（祖）细胞抗原CD34的表达升高，并伴有T淋巴系抗原CD3的表达降低。10例进行了细胞遗传学检查的患者中，5例有染色体核型异常，染色体核型异常的患者与染色体核型正常的患者在抗原表达上存在区别。结论：对MDS患者进行免疫表型检查有助于MDS的诊断分型研究。     

The implications of myelodysplastic syndrome – associated chromosomal abnormalities in the development of graft‐versus‐host disease

There is a growing body of evidences that acquired chromosomal abnormalities in bone marrow (BM) cells are associated with clinical manifestations of myelodysplastic syndrome (MDS). However, to our knowledge, there are no reports that describe the association between chromosomal abnormalities in MDS and graft‐versus‐host disease (GVHD) after allogeneic stem cell transplantation (allo‐SCT). Here, we describe two MDS cases with trisomy 8 and der(1;7)(q10;p10), who developed severe GVHD after allo‐SCT. We analyzed cytokine production and cell survival of monocytes from these patients with MDS before allo‐SCT, in comparison with healthy controls or an MDS patient with a different chromosomal abnormality, who has not developed GVHD. The monocytes from MDS patients with trisomy 8 and der(1;7)(q10;p10) produced a larger amount of pro‐inflammatory cytokine, tumor necrosis factor‐α, and a smaller amount of anti‐inflammatory cytokine, interleukin‐10, on stimulation with Toll‐like receptor (TLR) ligands. In addition, the monocytes from MDS cases with GVHD showed a decrease in apoptotic cell death upon stimulation with TLR ligands. We also detected host‐derived pro‐inflammatory antigen‐presenting cells (APCs) in skin GVHD lesions after allo‐SCT. These data suggest that trisomy 8 and der(1;7)(q10;p10) may be associated with the development of severe GVHD, by prolonging survival of pro‐inflammatory host‐derived APCs in GVHD lesions.     

Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells.

Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.     

Myeloid differentiation antigen defined by a monoclonal antibody IF10

A monoclonal antibody of IgM class that defines the antigen present on human peripheral blood granulocytes was produced and characterized. This monoclonal antibody (IF10) was made from a single fusion between P3-X63-Ag8-U1 (P3U1) myeloma cells and splenocytes from a BALB/c mouse immunized against human cultured monocytoid cell line THP-1 cells. IF10-defined antigen(s) was expressed on the cells of granulocyte lineage such as peripheral blood granulocytes and metamyelocytes, myelocytes, promyelocytes, and a part of myeloblasts in the normal bone marrow, whereas it was not detected on resting and activated T lymphocytes, B lymphocytes, adherent monocytes, and thymocytes. The IF10-defined antigen was also expressed on cultured monocytoid cell lines as well as myeloid and myeloid/erythroid (K-562) cell lines. T lymphoblastoid cell lines, particularly those representing the T cells at an early thymocyte level, and a part of null cell lines were also reactive to IF10. Therefore, IF10 may be a unique monoclonal antibody that defines an antigen(s) which is expressed on almost whole stages of granulocytes and early stages of macrophages and T-cell lineages, and possibly during very early stages of erythroid lineage.