Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TÜ 67)

Summary Anti-TÜ 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TÜ 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TÜ 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TÜ 67 antibody.Supported by the Deutsche Forschungsgemeinschaft (He 1380-2/1)     

Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.     

Separation of hemopoietic cells from adult mouse marrow by use of monoclonal antibodies

Primitive hemopoietic progenitor cells from adult mouse marrow have been substantially enriched by virtue of a negative selection procedure with monoclonal antibodies. It has been possible to segregate erythroid progenitor cells at distinct stages of differentiation on the basis of their cell surface antigens. This has been achieved with two monoclonal antibodies reactive with the mature elements of bone marrow. YBM 34.3 binds to a heat-stable antigen expressed on B lymphocytes, neutrophils, and cells of the erythroid lineage. YBM 6.1 reacts with cells of the neutrophil, eosinophil, and monocyte series but does not bind to colony- forming cells. Separation is achieved by indirect immunoadsorption (panning) with YBM 34.3 on Protein-A-coated plastic plates followed by FACS II cell sorting with YBM 6.1. The combined procedures yield a marrow population containing 58% immature cells (blasts, promyelocytes, and myelocytes) and 9.5% clonogenic cells. In addition, differential binding of YBM 34.3 can be used to segregate erythroid progenitor cells at distinct stages of differentiation (day 7 BFU-E, day 5 BFU-E and CFU- E) either by cell sorting or panning. It is shown that both techniques give a comparable degree of resolution of the different cell types with, however, an appreciable advantage of panning over cell sorting in allowing the rapid handling of large numbers of cells.     

Differential effect in vitro of tumor necrosis factor-alpha (TNF) on normal and virus-infected erythroid progenitors from Friend virus (FVA)-infected mice.

In vivo administration of tumor necrosis factor-alpha (TNF) suppresses both normal and Friend virus (FVA)-infected erythroid progenitor cells (CFU-E). To examine the mechanism of erythroid suppression by TNF, we examined TNF's direct effect on normal and virus-infected cells in vitro. Productively infected fibroblast cell lines, fresh acute virus-infected spleen cells, and virus-infected CFU-E were sensitive, whereas uninfected CFU-E were resistant to TNF cytotoxicity in vitro. When FVA-infected erythroblasts were depleted from the spleen cell population in vitro with antivirus antibodies, TNF suppression of the remaining (uninfected) cells was abrogated. In contrast, both normal and virus-infected macrophage progenitor cells and immature erythroid progenitor cells were equally sensitive to TNF cytotoxicity in vitro. Normal erythroblasts had significantly fewer TNF receptors than FVA-infected erythroblasts, which also were morphologically less mature. These results suggest that TNF can differentially suppress late-stage virus-infected erythroid progenitors in vitro.     

Surface antigenic determinants on human pluripotent and unipotent hematopoietic progenitor cells

RFB-1 is a monoclonal antibody previously shown to react with granulocyte-monocyte progenitors (CFU-GM) and immature lymphoid cells in human bone marrow. RFB-HLA-DR is a monoclonal antibody that reacts with HLA-DR (la-like) antigens. The present study shows that the bone marrow subset reactive with both RFB-1 and RFB-HLA-DR contains all the cells that give rise to mixed hematopoietic colonies (derived from CFU- GEMM; a pluripotent human progenitor cell) as well as to megakaryocytic (megakaryocyte-CFU-derived) and erythropoietic (derived from erythroid burst-forming units, BFU-E) colonies, as shown by fluorescence- activated cell sorting and complement-mediated cytotoxicity. These results indicate that CFU-GEMM, BFU-E, and megakaryocyte-CFU express RFB-1 and la-like antigens. RFB-1 antigen is also expressed on erythroid colony-forming units (CFU-E). RFB-1 and RFB-HLA-DR are useful reagents in the study of hematopoietic stem cells.     

Hematopoietic progenitor cell expression of the H-CAM (CD44) homing-associated adhesion molecule.

We explored the expression of a lymphocyte homing-associated cell adhesion molecule (H-CAM, CD44) on hematopoietic progenitors. We demonstrate that immature myeloid and erythroid leukemic cell lines stain intensely with monoclonal antibodies Hermes-1 and Hermes-3, which define distinct epitopes on lymphocyte surface H-CAM, a glycoprotein involved in lymphocyte interactions with endothelial cells. Using fluorescence-activated cell sorting (FACS), human marrow cells were fractionated into Hermeshi, Hermesmed, and Hermeslo populations according to the expression of both the Hermes-1 and Hermes-3 epitopes. Granulocyte-macrophage colony-forming unit and erythroid burst-forming unit precursors were found predominantly in the brightly positive fractions. Two-color FACS analysis confirmed that the My10 (CD34) positive populations of cells in bone marrow, which contain most of the progenitor cell activity, are brightly positive for Hermes-1. Finally, we demonstrate that among bone marrow cells, the highest levels of H-CAM are expressed on myeloid and erythroid progenitors as well as mature granulocytes and lymphocytes. Thus we provide evidence that molecules related or identical to the H-CAM homing receptor are expressed on marrow progenitor cells. H-CAM may contribute to progenitor cell interactions with marrow endothelial and stromal cell elements important to the maintenance and regulation of hematopoiesis.     

Identification and characterization of a differentiation-specific antigen on normal and malignant murine erythroid cells

Rauscher murine erythroleukemia cells grow continuously in vitro and undergo terminal differentiation in response to the physiological inducer erythropoietin. In the course of this developmental process they express many erythroid-specific markers. In order to investigate the expression of cell surface determinants during Rauscher cell differentiation we generated monoclonal antibodies to uninduced cells. Using an anti-Rauscher cell monoclonal antibody, we have identified a cell surface determinant, designated ERY-1, that is present on normal murine erythroid cells. This determinant is apparently absent from the early progenitor BFU-E, but is present on the more mature progenitors CFU-E and CFC-E. It disappears during erythroid maturation and is absent from the mature erythrocyte. This pattern of ERY-1 expression is exhibited with remarkable fidelity during the erythropoietin-induced differentiation of Rauscher cells. Such differentiation-specific expression of the ERY-1 determinant suggests that it may play a functional role in erythropoiesis.     

Expression of two natural killer cell antigens, H-25 and H-366, by human immature myeloid cells and by erythroid and granulocytic/monocytic colony-forming units

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

Phenotyping of peripheral blood hemopoietic progenitor cells--in vitro cultures using CD34-/CD33-immunomagnetic purging.

In contrast to many detailed studies on the antigenic profile of hemopoietic progenitor cells from human bone marrow, sparse information, so far, has been gathered with regard to the antigen expression of hemopoietic progenitors present in peripheral blood. Previous studies by multiparameter flow-cytometry have revealed substantial differences of the coexpression of the CD33-, CD19-, and CD74- antigens, respectively, on CD34-positive cells from blood versus those from bone marrow, respectively. Immunomagnetic purging with monoclonal antibodies detecting the CD34-, and the CD33- antigen, respectively, has been used to further characterize the expression of these antigens on day 8 and d-14 granulocyte/macrophage and erythroid colonies as grown from circulating progenitor cells. Purging with CD34 monoclonal antibody abrogated all colony formation, whereas purging with CD33 antibody led to differential inhibition of the various progenitors. Purging bone marrow cells with CD34 antibody, an inhibition of only about 25% was observed with regard to erythroid colonies, whereas an inhibition of about 85% was observed for CFU-GM. These findings reinforce the view that circulating progenitor cells represent relatively immature stages of differentiation, when compared to bone marrow progenitors. Particularly, d-8 erythroid colonies from blood do not represent the equivalent of the genuine CFU-E as described from bone marrow, but they seem to be early stages of BFU-E development.     

Expression of CD11, CDw15, and transferrin receptor antigens on human hematopoietic progenitor cells

The expression of transferrin receptor-associated antigens and of CD11 and CDw15 antigens was investigated on myeloid committed progenitor cells (CFU-GM day 10, CFU-GM day 7, and cluster-forming cells [CFC] day 4), on erythroid committed progenitor cells (BFU-E and CFU-E), and on multilineage progenitor cells (CFU-GEMM). Both complement-dependent cytotoxicity and fluorescence-activated cell-sorting assays were performed. Complement-dependent cytotoxicity appeared to be the more sensitive assay. Transferrin receptor-associated antigens appeared to be clearly present on all myeloid and erythroid committed progenitor cells, but were found to be only weakly expressed on CFU-GEMM. CD11 antigens appeared to be strongly expressed only on mature granulocytes, monocytes, and certain lymphocytes, but not significantly on myeloid committed precursor cells. Surprisingly, CD11 antigens were weakly, but significantly, present on CFU-E. CDw15 antigens appeared to be restricted to myeloid differentiation and were increasingly expressed from CFU-GM day 10 to CFC day 4. Thus, antitransferrin receptor, CD11, and CDw15 antibodies can be used to separate hematopoietic progenitor cells and may be useful tools in the study of hematopoietic differentiation.     

A and B blood group antigen expression on mixed colony cells and erythroid precursors: relevance for human allogeneic bone marrow transplantation

Using anti-A and anti-B blood group monoclonal antibodies and fluorescent activated cell sorting of human bone marrow, A (or B) blood group antigen was shown to be on 5.2 ± 5.9 (meanfSD) % of CFU-GEMM and 12 ± 5 ± 19.6% of the erythroid burst forming cells (designated BFU-GEMM) as defined by the mixed colony assay, and 49.5±20% of the BFU-E and 83.5±9.9% of the CFU-E as defined by the erythroid colony assay. This antigen expression on the BFU-GEMM is consistent with the concept that erythroid bursts stimulated by leucocyte conditioned medium are less mature, and are closer in development to the pluripotent stem cell than the BFU-E. These results help to explain the delayed erythropoiesis, and perhaps impaired engraftment of all cell lineages, that may occur in some recipients of ABO incompatible bone marrow transplants, with persistent and high anti-A titres.     

Myeloid-associated differentiation antigens on stem cells and their progeny identified by monoclonal antibodies

Within the hematopoietic system, monoclonal antibodies reactive with antigenic determinants, expressed in a lineage- and stage-restricted fashion, can be used to map myeloid differentiation. We have generated a series of monoclonal antibodies that reacts with myeloid-associated determinants on committed myeloid stem cells and their progeny. Their reactivity with peripheral blood cells was identified by immunofluorescence assays, with bone marrow cells by fluorescence- activated cell sorting, and with committed hematopoietic progenitor cells by both cytotoxic assays and fluorescence-activated cell sorting. Antibody 1G10, which has previously been reported to react with cells of the granulocytic lineage and with a minor subset of mature monocytes, was shown to react with granulocyte-macrophage colony- forming units (CFU-GM). Three antibodies not previously characterized (T5A7, L4F3, L1B2) were shown to react with both granulocytic and monocytic cells and in fluorescence-activated cell sorting studies to detectably stain granulocytic cells at different stages of maturation. These three antibodies also react with CFU-GM, two (L4F3 and L1B2) reacting with all CFU-GM, while T5A7 reacts with only a portion of the day 7 CFU-GM. Antibody L4F3 also reacts with a portion of erythroid burst-forming units (BFU-E). In contrast, the previously reported antibody 5F1, which reacts with monocytic cells, nucleated erythroid cells, and platelets, was shown to react with erythroid colony-forming units (CFU-E). Potential applications of these antibodies to studies of normal and malignant hematopoiesis are discussed.     

Enrichment of myeloid progenitor cells from normal human bone marrow using an immune-rosette technique

In this study we have developed methods for purification of myeloid progenitor cells (CFU-Cs) from normal human bone marrow cells. Bone marrow aspirates were obtained from volunteers, and mononuclear cells (MNCs) were separated by Ficoll-Hypaque gradient centrifugation. T- and B-lymphocytes, monocytes, mature granulocytes, and erythroid precursors were eliminated by an immune-rosette technique using a panel of murine monoclonal antibodies and immunoglobulin (Ig)-coated sheep red blood cells (SRBCs). MNCs were treated with OKT3, B1, M3, Mo5, and EP1 monoclonal antibodies, which are reactive with T cells, B cells, monocytes, granulocytes, and erythroid precursors, respectively. Antibody-treated MNCs were incubated with SRBCs that had been coated with goat antirabbit IgG F(ab')2 and rabbit antimouse Ig for immune rosetting. Rosetted cells were then separated from nonrosetted cells in Ficoll-Hypaque. Nonrosetted cells were, in the second step, treated with an OKIa1 monoclonal antibody and again separated into an Ia+ and Ia- cell fraction by the same manner; 39% +/- 19.2% (mean +/- 1 SD, range 16.3%-75.4%) of CFU-Cs (colonies plus clusters) were recovered in the OKT3-, B1-, M3-, Mo5-, EP1- cell fraction, and the number of CFU-Cs grown in semisolid agar was 149.6 +/- 73.0 (64.0-309.0)/10(4) plated cells in this purified fraction, representing an enrichment of 14.2 +/- 6.4 (6.0-27.3)-fold when compared with unseparated marrow cell fractions. CFU-Cs were enriched 17.7 +/- 8.6 (6.1-28.3)-fold in the Ia+ cell fraction. These purified myeloid precursors would be of value for in-depth studies of the interactions between hematopoietic progenitor cells and regulatory factors that influence their proliferation and differentiation and also of drug metabolism and determinants of cytotoxicity.     

Surface phenotypes of human hemopoietic progenitor cells defined by monoclonal antibodies

A panel of ten monoclonal antibodies which react with antigens present on the surface of myeloid leukemic cells was used to investigate the distribution of these antigens on normal hemopoietic stem cells and progenitor cells at various stages of maturity. A population of immature cells, possibly stem cells, that are capable of regenerating CFU-GM in long-term marrow cultures reacts with four antibodies recognizing antigens abundantly expressed in leukemic cells, but does not react with antibodies against Ia-like molecules or against carbohydrate determinants specific for myeloid cells. Progenitor cells that form mixed colonies in semisolid medium (CFU-GEMM), early erythroid (BFU-E) and early myelomonocytic (type 1 CFU-GM) progenitors retain the antigens present on the hypothetical stem cell population and begin to express Ia-like antigens. As they differentiate, myeloid and erythroid progenitors undergo a series of quantitative and qualitative shifts in surface phenotype. They begin to express stage- related, lineage-specific antigens and cease expressing antigens common to early cells of different lineages. The identification of antigens present on very immature normal progenitor cells should be valuable in future studies aimed at the detailed characterization of this relatively little-known hemopoietic cell population.     

CD64/Fc gamma RI is a granulo-monocytic lineage marker on CD34+ hematopoietic progenitor cells

The aim of this study was to identify markers specific for granulo- monocytic commitment of progenitor cells. Large panels of antibodies were screened for selective staining of subsets of CD34+ cells from fetal and adult bone marrow. Flow cytometric analysis showed that CD64/fc gamma RI was undetectable on noncommitted progenitor cells (CD34++, CD38-/lo, HLA-DR+) and expressed on a subset of lineage- committed progenitors (CD34+, CD38+) with higher mean orthogonal light scatter than the remaining CD34+ cells. The CD34+, CD64+ cells were CD19- and the majority were CD45RA+, CD71lo, suggesting that CD64 recognized granulomonocytic progenitor cells. Specificity of CD64 for the granulo-monocytic lineage was shown by demonstrating that colonies arising from CD34+, CD64+ cells consisted of 98% +/- 2% colony-forming unit-granulocyte-macrophage (CFU-GM) in semisolid medium containing stem cell factor (SCF), interleukin-3 (IL-3), IL-6, granulocyte- macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). In contrast, 63% +/- 15% of the colonies from the CD34+, CD64- cells were burst-forming unit-erythroid/colony-forming unit-erythroid (BFU-E/CFU-E). Furthermore, four-color immunofluourescence and cell sorting was used to analyze the progeny of cells cultured in liquid medium containing identical cytokines as used in the semisolid medium. This analysis showed that CD34+, CD64+ cells gave rise to 83% +/- 10% granulo-monocytic cells whereas progeny of the CD34+, CD64- cells contained 81% +/- 11% erythroid cells. Neutrophils as well as basophils and monocytes/macrophages were present in the cultures from CD34+, CD64+ cells, showing that this population contains progenitors of most types of granulo-monocytic cells. Two widely used myeloid markers, CD13 and CD33, were not myeloid-specific, because both were clearly positive on noncommitted progenitor cells. Of 40 antigens tested, CD15 was the only other marker fulfilling the criteria of a myeloid-specific marker. However, at concentrations of CD15 that did not induce aggregation, CD15+ cells constituted less than 50% of the CD34+, CD64+ cells. Furthermore, the CD34+, CD15- cells showed more than 50% higher CD34 mean fluorescence intensity than the CD64+, CD15+ cells, indicating that CD64 appears earlier than CD15 during differentiation. Thus, among a large number of antigens screened, CD64 was the most useful for the identification and purification of granulo-monocytic progenitor cells.     

SFL 23.6: a monoclonal antibody reactive with CFU-E, erythroblasts, and erythrocytes

A cytotoxic (IgG2b) monoclonal antibody (McAb) for a novel erythroid differentiation antigen was generated by hyperimmunizing young mice with mononuclear cells obtained from livers of 20- to 22-week-old fetuses. This McAb, designated SFL 23.6, shows an extremely well- defined reactivity with the cells of the erythroid lineage at all stages of maturation as evident from the labeling of morphologically identifiable erythroid precursors and of erythrocytes present in peripheral blood, bone marrow, and fetal liver, and from its reactivity with culture-derived erythroblasts. The nonerythroid cells present in these and other tissue preparations were not labeled by SFL 23.6. The erythroid lineage specificity of McAb SFL 23.6 was confirmed by a cell- sorting experiment in which 97% of the cells in the fluorescent fraction sorted from SFL 23.6-treated bone marrow cells were erythroid precursors at various stages of maturation. Complement-mediated cytotoxicity and progenitor cell-sorting experiments showed that most (greater than 90%) of the late erythroid progenitors (CFU-E) and only a small proportion of the early erythroid progenitors (BFU-E) express the antigenic determinant identified by SFL 23.6. The myeloid progenitors (CFU-GM) and multilineage progenitors (CFU-GEMM) were negative for the SFL 23.6 antigenic determinant. The antigen recognized by SFL 23.6 has not been determined as yet. Because of the pattern of its reactivity and its dependence on sialic acid residues, the possibility of its relationship to glycophoria A was entertained. However, previous work using antiglycophorin McAbs (R-10) has shown that this determinant is not expressed in CFU-E. Therefore, among the erythroid lineage-specific McAbs described thus far, SFL 23.6 is unique in its reactivity with CFU- E and the mature erythron. Reagents with such specificity may be useful in studies of erythroid differentiation and commitment.     

Changes in cell surface antigen expression during hemopoietic differentiation

Human bone marrow cells were separated on a fluorescence activated cell sorter (FACS) according to their binding of a series of monoclonal antibodies; the positive and negative fractions were cloned for erythroid burst and colony-forming units (BFU-E and CFU-E) and myeloid colony-forming units (CFU-GM), and cytocentrifuge slides were prepared for microscopy of maturing precursors. The pattern of antigen expression on hemopoietic progenitor and precursor populations has been established using antibodies defining blood group (A, I/i), HLA- associated (*A, B, C, DR, DC1), lineage specific, and transferrin receptor antigens. Like monomorphic HLA-DR, the antigen defined by monoclonal antibody OKT10 is expressed on the earliest progenitors and lost during differentiation, suggesting a role in interactions regulating the differentiation of these cells. The HLA-linked DC1 determinant, in contrast to HLA-DR, is not expressed at a detectable level on progenitor cells. Although a lineage-specific early antigen has not been identified, the transferrin receptor is expressed on the majority of erythroid progenitors, but only weakly on myeloid progenitors, and may provide an approach to isolating erythroid progenitors. These and earlier studies with monoclonal antibodies against HLA-DR and glycophorin now provide a detailed "map" of antigen expression during hemopoietic differentiation.     

Phenotypic heterogeneity of erythroblasts in erythroblastic leukemia revealed by monoclonal antibodies

Eight cases each of erythroleukemia (AML-M6) and erythroblastic crisis of chronic granulocytic leukemia (CGLBC-E) were immunophenotyped with the help of a panel of lineage-associated monoclonal antibodies (McAbs). The latter included those reactive with erythroid progenitor (BFU-E and CFU-E) and erythroid precursors at different stages of maturation. In six of eight cases of AML-M6, erythroblasts revealed an immature phenotype, as evident from reactivity of the blast cells with McAbs directed against the earlier stages of erythroid maturation. One case had the phenotype of CFU-E, and in the remaining case of AML-M6 the erythroblasts showed a "mature" surface antigenic profile. This immunophenotypic spectrum was unrelated to the morphologic maturity of the erythroblasts. In two cases of CGLBC-E, an early erythroblastic phenotype was observed, while in as many cases a "mature" phenotype was present. Four of eight cases, however, revealed a mixed, erythroid plus myeloid phenotype. In one of the four cases, two separate blast populations, which represented erythroblasts and myeloblasts, could be identified. In the remaining three cases the blasts were morphologically homogeneous and undifferentiated. High incidence of HLA-DR positivity in the latter three cases suggests the primitive nature of blasts cells and their closeness to the putative "bipotent" myeloid stem cell. Our study has shown phenotypic heterogeneity of blast cells in AML-M6 and CGLBC-E.     

Natural killer cells suppress human erythroid stem cell proliferation in vitro

To determine the role of natural killer (NK) cells in the regulation of human erythropoiesis, we studied the effects of NK-enriched cell populations on the in vitro proliferation of erythroid stem cells at three different levels of maturation (day 14 blood BFU-E, day 5-6 marrow CFU-E, and day 10-12 marrow BFU-E). NK cells were enriched from blood by Percoll density gradient centrifugation and by fluorescence- activated cell sorting (FACS), using the human natural killer cell monoclonal antibody, HNK-1. The isolated enriched fractions were cocultured with autologous nonadherent marrow cells or blood null cells and erythropoietin in a methylcellulose erythroid culture system. Cells from low-density Percoll fractions (NK-enriched cells) were predominantly large granular lymphocytes with cytotoxic activity against K562 targets 6-10-fold greater than cells obtained from high- density Percoll fractions (NK-depleted cells). In coculture with marrow nonadherent cells (NA) at NK:NA ratios of 2:1, NK-enriched cells suppressed day 5-6 CFU-E to 62% (p less than 0.025) of controls, whereas NK-depleted cells slightly augmented CFU-E to 130% of controls (p greater than 0.05). In contrast, no suppression of day 10-12 marrow BFU-E was observed employing NK-enriched cells. The NK CFU-E suppressor effects were abolished by complement-mediated lysis of NK-enriched cells with the natural killer cell antibody, HNK-1. Highly purified HNK- 1+ cells separated by FACS suppressed marrow CFU-E to 34% (p less than 0.025) and marrow BFU-E to 41% (p less than 0.025) of controls. HNK- cells had no significant effect on either BFU-E or CFU-E growth. NK- enriched cells were poor stimulators of day 14 blood BFU-E in comparison to equal numbers of NK-depleted cells or T cells isolated by E-rosetting (p less than 0.01). Interferon boosting of NK-enriched cells abolished their suboptimal burst-promoting effects and augmented their CFU-E suppressor effects. These studies provide evidence for a potential regulatory role of NK cells in erythropoiesis. The NK suppressor effect is maximal at the level of the mature erythroid stem cell CFU-E. These findings may explain some hypoproliferative anemias that develop in certain NK cell-activated states.     

Agranulocytosis induced by propylthiouracil: evidence of a drug dependent antibody reacting with granulocytes, monocytes and haematopoietic progenitor cells

A patient with agranulocytosis and myeloid marrow hypoplasia following a second exposure to propylthiouracil (PTU) was studied for antibodies against mature blood cells and bone marrow precursor cells. During the acute phase of the agranulocytosis, significant growth inhibition of the myeloid committed progenitor cells (CFU-GM) was found following incubation with complement, indicating the presence of in-vivo cell bound cytotoxic antibodies. Using immunofluorescence and complement dependent cytotoxicity techniques it was demonstrated that acute phase and recovery phase sera contained circulating antibodies, reactive not only with differentiated granulocytes and monocytes but also with myeloid and erythroid (BFU-E/CFU-E) progenitor cells. Complement dependent lysis of the progenitor cells was facilitated by preincubation with PTU. These results indicate that the agranulocytosis was mediated by a PTU dependent antibody that affected both mature blood cells and bone precursor cells.