Transgene expression of bcl-xL permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells.

Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.

Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumour cells.

Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8(+) T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer(+) cell lines were CTL and efficiently killed HLA-A*0201(+) melanoma cells, whilst sparing HLA-A*0201(+) B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo-HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour-associated antigens.

The monoclonal antibody 6B9 recognizes CD44 and not cell surface transglutaminase 2.

The multifunctional enzyme transglutaminase 2 (TG2) can be located intracellularly, in the extracellular matrix (ECM) and on the cell surface. Cell surface TG2 (csTG2) is poorly recognized both by most TG2-specific commercial antibodies and celiac disease-associated anti-TG2 autoantibodies. The recent characterization of a csTG2-specific monoclonal antibody (mAb), which did not recognize ECM-associated TG2, suggested major conformational differences between csTG2 and TG2 found in the ECM. Subsequent findings based on this antibody indicated ubiquitous abundance and novel roles of csTG2 in innate immune responses. We wished to identify the epitope of 6B9 so as to shed light on the disparate antibody binding properties of csTG2- and ECM-associated TG2. Surprisingly, and despite thorough effort, we were unable to isolate TG2 as the antigen of 6B9. We found that 6B9 does not react with recombinant human TG2. In immunoprecipitation experiments, 6B9 pulled down an 85 kDa protein which was identified as CD44 by mass spectrometry. Several flow cytometry experiments including the testing of CD44s transfectants indicated that CD44, and not csTG2, is the antigen of 6B9. We conclude that 6B9 does not recognize csTG2 but rather the cell surface glycoprotein CD44. Thus, recent knowledge of csTG2 gained through the use of 6B9 should be reevaluated.

Differential surface expression of cell adhesion molecules during granulocyte maturation.

Individual steps of granulocyte maturation, such as lineage commitment, proliferation, maturation, and migration from the marrow to the peripheral blood, may be influenced by distinct interactions with the bone marrow stroma. To identify candidates of membrane components involved in maturational stage-specific interactions, we studied changes in the expression of cell adhesion molecules along the granulocyte maturational pathway. Three-color flow cytometric measurements were used to measure levels of cell adhesion molecules along this pathway. The alpha chains of VLA-4 (CD49d) and VLA-5, the integrin beta 1 chain (CD29), and CD31 (PECAM-1) were expressed in high density on all early myeloid cells but down-modulated during postproliferative maturation. CD44 and L-selectin were expressed on CD34+ myeloid progenitor cells and mature granulocytes but down-modulated during the intermediate stages of maturation. The granulocyte receptor for endothelial selectins, sLex, was specifically expressed by myeloid progenitor cells. sLex was down-modulated during the intermediate stages of granulocyte maturation but up-regulated again during terminal maturation. In contrast, CD67, a putative granulocyte adhesion molecule, was negative on progenitors, transiently up-regulated during the intermediate stages of maturation, and almost absent from the surface of mature granulocytes. These results show that each stage of granulocyte maturation is associated with the expression of a unique combination of cell adhesion molecules. L-selectin, CD44, and beta 1 integrins were regulated as previously described for immature lymphopoietic cells and may therefore play general roles in the compartmentalization and development of leukocytes. In contrast, sLex and CD67 were specifically expressed by myeloid cells and could be specifically important for compartmentalization of distinct phases of granulocyte maturation.

Expression of leukocyte differentiation antigens during the differentiation of HL-60 cells induced by 1,25-dihydroxyvitamin D3: comparison with the maturation of normal monocytic and granulocytic bone marrow cells.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces monocytic differentiation of the HL-60 leukemic cell line. The present study investigated whether and to what extent this differentiation resembles the normal maturation of monocytic cells in the bone marrow. Multidimensional flow cytometry was used to identify changes in antigen expression that occur in normal bone marrow cells at distinct stages of monocytic and granulocytic maturation. HL-60 cells were analyzed in the same manner after exposure to 1,25-(OH)2D3 to determine whether the hormone induces a similar sequence of phenotypic changes. In the leukemic cells, monocytic features were sequentially induced and several maturational steps could be resolved. CD14, CD32, CD53, CD15, CDw65, CD29, CD16, and CD66b were modulated in 1,25-(OH)2D3-induced HL-60 cells as in the normal monocytic maturational pathway. Differences were observed for CD15s and CDw17. The expression pattern of CD44 during differentiation of HL-60 cells resembled that in granulocytic cells. The results therefore suggest that 1,25-(OH)2D3 induces a differentiation program in HL-60 cells that in many ways resembles that of normal monocytic cells in the bone marrow but also carries elements of the granulocytic pathway.

Primitive human hematopoietic progenitor cells express receptors for granulocyte-macrophage colony-stimulating factor.

Most cytokines act only synergistically in assays of primitive progenitor cell proliferation, and effects have usually been observed first after prolonged cell culture. Studies reporting that primitive progenitors lack receptors for a number of cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF), could indicate that several "synergistic" cytokines primarily affect cells that have differentiated in vitro. Here, however, we show that freshly isolated primitive progenitor cells (CD34hi CD38-) express receptors for GM-CSF at levels 20%-30% of granulo-monocytic progenitors. Although GM-CSF had minimal effects on the survival or proliferation of primitive progenitors when added alone, the cytokine enhanced stem cell factor (SCF) induced cell cycle entry in the first generation. The effect was not observed when cells were incubated sequentially with SCF and GM-CSF. The results suggest that the synergistic effects of GM-CSF are mediated directly on primitive progenitor cells and that the cytokine may be useful to enhance cell cycle entry of hematopoietic stem cells.

Signal transduction in human monocytes and granulocytes through the PI-linked antigen CD14.

A possible role for the PI-linked CD14 molecule in human monocyte and granulocyte signal mediation was investigated. Using flow cytometry and the fluorescent indicators Fluo-3 and dihydrorhodamine-123 it was shown that crosslinking of the CD14 molecule induces an increase in monocyte and granulocyte cytoplasmic calcium concentration and monocyte H2O2 production. These responses were found to be independent of IgG Fc receptors and suggest an intrinsic signal mediating capacity of the CD14 molecule.

Negative selection of human monocytes using magnetic particles covered by anti-lymphocyte antibodies.

We have developed a standardized procedure for the isolation of monocytes from peripheral blood by negative selection using magnetic polymer particles coated with monoclonal antibodies against T and B lymphocytes. The average purity of the monocyte suspension was 85%, and monocyte recovery was 72% from Ficoll-Hypaque gradient separated mononuclear cells and 32% from whole blood. In a lucigenin enhanced chemiluminescence assay there was no significant difference between cells separated immunomagnetically and those separated on a gradient. Nor did electron microscopy show any significant difference in morphology between such monocytes. Negative selection using magnetic polymer particles is an efficient method for the separation of monocytes with intact morphology and function as measured by chemiluminescence.

Expression of cell surface antigens during the differentiation of HL-60 cells induced by 1,25-dihydroxyvitamin D3, retinoic acid and DMSO.

HL-60 cells were induced to differentiate by 1,25-dihydroxyvitamin D3, retinoic acid or DMSO. In order to investigate to which extent this maturation mimics the in vivo monocytic or myeloid differentiation, we compared induced HL-60 cells with peripheral blood monocytes and granulocytes by using a panel of mAbs directed against myeloid cell surface antigens. Upon exposure to 1,25-(OH)2D3, HL-60 cells acquired a differentiation phenotype close to that of mature monocytes. The changes in myeloid cell surface antigens induced by retinoic acid or DMSO paralleled the expression pattern of these molecules in normal granulopoiesis, although maturation was not achieved and partially defective.

Targeting B cell leukemia with highly specific allogeneic T cells with a public recognition motif.

The possibility that allogeneic T cells may be targeted to leukemia has important therapeutic implications. As most tumor antigens represent self-proteins, high-avidity tumor-specific T cells are largely deleted from the repertoire of the patient. In contrast, T cells from major histocompatibility complex (MHC)-mismatched donors provide naïve repertoires wherein such cells have not been systematically eliminated. Yet, evidence for peptide degeneracy or poly-specificity warrants caution in the use of foreign human leukocyte antigen (HLA) or peptide complexes as therapeutic targets. Here, we cocultured HLA-A(*)0201-negative T cells with autologous dendritic cells engineered to present HLA-A(*)0201 complexed with a peptide from the B cell antigen CD20 (CD20p). HLA-A(*)0201/CD20p pentamer-reactive CD8(+) T cells were readily obtained from all donors. The polyclonal cells showed exquisite peptide and MHC specificity, and efficiently killed HLA-A(*)0201-positive B cells, including primary chronic lymphocytic leukemia cells. The T cell receptor (TCR) sequences displayed a novel type of conservation, with extensive homology in the TCR ß chain complementarity-determining region 3 and in J, but not V, region. This is surprising, as the donors were HLA disparate and their TCR repertoires are expected to show little overlap. The results demonstrate the first public recognition motif for an allogeneic HLA/peptide complex. The allo-restricted T cells or TCRs could provide graft-versus-leukemia in the absence of graft-versus-host disease.

High expression of CD7 on CD34+ cells is not linked to deletion of derivative chromosome 9 or lack of dendritic cells in chronic myeloid leukaemia.

OBJECTIVE: High expression of CD7 on CD34+ cells (>20 %) has been shown to be associated with inferior prognosis in chronic myeloid leukaemia (CML), but the reason has not been unravelled. We set out to investigate whether lack of dendritic cells or der(9)t(9;22)(q34;q11) deletions might be correlated with increased CD7 expression on CD34+ cells in CML. MATERIAL AND METHODS: We identified 43 patients in our cohort of CML patients in the first chronic phase in whom we were able to assess the expression of CD7 on CD34+ cells. der(9)t(9;22) deletions were evaluated by FISH (fluorescent in situ hybridization) analyses and the proportions of plasmacytoid and myeloid dendritic cells were assessed by flow cytometry. RESULTS: High and low expressions of CD7 on CD34+ cells were found in 19 and 24 patients, respectively. Two out of 20 patients examined had a der(9)t(9;22)(q34;11) deletion, one patient with high expression and one with low expression of CD7 on CD34+ cells. The proportions of plasmacytoid dendritic cells (PDCs) and myeloid dendritic cells (MDCs) were reduced in a majority of patients in our cohort, but no correlation was found between high or low expression of CD7 on CD34+ cells and the proportion of dendritic cells. CONCLUSIONS: A high proportion of CD34+CD7+ cells in patients with CML is not associated with der(9)t(9;22)(q34;q11) deletions. Nor did we find any correlation between CD7 expression on CD34+ cells and lack of dendritic cells. High expressions of CD7 on CD34+ cells and der(9)t(9;22)(q34;q11) deletions seem to be independent prognostic markers in CML.

Assay for monitoring in vitro selective depletion strategies in allogeneic stem cell transplantation.

BACKGROUND: GvHD is a serious and potentially life-threatening side-effect of allogeneic BMT, caused by alloreactive cells attacking normal host cells. A number of different approaches have been attempted to remove allo-activated cells from the graft prior to transplantation. When developing such assays, there is a need to control for unwanted removal of cells, as well as depletion efficiency related to activation kinetics. METHODS: The specific activation induced by the superantigens SEB and TSST-1 of T cells with defined Vbeta chains was utilized to follow activation of bystander cells and the kinetics of specific cellular activation by flow cytometry. RESULTS: The activation marker CD69 was up-regulated on bystander T cells, and was only transiently highly expressed on the specific T cells, making this marker unreliable for removal of alloreactive cells. In contrast, CD25 was found only on specifically activated T cells and was stably expressed over several days. However, it was not detected on all specific cells until day 6. Likewise, proliferation occurred only in T cells expressing the expected Vbeta chains, with all activated cells having undergone at least one cell cycle by day 4. DISCUSSION: In conclusion, our assay demonstrates that only temporary bystander activation occurs when polyclonally activating T cells by SEB or TSST-1, and that CD25, but not CD69, can be used for removal of specifically activated cells. Furthermore, this assay is useful for monitoring methods aiming at specific removal of cycling cells.

Plasmacytoid dendritic cells induce a distinct cytokine pattern in virus-specific CD4+ memory T cells that is modulated by CpG oligodeoxynucleotides.

Inherent properties of dendritic cell (DC) subsets are important in the regulation of naïve T-cell differentiation (e.g. Th1 versus Th2 cells), whereas effector memory T cells are believed to produce a fixed cytokine repertoire independent of the type of antigen presenting cell. Here we show that two distinct human DC subsets, plasmacytoid DC (PDC) and myeloid CD11c+ DC, induced autologous mumps virus-specific memory CD4(+) T cells to produce markedly different cytokine patterns upon antigen stimulation. PDC stimulated the T cells to produce gamma-interferon (IFN-gamma) and interleukin-(IL)-10, whereas CD11c+ DC induced lower levels of IFN-gamma, virtually no IL-10, but significant levels of IL-5. Analysis of intracellular cytokine production showed simultaneous production of IL-10 and IFN-gamma in mumps-specific T cells activated by PDC, a cytokine pattern similar to that described for Th1-like regulatory cells. Introduction of CpG oligodeoxynucleotides in PDC/T-cell co-cultures had synergistic effect on virus-dependent IFN-gamma production, whereas the other cytokines remained unchanged. Together, our results show that the type of DC involved in reactivation of previously primed T cells may have significant impact on the resulting cytokine response and suggest that targeting of viral antigens and adjuvant to specific DC subsets should be considered in the design of therapeutic antiviral vaccines.

Signal transduction in monocytes and granulocytes measured by multiparameter flow cytometry.

The novel calcium indicator fura red and the oxidative burst indicator dihydrorhodamine (both excited at 488 nm) were used in combination with multiparameter flow cytometry to allow simultaneous kinetic measurements of calcium fluxes and oxidative bursts in monocytes and granulocytes. Using this method it was possible to obtain direct evidence for the following cell type- and stimulus-specific differences in signal transduction pathways: 1) n-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B-induced oxidative burst is several-fold higher in granulocytes than in monocytes although the calcium fluxes have similar amplitudes in the two cell types; 2) stimulus-induced calcium fluxes in granulocytes are mainly due to release from intracellular stores, whereas monocytes mobilize calcium mainly by influx from the medium; 3) the FMLP/cytochalasin B-induced calcium flux in monocytes is less sensitive to the G-protein inhibitor pertussis toxin than the flux in granulocytes; 4) in contrast to FMLP/cytochalasin B, the protein kinase C activator phorbol myristate acetate (PMA) induces an oxidative burst that is not preceded by a cytoplasmic calcium flux; 5) the PMA-induced oxidative burst can be triggered in monocytes and granulocytes that are depleted of intracellular calcium ions, whereas that induced by FMLP/cytochalasin B can not; 6) the G-protein inhibitor pertussis toxin blocks an early event in the signal transduction pathway of FMLP/cytochalasin B, as shown by inhibition of both calcium fluxes and oxidative burst; and 7) 100 nM of the protein kinase inhibitor staurosporine blocks the FMLP/cytochalasin B-induced respiratory burst by interfering with a step downstream to cytoplasmic calcium fluxes, whereas only 10-20 nM is necessary to block PMA-induced oxidative burst.

[Dendritic cells--strong candidates for immunotherapy]. / Dendrittiske celler--sterke kandidater for immunterapi.

Why are immune responses primarily directed towards infectious agents, and how can the immune system be manipulated to attack for instance malignant cells? The role of the dendritic cells in the immune system may provide the answers. We present a review of a field in which results from basic science are rapidly applied in clinical trials. We searched the Medline database using the terms dendritic cells combined with ontogeny, subpopulations, vaccine or review. Results from our own experimental work are also described. The cited studies show that dendritic cells take up material from their surroundings and migrate to lymphoid tissue where the material is presented to T-cells. Dendritic cells have the ability to selectively direct immune responses towards potentially harmful agents such as bacteria and viruses. Clinical trials show that vaccines based on the use of dendritic cells induce tumor-specific immunity and clinical remission. Experiments conducted by the authors and others indicate the existence of subpopulations of dendritic cells with specialized functions. Dendritic cells play a central role in the initiation of immune responses and may be used to manipulate the immune system. Their use in the treatment of diseases such as cancer is highly promising.

CD53, a protein with four membrane-spanning domains, mediates signal transduction in human monocytes and B cells.

CD53 is a member of a novel family of molecules with four presumably membrane-spanning domains. The structure and functional characteristics of these molecules indicate that they may play an important role in transmembrane communication. We therefore investigated whether CD53 is involved in activation of human leukocytes. Cross-linking of cell-bound F(ab')2 fragments of two different anti-CD53 mAb with F(ab')2 anti-mouse Ig led to cytoplasmic calcium fluxes in B cells, monocytes, and granulocytes and activation of the monocyte oxidative burst. These responses were specific for CD53, as cross-linking of CD11a, CD18, CD35, CD43, CD44, CD45, or CDw50 did not induce leukocyte activation. Low concentrations of staurosporine (10 to 20 nM) completely inhibited PMA-mediated activation, but had no effect on CD53-mediated calcium fluxes and inhibited only partially CD53-mediated oxidative burst. This suggests that CD53-mediated signaling is largely independent of protein kinase C. CD53-mediated calcium fluxes were inhibited by high concentrations of staurosporine (300 to 500 nM) but not by ADP-ribosylating toxins, suggesting dependence on tyrosine kinases rather than GTP-binding proteins. The results indicate that CD53, like several other leukocyte Ag with four membrane-spanning regions, has the ability to mediate cell activation, and support the view that these molecules are involved in transmembrane communication.

Flow cytometric assay for the measurement of human bone marrow phenotype, function and cell cycle.

A flow cytometric assay for the measurement of human bone marrow and blood leukocyte antigen expression, phagocytosis, and proliferation is described. Subpopulations of leukocytes were identified by their light scatter characteristics, and the expression of a myeloid differentiation antigen (designated CDw65) determined following incubation with CDw65 specific fluorescein-isothiocyanate (FITC) conjugated monoclonal antibodies (VIM2). Incubation of leukocytes with ethidium monoazide (EMA) labeled Candida albicans followed by staining with FITC conjugated VIM2 allowed the combined determination of cellular CDw65 expression and phagocytic capacity. In addition, immunostained leukocytes were fixed, and their DNA labeled with propidium iodide (PI), before CDw65 expression was measured for cells in different phases of the cell cycle. The method allows evaluation of phenotypic and functional heterogeneity, as well as cell cycle parameters, within subpopulations of cells during hematopoietic differentiation.

Expression of cell surface markers during differentiation of CD34+, CD38-/lo fetal and adult bone marrow cells.

Even though there has been considerable progress in the phenotypic characterization of CD34+ bone marrow cells, there is still limited knowledge about the cell phenotypes corresponding to functional terms such as colony-forming cells, burst-forming cells, long-term culture-initiating cells, and high-proliferative potential cells. In this study, we performed a detailed analysis of phenotypic characteristics of subsets of CD34+ cells. We compared cells from adult and fetal bone marrow to investigate whether reported functional differences are reflected in the cellular phenotypes. CD34+, CD38-/lo, HLA-DR+ cells, which have been shown to contain the most immature hematopoietic progenitor cells, stained as a homogeneous population with most monoclonal antibodies (mAbs). The antigens sLex, CD32, and CD7 were, however, heterogeneously expressed in the CD38-/lo population. Phenotypic differences in the CD34+, CD38-/lo population was found when comparing adult and fetal bone marrow cells. Adult bone marrow CD34+, CD38-/lo cells stained more brightly with CD4, Thy-1, and CD49b and more dimly with CD32 than the same population in fetal bone marrow. Certain antigens that have previously been regarded as lineage-specific were found on the CD34+, CD38-/lo, HLA-DR+ cells in both fetal and adult bone marrow. These included CD52, CD13, and CD33. The markers that were found to be most useful in discriminating between subsets of lineage-committed cells within the CD34+, CD38+ population included the B cell marker CD19 and the granulomonocytic marker CD64. The phenotypic analysis presented here should provide a basis for establishing a better link between functional and phenotypic characteristics of hematopoietic progenitor cells.

The IgG FcRII and the PI-linked IgG FcRIII trigger cytoplasmic calcium fluxes independently in human granulocytes.

The aim of this study was to investigate signal transduction through the two Fc receptors for IgG on human granulocytes. Using flow cytometry and the calcium indicator Fluo-3, we measured changes in leucocyte cytoplasmic calcium concentrations following cross-linking of cellular Fc receptors with specific antibodies. Two different approaches were used in order to study the two Fc receptors independently of each other. One was to avoid the presence of IgG Fc fragments, capable of binding to both types of receptors. The other was to use leucocytes from a patient with paroxysmal nocturnal haemoglobinuria (PNH) deficient in granulocyte FcRIII. In contrast to earlier reports, both approaches showed that the two types of IgG Fc receptors on granulocytes are capable of increasing cytoplasmic free calcium concentrations independently of each other. The results suggest that free cytoplasmic calcium ions are involved in the signal transduction pathway of both types of IgG Fc receptors on human granulocytes.

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.