Evolutionary conservation of circulating soluble low density lipoprotein receptor-related protein-like ("LRP-like") molecules.

Low density lipoprotein receptor family members characteristically bind 39-kDa receptor associated protein (RAP). Soluble forms of these receptors have been described in humans including the 515/85-kDa dimeric receptor, low density lipoprotein receptor-related protein (LRP/alpha2MR), which is involved in multiple processes including lipoprotein and protease metabolism. Here we demonstrate evolutionary conservation in the generation of these soluble RAP-binding proteins of high molecular weight, by identifying their presence in mammalian, avian, and reptilian sera as well as in the circulating haemolymph of a mollusc. Sera extracted on immobilized RAP, produced bands at approximately 500 kDa in radiolabeled ligand blots by using the LRP/alpha2MR-specific ligand, Pseudomonas exotoxin A (PEA). These findings suggest that circulating RAP-binding proteins with high molecular weight in vertebrates share features of LRP/alpha2MR (LRP-like molecules). RAP-binding molecules in the mammalian serum extracts were further characterized as LRP/alpha2MR homologues in Western blots by using antibodies against the 515-kDa alpha-chain of LRP/alpha2MR. Western blots of mammalian serum extracts using two monoclonal antibodies recognizing the 85-kDa transmembrane beta-chain suggested that a portion of the beta-chain's ectodomain remains associated with the alpha-chain, but the beta-chain's intracellular carboxy terminus is absent. These results are consistent with evolutionary conservation in the generation, composition, and ligand-binding ability of soluble LRP-like receptors and suggest that their presence is a necessary aspect of the receptor's function.

In vitro invasiveness of human breast cancer cells is promoted by low density lipoprotein receptor-related protein.

Low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor (LRP) is a surface membrane endocytic receptor, one of whose many functions is the regulation of plasminogen activator-mediated cell migration. LRP is known to have a role in migration and invasion, but its direct involvement has been demonstrated only in non-tumour cells. We investigated six breast cancer cell lines and a normal mammary epithelial cell clone for surface and total cellular LRP expression, and confirmed that its presence corresponds to the ability to invade and migrate in vitro. We showed that LRP in the tumour cell lines is expressed at a wide range of levels: from approximately 300 to approximately 6,300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1,000 sites/cell and were markedly invasive in our assay, the remainder of the cell lines and the normal clone having far fewer LRP sites and lacking invasive ability. We further showed that the migratory and invasive abilities of a highly invasive breast cancer cell line are both inhibited by receptor-associated protein, a unique LRP ligand which normally has a solely intracellular distribution but which, when added to culture medium, can inhibit all other ligand interactions with this receptor.

Soluble low-density lipoprotein receptor-related protein.

Soluble forms of receptors can influence the activity of their membrane-bound counterparts by affecting their interactions with ligands. Low density lipoprotein (LDL) receptor-related protein (LRP), a member of the LDL receptor family, binds multiple classes of ligands and has been implicated in a broad range of normal and disease processes involving lipid metabolism, protease clearance, and cell migration. We recently identified a soluble form of LRP (sLRP) in human plasma and showed that it retains LRP-ligand binding ability. These findings open potentially important additional aspects in the biology of this multifunctional receptor. This review summarizes characteristics of soluble LRP and relates these to the membrane-bound form of the receptor.

Soluble low density lipoprotein receptor-related protein (LRP) circulates in human plasma.

Our studies have identified a soluble molecule in normal human plasma and serum with the characteristics of the alpha-chain of the low density lipoprotein receptor-related protein (LRP). LRP is a large multifunctional receptor mediating the clearance of diverse ligands, including selected lipoproteins, various protease inhibitor complexes, and thrombospondin. A soluble molecule (sLRP) has been isolated from plasma using an affinity matrix coupled with methylamine-activated alpha2-macroglobulin, the ligand uniquely recognized by LRP, and eluted with EDTA. This eluate contains a protein that co-migrates on SDS-polyacrylamide gel electrophoresis with authentic human placental LRP alpha-chain, is recognized by anti-LRP alpha-chain monoclonal antibodies, and binds the 39-kDa receptor-associated protein (RAP) and tissue plasminogen activator-inhibitor complexes. A similar RAP-binding molecule was detected in medium conditioned for 24 h by primary cultures of rat hepatocytes, suggesting that the liver may be the in vivo source of sLRP. In contrast, immunoprecipitation experiments failed to detect the production of sLRP by cultured HepG2 hepatoma and primary human fibroblast cells. Addition of a soluble form of LRP to cultured HepG2 cells resulted in a significant inhibition of capacity of these cells to degrade tPA, a process that has been demonstrated to be mediated by cell surface LRP. Preliminary data indicate that the concentration of sLRP is altered in the plasma of patients with liver disease. Increased levels of sLRP may antagonize the clearance of ligands by cell bound LRP perturbing diverse processes including lipid metabolism, cell migration and extracellular proteinase activity.

Low density lipoprotein receptor-related protein (LRP) expression varies among Hep G2 cell lines.

The multiligand receptor, low density lipoprotein receptor-related protein (LRP), is implicated in processes such as atherosclerosis and fibrinolysis through its mediation of the catabolism of lipoproteins, proteases, and protease inhibitor complexes. The hepatoma cell line Hep G2 expresses LRP and has been used widely to investigate the catabolism of LRP ligands including tissue-type plasminogen activator (tPA). However, the mechanism and degree by which tPA interacts with Hep G2 has been reported with some inconsistencies which may reflect variation in their level of LRP expression. To address this possibility we characterized, antigenically and functionally, LRP expression in high and low passage Hep G2 cells both from the parental line (ATCC sourced) and a cloned subline, a16. The LRP contribution to 125I-tPA binding varied from 65% for high passage a16 cells, to 20% for low passage parent cells as quantified by inhibition in the presence of 39-kD receptor associated protein (RAP) which prevents binding of all known LRP ligands. The same trend in LRP expression among Hep G2 sublines was further evident in their ability to degrade 125I-tPA and survive Pseudomonas exotoxin A challenge. These results imply wide variability in basal LRP expression among Hep G2 lines dependent on cell lineage and long-term culture conditions.

Urokinase binding and catabolism by Hep G2 cells is plasminogen activator inhibitor-1 dependent, analogous to interactions of tissue-type plasminogen activator with these cells.

The adherent human hepatoma cell line Hep G2 exhibits receptor mediated endocytosis and catabolism of tissue-type plasminogen activator.plasminogen activator inhibitor type-1 (t-PA.PAI-1) complexes formed when exogenous t-PA combines with endogenous PAI-1 in the extracellular matrix. To determine whether the other major PA, urokinase (u-PA), which also complexes with PAI-1, is metabolised via the same mechanism, 125I-labelled high (hmw) and low (lmw) molecular weight forms of u-PA were incubated with Hep G2 cells at 4 degrees C for 2 hr in the absence and presence of a 100-fold excess of unlabelled ligand in order to detect specific binding. Both hmw and lmw 125I-u-PA formed complexes with PAI-1 and these bound specifically and with high affinity (apparent Kd 3.9 and 4.1 nM, with Bmax 78 x 10(3) and 83 x 10(3) binding sites/cell respectively). Binding by each form of radiolabelled u-PA was inhibited in a dose-dependent fashion by unlabelled t-PA, hmw-u-PA, lmw-u-PA, and by monoclonal anti-PAI-1 antibody. At 37 degrees C, bound hmw and lmw 125I-u-PA.PAI-1 complexes were internalised and degraded rapidly. These findings indicate that the specificity of the previously described receptor which mediates PAI-1 dependent catabolism of t-PA by Hep G2 cells extends to complexes of u-PA with this inhibitor.

Functional and phenotypic assessment of neonatal human leucocytes expressing natural killer cell-associated antigens.

A subpopulation of mononuclear leucocytes was prepared from umbilical cord venous blood by immunomagnetic depletion of lymphocytes and monocytes using monoclonal antibodies to CD2, CD3, CD14 and CD19 antigens, and examined for NK cell-associated phenotypic and functional properties. The depleted population was enriched for the NK markers CD16 (mean 53.6% positive) and CD56 (mean 42.7% positive). While there was considerable overlap of these two markers, approximately one-third of CD16+ cells were CD56-; in contrast, few CD56+ CD16- cells were found. CD16+/CD56+ cells also co-expressed CD7 and CD45RA antigens, while a minority weakly expressed CD8. Another marker of adult NK cells, CD57, was virtually absent from CD16+/CD56+ cells, as was MHC Class 2. Freshly depleted cord cells had virtually absent natural cytotoxicity to K562 targets in a chromium release assay, but NK activity could be induced after 18 h exposure to recombinant human IL-2, without significant change in phenotype. These findings confirm the phenotypic differences and functional defects of NK cells in cord blood as compared to adult blood, and identify a subset of cells with unique phenotype (CD2- CD3- CD7+ CD16+ CD56- CD57-). The precise relationship of this subset of cells to NK lineage remains to be defined.

Rapid positive selection of CD34+ cells using magnetic microspheres coated with monoclonal antibody QBEND/10 linked via a cleavable disulphide bond.

Positive selection of CD34+ cells has applications in diagnostic pathology, in peripheral blood and bone marrow transplantation, and in studies on the function and regulation of primitive haemopoietic stem cells. Antibody-coated magnetic microspheres (dynabeads) can be used to isolate these cells by positive selection procedures. However, the advantages of using dynabeads in some positive selection protocols are compromised by the retention of the beads on the cells. We present a protocol which allows the rapid chemical release of the beads from positively sorted cells. The murine immunoglobulin (Ig) G1 CD34 antibody, QBEND/10, was immobilised onto dynabeads as part of a three-layered immune complex: QBEND/10 was attached to F(ab')2 anti-mouse immunoglobulin antibody fragments, which were immunologically bound to a mouse IgG1 myeloma protein. The myeloma protein covalently bonded the triplex to the beads. Thus, disulphide bonds in the hinge region of the F(ab')2 could be reduced with 10 microM dithiothreitol and CD34+ cells released within 20 min. Purified cells can be re-phenotyped by multiple markers and subsets identified. Purity of 97%, recovery of > 50%, and viability over 90% of the CD34+ cells was readily achieved. Furthermore, granulocyte-macrophage colony-forming cells were retained in the positive fraction. This methodology can be used to purify other cell types, including T and B lymphocytes.

Circulating stem cells in mice treated with cyclophosphamide.

Chemotherapy has been used clinically to mobilize hematopoietic progenitor cells into the peripheral blood so that they can be harvested for autologous transplantation. In humans, this is demonstrated by the presence of circulating granulocyte-macrophage colony-forming cells (CFU-GM) and CD34-positive cells, but it has not been possible to confirm the presence of marrow-repopulating stem cells. In this study, we treated mice with 200 mg/kg cyclophosphamide (CY) and measured the numbers of white blood cells, day 12 CFU-S (CFU-S12), and CFU-GM in the peripheral blood. There was a peak in the numbers of CFU-S12 and CFU-GM 8 days after treatment with cyclophosphamide. Peripheral blood cells taken at this time rescued lethally irradiated mice and engraftment of donor cells was confirmed after 140 days in sex mismatched recipients using a Y chromosome-specific probe. In vitro culture of the blood cells harvested after cyclophosphamide showed that they proliferated in suspension cultures for at least a year in the presence of interleukin-3. The cultured cells rapidly lost their abilities to rescue irradiated mice and to form colonies in vitro, but they did not become leukemic. Also, CY-treated mice were irradiated with a leukemogenic dose of x-rays to coincide with peak circulating cell numbers but these animals did not develop an excess of leukemias over mice given irradiation alone.

Deficiency of a phosphatidylinositol-anchored cell adhesion molecule influences haemopoietic progenitor binding to marrow stroma in chronic myeloid leukaemia.

The interactions between haemopoietic progenitor cells and marrow stromal cells that are essential for the regulation of normal haemopoiesis are defective in chronic phase chronic myeloid leukaemia (CML). The presence of primitive progenitor cells (blast colony-forming cells, Bl-CFC) in the blood of patients with CML is reflected by their reduced capacity to bind to marrow derived stromal layers in vitro. Whereas normal bone marrow Bl-CFC bind irreversibly to cultured stromal layers (and none are found in normal blood), the Bl-CFC in CML bind transiently and then detach. The normal cell adhesion mechanism is partially sensitive to treatment with phosphatidylinositol-specific phospholipase C (Pl-PLC), indicating the participation of a phosphatidylinositol (Pl)-linked structure; however, when CML cells were treated with Pl-PLC it had no effect on progenitor binding. Two other Pl-linked structures, decay-accelerating factor (DAF) and lymphocyte function associated antigen-3 (LFA-3) were normally expressed on CD34 positive CML cells and normally susceptible to Pl-PLC treatment. The treatment of normal cells with Pl-PLC, to mimic the situation in CML, resulted in the indiscriminate and inefficient binding of Bl-CFC to stroma. Moreover, treatment of the normal cells with 5637 conditioned medium (CM), which contains haemopoietic growth factors, also reduced the binding capacity of normal Bl-CFC; 5637CM treatment did not alter the expression of DAF. It is proposed that a Pl-linked cell adhesion molecule (CAM) is deficient in CML as a consequence of the constitutive activation of ABL kinase whilst, in normal cells, CAMs attached in this manner are responsible for efficient adhesion to stroma and are regulated by growth factors.

A novel human leucocyte surface membrane antigen defined by murine monoclonal antibody.

A murine monoclonal antibody has been produced which identifies a novel human leucocyte differentiation antigen. The antibody, designated WM-66, of IgM subclass, was cytolytic with human complement. WM-66 was shown to react with virtually all normal T and B lymphocytes from peripheral blood and lymphoid tissues, as well as blood monocytes and approximately 40% of bone marrow mononuclear cells. The antibody also bound to the majority of cases of chronic B-cell malignancies, including chronic lymphatic leukaemia and non-Hodgkin's lymphoma, but not to cases of acute leukaemia or to the majority of leukaemic and lymphoblastoid cell lines. WM-66 also reacted with epithelium of bronchus and salivary gland ducts. A single band of relative molecular mass 65,000 Daltons was immunoprecipitated from membrane extracts of normal lymphocytes and the B-cell line Daudi. Treatment of a number of WM-66-negative B-cell lines with neuraminidase resulted in WM-66 binding, indicating that the antigen exists in a covert form masked by sialic acid residues on a wider spectrum of cell types than was initially apparent. The reactivity pattern of WM-66 indicates that it recognises a previously undescribed surface membrane molecule with broad non-lineage-specific distribution on leucocytes. This has recently been confirmed at the Fourth International Workshop on Human Leucocyte Differentiation Antigens. Although the biological function of the molecule recognised by WM-66 is unknown, the lytic properties of the antibody suggest a possible in vivo therapeutic role as an immunosuppressant or for treatment of lymphoid malignancy.

A novel non-lineage antigen on human leucocytes: characterization with two CD-48 monoclonal antibodies.

Two murine monoclonal antibodies have been produced which identify a novel surface antigen expressed on human leucocytes in a non-lineage-restricted distribution. Antibodies WM-63 and WM-68 were derived after immunization of mice with human T-CLL cells and the leukaemic cell line HSB-2. Both antibodies were shown to react with over 90 per cent of normal T and B lymphocytes from peripheral blood and tonsil, and also with monocytes from peripheral blood. A subset of bone marrow leucocytes, including granulocyte-macrophage progenitors, were also reactive. No activity with non-haemopoietic cells or tissues could be identified, however WM-63 and WM-68 showed binding to virtually all cases of chronic B cell malignancy, including chronic lymphatic leukaemia and non-Hodgkin's lymphoma, as well as a proportion of cases of acute leukaemia. Although the antigen recognized by these antibodies could not be immunoprecipitated from membrane extracts, it was removed from the surface of intact cells using the proteolytic enzymes protease and papain. Re-expression on cultured cells was inhibited by incubation with puromycin, cycloheximide, and tunicamycin, indicating that the epitopes detected by WM-63 and WM-68 are likely to be carbohydrate moieties on a protein backbone. Removal of the antigen from the cell surface by treatment with the enzyme phosphatidyl-inositol phospholipase C indicates that it is linked by a phosphatidyl-inositol bond. WM-63 and WM-68 were both recently clustered at the Fourth International Workshop on Human Leucocyte Differentiation Antigens into CD-48, together with four other monoclonal antibodies. Although no biological function has been ascribed to the molecule detected by these antibodies, its restriction to the haemopoietic lineage suggests a role in regulation of leucocyte function.

Lack of correlation between immunoglobulin and T cell receptor gene rearrangements and TdT expression in acute myeloid leukaemia.

Thirty-two cases of acute myeloid leukaemia (AML) were examined for expression of terminal deoxynucleotidyl transferase (TdT) and rearrangements of the genes coding for the immunoglobulin heavy chain and the beta chain of the T cell receptor, in order to establish whether these two forms of lineage infidelity are linked. In 17 cases of AML with greater than or equal to 10% TdT+ cells, three cases showed evidence of gene rearrangement, two having clonal rearrangements in the immunoglobulin gene and one with a rearranged T cell receptor gene. Among 15 AML cases without significant numbers of TdT-positive blasts, three cases had rearrangements in both immunoglobulin and T cell receptor genes, while a fourth case had an immunoglobulin gene rearrangement. No relationship was seen between lymphoid gene rearrangements and expression of the lymphoid surface antigens CD7 and CD10. The lack of association between TdT expression and gene rearrangements does not support the concept of an orderly activation of the recombinase machinery in those cases of AML with features of early lymphoid differentiation.

Unusual immunophenotypes in acute leukaemias: incidence and clinical correlations.

The incidence and clinical implications of unusual patterns of expression of leucocyte differentiation antigens in acute leukaemia were assessed on 568 newly diagnosed paediatric and adult cases undergoing immunophenotyping with a panel of monoclonal antibodies at a single centre. Among patients with the precursor B (common) form of acute lymphoblastic leukaemia (ALL), the major variant seen was the group of 15 cases with expression of myeloid surface antigens. 4.5% of ALL cases tested with antibody to CD-11b were positive, 5.1% were CD-13+, and 10.8% CD-33+. All 15 patients achieved a complete remission with chemotherapy, with six of eight children and four of seven adults remaining disease free. A smaller proportion (1.5%) of precursor B ALL patients showed expression of the T lineage marker, CD-7. The only significant variant seen in the precursor T-ALL group was expression of HLA-DR antigen, which was found in five of 35 cases; although all responded to treatment, only one remains a disease-free survivor. Among patients with acute myeloid leukaemia (AML), expression of the lymphoid markers terminal transferase (TdT) and CD-7 were commonly seen (22.2% and 28.4% respectively of cases tested). Other lymphoid markers detected on AML cases were CD2 (11.1%), CD-10 (1%) and CD-19 (4.4%). These results confirm that examples of lineage infidelity are regularly seen in large series of patients with acute leukaemia. Prospective studies using uniform treatment protocols are required to establish whether these patients have significantly different disease outcomes.

Expression of a novel human pan leucocyte differentiation antigen on leukaemic cells.

A murine monoclonal antibody has been produced which identifies a novel surface membrane antigen present on virtually all normal human leucocytes and leukaemic cells. The antibody, designated WM-65, reacted with over 95% of peripheral blood, tonsil and thymic lymphocytes, and with a similar proportion of monocytes and granulocytes. A majority of nucleated normal bone marrow cells were also reactive with WM-65; however, these included only a small proportion of myeloid progenitor cells. WM-65 reacted with a wide range of acute and chronic leukaemias of both myeloid and lymphoid types, and with corresponding cell lines, but did not react with non-haemopoietic cells. By immunoprecipitation and SDS-PAGE, WM-65 identifies a heavily glycosylated surface protein of molecular weight between 40 and 50 kD. This property, and the broad non-lineage-specific distribution of the antigen on haemopoietically-derived cells, indicates that WM-65 is different from other monoclonal antibodies with "leucocyte common" reactivity patterns. The extensive reactivity of WM-65 with leukaemic cells raises the possibility of therapeutic applications of the antibody in haematological malignancies.

Phenotypic characterization of immature lymphoid cells in human umbilical cord blood.

The antigenic phenotype of neonatal lymphoid cells isolated from umbilical cord blood was investigated using monoclonal antibodies and flow cytometry. Although the majority of cells expressed mature T or B cell differentiation antigens, small subpopulations of phenotypically immature lymphocytes were detected. A small proportion (mean 2.8%) of cells expressed the common acute lymphoblastic leukaemia antigen (CD-10), a significantly higher figure than that detected on adult peripheral blood lymphocytes. The cortical thymocyte antigen (CD-1) was detected on a very small subset of cord lymphoid cells, but was also present on adult lymphocytes at approximately the same frequency. The nuclear enzyme terminal deoxynucleotidyl transferase (TdT), a marker of early lymphoid differentiation, was detected by immunofluorescence on 0.031% of mononuclear cells in cytocentrifuge preparations, representing an approximate 10-fold increase in frequency over expression in childhood or adult blood. These circulating TdT+ cells were shown in double labelling experiments to predominantly express markers of B cell differentiation (CD-24, CD-10, MHC Class 2), although occasional cells co-expressing the T lineage marker CD-2 were also seen. These findings are consistent with the circulation of B cell precursors in neonatal blood. The nature of the CD-1+ cells is unclear, although the absence of CD-1+ TdT+ double labelled cells mitigates against the possible presence of immature thymus-processed lymphocytes in these samples.

Further characterization of human myeloid antigens (gp160,95; gp150; gp67): investigation of epitopic heterogeneity and non-haemopoietic distribution using panels of monoclonal antibodies belonging to CD-11b, CD-13 and CD-33.

We have investigated the binding of over 30 different monoclonal antibodies (MAB) belonging to three distinct clusters of differentiation (CD-11b; CD-13; CD-33; as defined by the Third International Workshop on Leucocyte Differentiation Antigens (ILWS), 1986), and which are reactive with three distinct myeloid restricted surface antigens ('gp160,95'; 'gp150'; 'gp67'). By investigating reactivity with non-haemopoietic cells, we have confirmed that CD-11b and CD-33 MAB reactivity is largely restricted to haemopoietic cells, whilst CD-13 MAB showed additional binding to a wide range of non-haemopoietic cells. Epitopic heterogeneity was also investigated within each cluster of differentiation. Tested anti-CR3 (CD-11b) MAB varied in their ability to block the binding of complement coated sheep red blood cells and zymosan particles. A more detailed analysis of MAB binding heterogeneity was performed by competitive inhibition assays. It was demonstrated that MAB from both CD-11b and CD-13 bind to several distinct epitopes (at least six and five respectively) on their respective antigen molecules. In contrast, CD-33 MAB appear to bind to only a single site on 'gp67'. These data may allow for a clearer appreciation of the disparate functional effects obtained using different MAB reagents to individual myeloid antigens, as reported by a number of workers.

An acquired Bernard-Soulier-like platelet defect associated with juvenile myelodysplastic syndrome.

Bernard-Soulier syndrome is an inherited bleeding abnormality characterized by thrombocytopenia with large platelets and deficiency of the platelet membrane glycoprotein (GP) Ib-IX complex. We have identified a young female with an acquired Bernard-Soulier-like platelet defect and a coexisting primary myelodysplastic disorder. Abnormal bruising had developed at age 5. A normal platelet count with some giant platelets was noted at age 7. At age 9 she developed a large haematoma following surgery. Laboratory investigation revealed thrombocytopenia and large platelets. Platelet membrane glycoprotein analysis showed a marked deficiency of the components of the GP Ib-IX complex (approximately equal to 25% of normal). Flow cytometry revealed two populations of platelets: a predominant population of large platelets lacking the GP Ib-IX complex and a minor population of normal-sized platelets with normal GP Ib-IX expression. The patient developed progressive anaemia, more severe thrombocytopenia and neutropenia, and circulating blast cells were seen. A bone marrow showed gross hypercellularity with marked dysplasia of all three lineages and increased blasts. Marrow cytogenetic studies showed the presence of monosomy 7 in all metaphases, with an additional trisomy 21 in 10%. Peripheral blood cells were normal 46XX. The above data are consistent with an acquired myelodysplastic syndrome associated with a Bernard-Soulier-like platelet defect.

Immunophenotype of clonogenic cells in myeloid leukaemia.

Leukaemic clonogenic cells, capable of forming colonies of blast cells in an in-vitro assay, were examined for surface antigen expression using a panel of monoclonal antibodies (Mabs) to stem cell and myeloid differentiation antigens in nine cases of acute myeloid leukaemia (AML) and four cases of chronic myeloid leukaemia in myeloid blast crisis (CML-MBC). Clonogenic cells were found to be most frequently positive with anti-HLA-DR (positive in 100% cases) and RFB-1 (71%) Mabs, with significant reactivity also being seen with CD-33 (69%) and CD-13 (61%) myeloid specific antibodies. CD-11b and CD-15 antigens, expressed predominantly on mature leucocytes, were not significantly expressed on the clonogenic population. Interestingly, the CD-34 antigen, detected by MY-10 Mab on normal myeloid progenitor cells, was demonstrated on the clonogenic fraction of only one of seven cases tested. A discrepancy between antigen expression of clonogenic cells and immunophenotype of the total leukaemic population was frequently seen, with "early" markers (CD-33, HLA-DR, RFB-1) expressed on a higher proportion of the clonogenic fraction than the overall population, while the converse was the case for the "later" marker, CD-11b. Based on the known normal distribution of differentiation antigens, particularly the CD-13 antigen, cases could be ranked according to clonogenic phenotype into immature (CD-13- HLA-DR+ CD-33+ or CD-33-; five cases), and mature (CD-13+ HLA-DR+ CD-33+; eight cases), levels. However, there was no correlation between these maturation levels and the morphology according to the FAB classification. Of note, the mature group included three CML-MBC, as well as two AML cases with a history of myelodysplasia or myeloproliferative disorder. These immunophenotypic findings indicate a heterogeneity in the level of maturation of the clonogenic population, not only in cases of de-novo AML, but also in AML thought to derive from multipotential stem cells.

Characterization of monoclonal antibodies to the human myeloid-differentiation antigen, 'gp67' (CD-33).

This report describes the production and characterization of two new monoclonal antibodies (MoAb), WM-53 and WM-54, reacting with a human myeloid differentiation antigen, which has recently been assigned by the Third International Leucocyte Workshop on Human Differentiation Antigens into cluster CD-33 ('gp67'). To date, only three other MoAb (MY-9, L1B2, L4F3) have been reported to react with this antigen. In peripheral blood, WM-53 and WM-54 were found to bind to monocytes, but failed to react with erythrocytes, platelets and lymphoid cells. WM-54 was also faintly reactive with granulocytes. Myeloid 'specificity' was also observed with leukaemias as both WM-53 and WM-54 were reactive with most cases of acute myeloid leukaemia (AML), but with only a minority of lymphoid leukaemias. Fluorescence activated cell sorting of normal bone marrow cells demonstrated that both MoAb bound to the majority of CFUgm and CFUmix progenitor cells. Immunoprecipitation studies confirmed that these MoAb, in parallel with MY-9, bound to a protein of approximately 70 KD molecular weight, together with a previously undescribed higher molecular weight component of approximately 140 KD on non-reduced gels, possibly representing a disulphide linked dimer of the lower molecular weight protein. Competitive binding assays, using MoAb WM-53 and WM-54 as well as MY-9, L1B2, and L4F3, demonstrated that all five CD-33 MoAb are capable of competing with each other for binding onto HL-60 cells, suggesting that all recognize a single epitopic site on 'gp67'.