Expression of the three myeloid cell-associated immunoglobulin G Fc receptors defined by murine monoclonal antibodies on normal bone marrow and acute leukemia cells

Monoclonal antibodies (MoAbs) have been prepared recently that recognize the three cell-surface receptors for the Fc portion of immunoglobulin (Ig), termed Fc gamma RI (MoAb 32.2), Fc gamma R II (MoAb IV-3), and Fc gamma R III (MoAb 3G8) that are expressed on selected subsets of non-T lymphocyte peripheral blood leukocytes. In the blood, Fc gamma R I is expressed exclusively on monocytes and macrophages, Fc gamma R II on granulocytes, mononuclear phagocytes, platelets, and B cells, and Fc gamma R III on granulocytes and natural killer (NK) cells. We have examined the expression of these molecules on normal bone marrow (BM) cells and on leukemia cells from the blood and/or BM in order to determine their normal ontogeny as well as their distribution on leukemic cells. BM was obtained from six normal volunteers and from 170 patients with newly diagnosed acute leukemia. Normal BM cells were found to express Fc gamma R I, II, and III with the following percentages: 40%, 58%, and 56%, respectively. Cell sorting revealed that both Fc gamma R I and Fc gamma R II were detectable on all subclasses of myeloid precursors as early as myeloblasts. Cell sorting experiments revealed that 66% of the granulocyte-monocyte colony-forming cells (CFU-GM) and 50% of erythroid burst-forming units (BFU-E) were Fc gamma R II positive with only 20% and 28%, respectively, of CFU-GM and BFU-E were Fc gamma R I positive. Acute myeloid leukemia (AML) cells expressed the three receptors with the following frequency (n = 146): Fc gamma R I, 58%; Fc gamma R II, 67%; and Fc gamma R III, 26% of patients. Despite the fact that Fc gamma R I is only expressed on monocytes among blood cells, AML cells without monocytoid differentiation (French-American-British [FAB]M1, M2, M3, M6) were sometimes positive for this receptor. However, Fc gamma R I was highly correlated with FAB M4 and M5 morphology (P less than .001). Fc gamma R II was also correlated with FAB M4 and M5 morphology (P = .003). Cells from 11 patients with acute lymphoblastic leukemia were negative for Fc gamma R I, but six cases were positive for Fc gamma R II and III (not the same patients). These studies demonstrate that Ig Fc gamma R are acquired during normal differentiation in the BM at or before the level of colony-forming units. In addition, we show that acute leukemia cells commonly express Fc gamma R.     

Acute myelomonocytic leukemia: an immunoelectron microscopic study

Acute myelomonocytic leukemia (M4; French-American-British classification) is light microscopically defined as the leukemia constituting leukemic cells in both granulocytic and monocytic lineages. Therefore, the characteristics of M4 have not been fully elucidated. The author previously indicated that normal neutrophilic granulocytes could be ultrastructurally differentiated from normal monocytes by the double staining of lactoferrin and lysozyme. In this investigation, the ultrastructural localization of both proteins were observed in order to make the outline of M4 clear. The leukemic cells in acute myeloid leukemia (M2) were also examined in comparison with those in M4. The leukemic cells in M4 showed the double stainability of lactoferrin and lysozyme, and the positive reactions were localized in the cytoplasmic matrix and in the granules. The staining pattern was similar to that in M2. The coexistence of lactoferrin and lysozyme in the leukemic cells in M4, which has ultrastructurally the monocytic characteristics, implied that the leukemic cells also possess the characteristics of the cells in the granulocytic lineage. This suggests that the presence of the various leukemic cells in the granulocytic lineage. This suggests that the presence of the various leukemic cells signifies the diversely abnormal maturations in vivo of the monocytes/granulocytes precursor cell and that M4 consists of not two kinds of distinguishable cells of granulocytic and monocytic lineages but various consecutive cells based on a malignant transformation of the precursor cell.     

Cytochemical and immunophenotypic heterogeneity in acute promyelocytic leukemia

The leukemic promyelocytes in 37 cases of acute promyelocytic leukemia (APML; FAB, M3) were examined for their cytochemical property. Thirty-two cases (86%) showed strong myeloperoxidase (MPO), chloroacetate esterase (Es-chl) and Sudan black B (SBB) positivity, suggesting a pure neutrophilic differentiation of the leukemic cells. However, in 5 out of 37 cases, a strong, diffuse alpha-naphthyl acetate esterase (Es-a) positivity, which was sensitive to sodium fluoride treatment was observed in addition to strong MPO, Es-chl and SBB positivity. This suggested monocytic differentiation of a proportion of APML cases. In 31 cases, surface marker studies were carried out with the help of a panel of monoclonal antibodies consisting of two panmyeloid antibodies (GM 58/8 and 1G10), one anti-HLA-DR antibody (7.2) and one 'myeloid' antibody (5F1) with restricted reactivity with monocytes (CD14). The purpose of including the monoclonal antibodies 5F1 and 7.2 was to determine if a correlation could be established between strong Es-a positivity and reactivity of the leukemic promyelocytes with 5F1 and 7.2 in individual cases. All the 5 cases with 'monocytoid' cytochemistry were unreactive with 5F1, and only one case in this group showed 15% 7.2-positive cells. The lack of immunophenotypic support for the monocytic cytochemistry of the 5 cases of APML suggests that the monocytic phenotype of leukemic promyelocytes is both aberrant and incomplete. Since normal promyelocytes are purely neutrophilic, this could be a manifestation of an 'intralineage infidelity' in APML, similar to that observed in other types of acute leukemia. The clinical significance of 'monocytic' phenotype of APML is not clear; cases with monocytic differentiation did not show different clinical and hematological features when compared to the more common, pure neutrophilic variety.     

Interleukin-1 producing ability of leukaemia cells and its relationship to morphological diagnosis

Interleukin-1 (IL-1) is a low molecular weight polypeptide produced by monocyte-macrophage lineage cells. IL-1 production by primary-cultured leukaemic cells of several FAB subtypes was estimated and compared with in vitro and in vivo lysozyme production. The results indicate that IL-1 production by monocytic leukaemia cells (M4 and M5) is significantly higher than that of myelocytic leukaemia cells (M1, M2 and M3). On the other hand, the serum lysozyme level was not correlated with the FAB subtypes and in vitro lysozyme production by monocytic leukaemia cells was higher than that of myelocytic leukaemia cells, but the M2 subtype was indistinguishable from monocytic leukaemia cells solely on the basis of lysozyme production. We concluded that measurement of IL-1 production by leukaemic cells, as a marker of monocytic leukaemias, was convenient and reliable, and might be useful for the diagnosis of morphologically or cytochemically atypical cases.     

Two murine monoclonal antibodies to peripheral blood monocyte differentiation antigens discriminate within M5 acute non-lymphoid leukemia (ANLL) cells

Two murine monoclonal antibodies (MoAbs), LAM3 and LAM7 of the IgG₁ isotype, which were produced by immunization with normal peripheral blood monocytes (PBM), were assayed in their specificity by indirect immunofluorescence against a panel of normal as well as leukemic cells. Both LAM3 and LAM7 were reactive with PBM while LAM3 also recognized platelets. Neither MoAb showed reactivity with erythrocytes, granulocytes, or resting and mitogen activated B and T lymphocytes. The reactivity with bone marrow cells correlated with the degree of monocyte contamination. Among the 62 cases of leukemia tested, which included three cases of B-CLL, 19 cases of ALL, and 40 cases of ANLL, both MoAbs reacted highly homogenously only with M5b ANLL cells. These findings indicate that the two MoAbs, which recognize two distinct epitopes, represent useful markers in the differential diagnosis of M5b ANLL.     

Monoclonal antibodies to the human CSF-1 receptor (c-fms proto-oncogene product) detect epitopes on normal mononuclear phagocytes and on human myeloid leukemic blast cells

The first monoclonal antibodies (MoAbs) to epitopes in the extracellular domain of the human c-fms proto-oncogene product (receptor for the macrophage colony stimulating factor, CSF-1) were used with flow cytometric techniques to study receptor expression on normal human peripheral blood monocytes, bone marrow cells, and leukemic blasts. On normal cells CSF-1 receptors were restricted in their expression to cells of the mononuclear phagocyte lineage. CSF-1 receptors were detected on leukemic blasts from 15 (30%) of 50 children with acute myeloid leukemia, compared with four (15%) of 26 adults. By contrast, detectable CSF-1 receptors were uniformly absent on blasts from 19 children with acute lymphoblastic leukemia. CSF-1 receptors on normal monocytes and myeloid leukemia cells could be induced to downmodulate by incubation with either human recombinant CSF-1 or phorbol esters, confirming that the receptors had functional ligand- binding sites and responded to transmodulation by inducers of protein kinase C. The numbers of receptors per cell and the percentage of positive cases were highest for leukemic blasts with cytochemical and morphological features of monocytes. However, CSF-1 receptors were also detected on a subset of leukemic blast cells with features of granulocytic differentiation (FAB subtypes M1 through M3). Southern blotting analyses of DNA from 47 cases of acute myeloid leukemia demonstrated no rearrangements within the 32 kb of genomic sequences that contain CSF-1 receptor coding exons or in the 50 kb upstream of the first coding exon. Analysis of the upstream region of the c-fms locus revealed that sequences representing the terminal 112 untranslated nucleotides of c-fms mRNA map 26 kb 5' to the first coding exon, suggesting that at least one c-fms promoter is separated from the receptor coding sequences by a very long intron. Whereas expression of the CSF-1 receptor in myeloid leukemic blasts is not restricted to cells with monocytic characteristics, the apparently aberrant pattern of receptor synthesis in a subset of cases with granulocytic features appears not to be due to chromosomal rearrangements within 50 kb upstream of sequences encoding the receptor.     

A case of acute monocytic leukemia with lysozyme-positive leukemic monocytes and normal serum lysozyme levels

We report a case of acute monocytic leukemia with normal serum lysozyme but with immunoperoxidase positivity for lysozyme in the leukemic blasts. The diagnosis of acute monocytic leukemia was made on the basis of cellular morphology, immunocytochemistry and cytochemistry. Discrepancy between serum lysozyme and intracytoplasmic lysozyme has not previously been reported in the literature. Levels of serum lysozyme may be misleading in cases of acute monocytic leukemia (FAB classification M5).     

The distribution of lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin in normal hematopoietic cells and in myeloid leukemias: an immunoperoxidase study on cytocentrifuge preparations, smears, and paraffin sections

Blood and bone marrow samples from 20 individuals with reactive conditions and 26 cases of acute and chronic myeloid leukemias were tested for the presence of lysozyme, alpha-1-antitrypsin (alpha-1-AT), and alpha-1-antichymotrypsin (alpha-1-ACT). We compared the reactivity of samples in smears, cytocentrifuge preparations, and paraffin sections. Lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin were found only in polymorphonuclear leukocytes and monocytes and their precursors. Lymphocytes, E-rosetting cells, Con A-activated lymphocytes, natural killer (NK) cells, red blood cells, erythroblasts, and megakaryocytes were consistently negative. Leukemic myeloblasts showed definite reactivity for both alpha-1-antitrypsin and alpha-1-ACT, but not for lysozyme. By contrast, lysozyme was present in poorly differentiated leukemic monoblasts, while alpha-1-antitrypsin and alpha-1-antichymotrypsin showed only weak reactivity. More mature myeloid and moncytic cells showed positive staining for all three antigens tested with differences in staining distribution and intensity. In four cases of chronic myeloid leukemia (CML), circulating mature polymorphonuclear leukocytes were deficient in both lysozymne and alpha-1-antitrypsin. The use of lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin identifies normal and leukemic cells of the myeloid-monocytic series at all stages of maturation and is applicable to a variety of sample preparations.     

Double minute chromosomes (DMs) in a case of acute myelomonocytic leukemia

A 71-year old male was admitted to our hospital because of general malaise and fever. Peripheral blood showed Hb 8.1 g/dl, platelet 7.0 X 10(4)/microliters, and WBC 18.100/microliters with 64% leukemic cells. Bone marrow showed normocellularity with 73.4% leukemic cells. They were positive for peroxidase and alpha-naphthyl butyrate esterase stainings. Serum and urine lysozyme levels were elevated. He was diagnosed as having acute myelomonocytic leukemia (M 4 in FAB classification). Chromosome analysis of bone marrow cells showed 45, XY, -17, t (9; 17) (q22; p13) and double minute chromosomes (DMs) were observed in the 50 cells analyzed. A complete remission (CR) was obtained by DCMP regimen, but he relapsed as acute monocytic leukemia (M 5 b in FAB classification) and died 5 months after diagnosis. DMs appear to be rare in acute leukemia and the clinical and etiologic implications of DMs are discussed.     

Analysis of acute myeloid leukemia cells by flow cytometry,introducing a new light-scattering classification

A combined flow-cytometric evaluation of light scattering and the immunophenotype of acute myeloid leukemia (AML) cells from 71 newly diagnosed consecutive patients was conducted. Light-scattering characteristic of AML cells examined by flow cytometry and multiple surface markers were also analyzed using the same samples, to enable a comparison with the French-American-British (FAB) classification. Our AML cases could be classified into three light-scattering classification (LSC) types according to their physical properties on flow cytometry. These were type A, where forward light scattering (FSC) of the leukemic cell population was larger than that of lymphocytes, while side light scattering (SSC) was the same or larger than that of lymphocytes but smaller than that of monocytes; type B, where FSC of the leukemic cell population was larger than that of lymphocytes and SSC spread toward that of monocytes; and type C, where both FSC and SSC of the leukemic cell population spread beyond those of monocytes. Although a clear relationship between the FAB classification and LSC classification by the light-scattering profile of AML was not established, we observed the following findings. The majority of cases were classified as type A (58%), while type B comprised 25% and type C comprised 17%. While CD7 expression on AML cells is considered to be an immature characteristic, CD7 was expressed more frequently among LSC type A cases. Furthermore, all but one of the FAB M1 cases were classified as type A. On the other hand, CD7 was not expressed on type C leukemic cells. The percentage of cases in which more than 60% of leukemic cells possessed another immature surface antigen, CD 34ö, was 13/18 (72%) among FAB M1 cases, much higher than among FAB M2 (35%) or FAB M4 (27%) cases. A negative correlation was observed between mature antigen CD33 and CD34 among the FAB M2 cases. The frequency of CD7 expression was 25% among the total cases, and CD7-positive cases were frequent among FAB M1 and M2, but not among FAB M3 cases. These findings concerning LSC and immunophenotyping indicate that the scattergram pattern analysis may contribute towards more precise immunophenotyping, in that it reflects the maturation stage of each AML case.Abbreviations AML acute myeloid leukemia - LSC light-scattering classification - FAB French-American-British classification - CD cluster of differentiation Partly supported by grants in aid from the Ministry of Education, Science and Culture of Japan (03670325, 04247102, 04454572 and 05670916) and from the Fukuoka Anti-cancer Society     

bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells.

The present study provides immunobiochemical and molecular data on the differentiation-linked expression of the bcl-2 proto-oncogene in normal and neoplastic myeloid cells. Using a recently developed monoclonal antibody (MoAb) to the bcl-2 molecule, staining of normal bone marrow myeloblasts, promyelocytes, and myelocytes, but neither monocytes nor most polymorphonuclear cells, was demonstrated. By two-color flow cytometric analysis, bcl-2 was evidenced in CD33+ and CD33+/CD34+ myeloid cells as well as in the more primitive CD33-/CD34+ population. The leukemic cell lines HL-60, KG1, GM-1, and K562 were bcl-2 positive together with 11 of 14 acute myeloid leukemias (AML) and three of three chronic myeloid leukemias (CML) in blast crises; six of seven CML were negative. Among myelodysplastic cases, augmentation of the bcl-2 positive myeloblastic compartment was found in refractory anemia with excess of blasts (RAEB) and in transformation (RAEB-t). Western blots of myeloid leukemias and control lymphocytes extracts evidenced an anti-bcl-2 immunoreactive band of the expected size (26 Kd). Moreover, the HL-60 and KG1 cell lines, both positive for the bcl-2 protein, exhibited the appropriate size bcl-2 mRNA (7.5 Kb). These findings clearly indicate that the bcl-2 gene is operative in myeloid cells and that the anti-bcl-2 MoAb identifies its product and not a cross-reactive epitope. Induction of HL-60 differentiation toward the monocytic and granulocytic pathways was accompanied by a marked decrease in bcl-2 mRNA and protein levels; bivariate flow cytometric analysis showed that the fraction becoming bcl-2 negative was in the G1 phase of the cell cycle. These data establish that the bcl-2 proto-oncogene is expressed on myeloid cells and their progenitors and is regulated in a differentiation-linked manner.     

BER-H2: a new anti-Ki-1 (CD30) monoclonal antibody directed at a formol-resistant epitope

The production and characterization of a monoclonal antibody (MoAb) designated Ber-H2, directed against a new epitope of the Ki-1 (CD30) antigen, are described. In comparison with the formerly reported Ki-1 MoAb whose reactivity with Hodgkin and Reed-Sternberg (H-RS) cells in frozen tissue sections is well-documented, the Ber-H2 MoAb showed new, important features: the labeling intensity of the Ber-H2 MoAb was much stronger, and the number of positively labeled cells was higher. Most important, however, was that the Ber-H2 MoAb could be applied in routinely processed, formaldehyde-fixed, paraffin-embedded tissue sections. Therefore, it was possible to investigate an unprecedented number of tumors received as frozen or formaldehyde-fixed material for expression of the CD30 antigen. Beside Hodgkin's disease, the Ber-H2 MoAb labeled a variable number of cells in lymphomatoid papulosis, peripheral T-cell lymphomas, and angoimmunoblastic lymphadenopathy. Among B-cell non-Hodgkin's lymphomas (NHLs), some cases containing large centroblast-like or immunoblast-like cells or displaying plasma-cellular differentiation were positive. This finding was in keeping with the reactivity of the Ber-H2 MoAb with activated B-cell blasts and a subpopulation of plasma cells in paraffin sections of normal lymphoid tissue. The diagnostic value of the Ber-H2 MoAb was most significant for a group of anaplastic large-cell (ALC) lymphomas (formerly frequently referred to as malignant histiocytosis or regressive atypical histiocytosis), of which more than 50 cases could be investigated, owing to applicability in paraffin sections. Although about one third of these ALC lymphomas did not express the leukocyte common (CD45) antigen, they were consistently reactive with the Ber-H2 MoAb in both frozen and paraffin-embedded tissue sections. Using the Ber-H2 MoAb, these Ki-1 lymphomas could be easily distinguished from other nonlymphoid anaplastic large-cell tumors.     

Role of the sialophorin (CD43) receptor in mediating influenza A virus- induced polymorphonuclear leukocyte dysfunction

Polymorphonuclear leukocytes (PMNLs) exposed to influenza A virus (IAV) undergo activation of the respiratory burst followed by depression of cell function when subsequently exposed to particulate or soluble stimuli. The effect of IAV on PMNLs is likely to be mediated through the attachment of IAV to one or more specific receptors. Recently, IAV has been shown to bind to the sialophorin protein (CD43) receptor on PMNL plasma membranes. The present study was performed to determine if the sialophorin receptor was responsible for IAV-induced PMNL dysfunction. When PMNLs were incubated with IAV or CD43 monoclonal antibody (MoAb) for 30 minutes and then exposed to a secondary particulate (opsonized zymosan) or soluble (FMLP or phorbol 12- myristate 13-acetate) stimulus, there was significant depression of the PMNL chemiluminescence response compared with the equivalent control (P < .05). When PMNL were incubated with the CD43 MoAb and then cross- linked with a goat antimouse IgG antibody, no depression of PMNL function occurred upon secondary stimulation. Exposure of cells to IAV aggregates also eliminated the PMNL dysfunction that normally occurs due to the virus. Similar to IAV, PMNL dysfunction due to the CD43 MoAb could be overcome by priming the cells with granulocyte-macrophage colony-stimulating factor. These findings indicate that IAV-induced PMNL dysfunction is mediated, at least in part, through the sialophorin receptor.     

Distinct histamine-induced cyclic AMP synthesis in acute leukemia

Histamine receptors of the H2 type are found on mature hemopoetic cells such as lymphocytes and monocytes. Since little is known about histamine receptors on immature cells, we studied histamine-induced cAMP synthesis in leukemic cells of 51 patients using 73 samples of peripheral blood and/or bone marrow. Histamine-induced cAMP synthesis was found in all cALL (FAB L1, L2), but only occasionally in AML. In 1 out of 11 myelomonocytic leukemias (M4), and also in 2 out of 5 monocytic leukemias (FAB M5), histamine-inducible cAMP synthesis was found, but no induction of cAMP synthesis by histamine was found in 19 cases from the remaining subgroups (FAB M1-M3, M6). The inhibiting action of H2 antagonists on cAMP synthesis indicates that the receptor is of the H2 type. Since H2 receptors were found in all cALL blasts, we assume a compulsory expression of this receptor on immature cells of the B-lymphocytic lineage.     

Reactivity profiles of leukemic myeloblasts with monoclonal antibodies directed to sialosyl-Le(x) and other lacto-series type 2 chain antigens:absence of reactivity with normal hematopoietic progenitor cells.

We investigated the expression profiles of lacto-series type 2 antigens in hematopoietic cells and their progenitors, in comparison with leukemic leukocytes. Reactivity profiles of various anti-type 2 chain monoclonal antibodies (MoAbs) with leukemic blasts from 12 patients with acute myeloblastic leukemia (AML) and those from two patients with acute unclassified leukemia (AUL) show that anti-sialosyl-Le(x) MoAb SNH3 reacted strongly with greater than 95% of leukemic blast leukocyte populations from all patients (14 of 14). Another anti-sialosyl-Le(x) MoAb, FH6, showed less reactivity than SNH3 (12 of 14 patients), while anti-Le(y) MoAb AH6 showed reactivity with only 8 of 14 patients. On the other hand, none of the anti-type 2 chain MoAbs reacted with CD34+ normal adult bone marrow (BM) mononuclear cells obtained independently from three healthy volunteers. MoAb SNH3, but not FH6 or AH6, showed complement-mediated cytotoxicity to leukemic blasts from these patients, as well as to myelogenous leukemia cell line HL60. Colony-forming unit granulocyte-macrophage (CFU-GM), but not burst-forming unit-erythroid (BFU-E), was incompletely inhibited by treatment of normal BM mononuclear cells with SNH3 and complement. The absence of type 2 chain antigen expression in hematopoietic progenitor cells and in in vitro hematopoietic colonies (CFU-GM and BFU-E) strongly suggests that application of anti-carbohydrate MoAbs, particularly anti-sialosyl-Le(x) could be useful for elimination of leukemic myeloblasts infiltrating in BM, for purging of leukemic blasts in BM, and for facilitation of autologous BM transplantation.     

A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors

A murine monoclonal antibody (MoAb) designated FA6-152 has been obtained by immunizing mice with fetal erythrocytes. This antibody agglutinates fetal but not adult erythrocytes. Among blood cells, this antibody bound to both adult and fetal monocytes, platelets, and reticulocytes, but did not react with lymphocytes and granulocytes. Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM, and CFU-MK-derived colonies has shown that the antigen defined by FA6-152 MoAb was absent from the granulocytic precursors and was detected on the megakaryocytic lineage at a later stage of differentiation than the platelet-specific markers. In contrast, the antigen appeared as a very early marker of the erythroid differentiation since all erythroblasts, including proerythroblasts, were labeled even before the expression of glycophorin A. Cells from adult marrow and fetal liver were sorted with the FA6-152 MoAb and studied by electron microscopy and cell culture. The negative fraction contained granulocytic, monocytic, and megakaryocytic precursors, whereas the positive fraction was devoid of these precursors and contained monocytes, erythroblasts at all stages of maturation, and a homogeneous population of blasts. Cultures have shown that the only hematopoietic progenitors present in this positive fraction were CFU-E and some BFU-E. The antigenic density was related to the differentiation stage of the erythroid progenitors. In conclusion, this antibody is similar to the previously described 5F1 MoAb (Bernstein and Andrews, J Immunol 128:876, 1982; and Andrews et al, Blood 62:124, 1983) and provides a useful probe for studies leading to improved understanding of normal and malignant erythroid differentiation.     

Activation signals leading to proliferation of normal and leukemic CD3+ large granular lymphocytes.

The activation signals leading to proliferation of normal and leukemic CD3+ large granular lymphocytes (LGL) were studied in vitro. Anti-CD3 monoclonal antibody (MoAb) alone (P less than .01) and recombinant interleukin-2 (IL-2) alone (P less than .01) caused significant stimulation of peripheral blood mononuclear cells (PBMC) from four CD3+ LGL leukemia patients, as measured in a 3H-thymidine incorporation assay. Recombinant interleukin-4 (IL-4) alone had no effect (P = .11). The combination signals of anti-CD3 MoAb and either IL-2 or IL-4 produced a proliferative response greater than anti-CD3 MoAb alone (P less than .01) or lymphokine alone (P less than .01). Leukemic LGL, purified by two-color sorting, were subsequently activated by anti-CD3 MoAb and IL-2 and assessed for DNA content by viable Hoechst No. 33342 (HO) staining. Results of these studies demonstrated that leukemic LGL were stimulated directly by anti-CD3 MoAb and IL-2, with the percentage of cells in cell cycle (S + G2/M) ranging from 16% to 72%. Normal CD3+ LGL were also stimulated to enter the cell cycle by anti-CD3 and IL-2. These results show that leukemic LGL proliferate in vitro after activation through the T-cell receptor and/or lymphokine.     

A new procoagulant in acute leukemia

To verify whether cancer procoagulant (CP), a cysteine proteinase procoagulant distinct from tissue factor (TF), is associated with leukemic cells, we assayed the procoagulant activity of blast cell extracts from 26 patients with different cytological subtypes of acute nonlymphoid leukemia (ANLL) according to the French-American-British classification. All the samples except two shortened the recalcification time of normal human plasma, the effect being significantly greater in the M3 subgroup. The two criteria used to distinguish between CP and TF, independence from factor VII in initiating blood coagulation and sensitivity to cysteine-proteinase inhibitors, were positive in 19 samples from M1, M2, M3, and M4 cytological subtypes. None of the M5 samples fulfilled these criteria. In addition, M1, M2, M3, and M4 samples immunoreacted with an anti-CP goat polyclonal antibody on an Ouchterlony immunodiffusion plate. This study provides the first evidence for a procoagulant other than TF that is associated with leukemic cells.