The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF

In human blood two monocyte populations can be distinguished, i.e., the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14(+)CD16(+) monocytes are major producers of TNF in human blood.     

Detection by flow cytometry of protoplast fusion and transient expression of transferred heterologous CD4 sequences in COS-7 cells

Transfer and expression of a plasmid containing the gene encoding the human T-cell antigen CD4 by protoplast fusion was measured by flow cytometry (FCM). Protoplasts were prelabeled with fluorescein isothiocyanate (FITC) and fused to COS-7 cells. Nonspecific protoplast adsorption to the plasma membrane was differentiated from successful protoplast fusion by the addition of an antibody directed against fluorescein to quench extracellular protoplast fluorescence. Transfection efficiencies were defined as both percent CD4 expressing cells and CD4 expression levels on a single cell basis in the transient immunofluorescence assay. Cell sorting studies indicated that intracellular protoplast-associated fluorescence immediately after fusion exhibited a good correlation with transient CD4 transfection efficiencies as measured by indirect immunofluorescence. Reconstruction experiments comparing CD4 transfer efficiencies of protoplast fusion and calcium phosphate transfection showed that fusion resulted in a higher percentage of CD4 expressing transfectants, while calcium phosphate transfection yielded higher CD4 expression levels on a single cell basis. Thus, FCM appears to be useful as a new tool for sensitive detection of transient expression of heterologous reporter genes in COS-7 cells.     

Triple labeling with two-color immunofluorescence using one light source: a useful approach for the analysis of cells positive for one label and negative for the other two

Many laboratories do not have access to a flow cytometer allowing three-color immunofluorescence analysis through the use of multiple light sources. In view of the usefulness of such analyses in the dissection of cell parameters, we describe an approach permitting the study of three labels by using one light source and the two-color immunofluorescence assay. It is useful for the enumeration of cell subpopulations positive for one label and negative for two or more others as well as for qualitative analysis concerning the expression of these labels. This approach is simple and rapid; it does not require additional material and technical steps other than that used in the two-color immunofluorescence assay. Briefly, it consists of the use of a label coupled to a dye (PE or FITC or instance) and two different labels coupled to the other dye. An argon ion laser, operating at 488 nm and 60 mW, excites both fluorescein and phycoerythrin conjugated antibodies. We provided a general example, using three hypothetical labels (X, Y, and Z), and four practical applications: CD3+CD4CD8- and CD8+CD16-CD3- peripheral blood lymphocytes, CD2+CD16-CD3- and CD56+CD16-CD3- peripheral blood, and decidual infiltrating lymphocytes.     

Immunofluorescence signal amplification by the enzyme-catalyzed deposition of a fluorescent reporter substrate (CARD)

Progress has been made in improving the immunohistochemical detection of antigens for imaging and flow cytometry. We report the synthesis of a novel fluorescent horseradish peroxidase substrate, Cy3.29-tyramide, and its application in an enzyme-based signal amplification system, catalyzed reporter deposition (CARD). The catalyzed deposition of Cy3.29-tyramide was used to detect cell surface markers such as CD8 and CD25 on tonsil tissue and human lymphocytes. We compared the fluorescence CARD method to standard indirect immunofluorescence detection methods and found that an amplification of up to 15-fold was possible with CARD. The detection of the intracellular protein myosin II in fibroblastic cells and rabbit serum proteins blotted onto nitrocellulose was also improved. Thus, fluorescent CARD is a simple modification that can be made to standard immunofluorescence staining protocols to enhance significantly the detection of antigens.     

Flow cytometric analysis of live cell proliferation and phenotype in populations with low viability.

BACKGROUND: Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of subpopulations to stimuli in mixed cell preparations; however, in low-viability cell preparations, dead cells interfere with accurate flow cytometric data analysis because of nonspecific binding of antibodies and altered DNA-staining profiles. Light scatter differences between nonviable and viable cells are unreliable, particularly after the cell permeabilization step that is necessary for DNA staining. We developed a method for identification of nonviable cells by fluorescence in cell preparations that are stained simultaneously for cell surface or intracellular immunofluorescence and DNA content. MATERIALS AND METHODS: Nonviable cells that have lost membrane integrity are identified by uptake of 7-amino-actinomycin D (7-AAD). Transfer of 7-AAD from stained nonviable cells to unstained viable cells after permeabilization is prevented by blocking DNA binding with nonfluorescent actinomycin D (AD). Pyronin Y(G) (PY) is used for DNA staining because the orange spectral emission of PY can be separated from the green fluorescein isothiocyanate (FITC) emission and the red emission of 7-AAD, respectively. RESULTS: Application of the method to the analysis of the T-cell leukemia cell line Molt-4f and of cultured human peripheral blood mononuclear cells is presented. In both cell preparations, 7-AAD staining permitted reliable dead cell exclusion. Live, 7-AAD-negative Molt-4f cells showed higher expression levels of cell surface CD4 and of intracellular CD3, showed a higher proportion of cells in the G1 phase of the cell cycle, and showed a lower coefficient of variation of the G1 peak compared with data obtained from all the cells in the preparation. Live, CD8+ lymphocytes from OKT3-stimulated cultures of human peripheral blood mononuclear cells showed a specific proliferative response as measured by DNA content analysis. CONCLUSIONS: The results show that cells stained with FITC-labeled antibodies can be analyzed by single-laser flow cytometry for DNA content combined with dead cell discrimination. Furthermore, they emphasize the need for exclusion of dead cells from the analysis of cell preparations with low viability to obtain reliable data on immunofluorescence and cell-cycle distributions.     

185例儿童急性白血病流式细胞术免疫分型研究

目的:探讨儿童急性白血病流式细胞术免疫分型的意义。方法:采用流式细胞术三色荧光标记技术和CD45/SSC双参数散点图设门,检测185例儿童急性白血病的免疫表型,对抗原表达情况进行分析。结果:流式细胞术免疫分型和FAB分型的符合率为89.19%。185例儿童急性白血病中,ALL为121例,占AL的65.41%,B-ALL为113例,主要表达B系的CD19(99.12%)、CD22(98.13%)、CD79a(96.19%)、CD10(86.73%)。T-ALL占8例;主要表达CD5(100%)、CD7(100%)、cCD3(100%)、CD8(87.5%)。AML为47例,占25.41%,主要表达CD33(93.62%)、CD15(78.72%)、CD64(76.6%)、MPO(76.6%)、CD13(74.47%)。在B-ALL,AML,T-ALL中,敏感性最高的抗体分别是CD19,CD33,CD5和CD7,特异性最强的抗体分别是CD79a,MPO,cCD3。AMLL为17例,占9.19%,其中B/M为9例,T/B为5例,T/M为3例。My十-ALL为54例,占ALL的44.63%,表达的髓系抗原为CD13、CD15、CD33、CD64。Ly+-AML为18例,占AML的38.30%,表达的淋系抗原为CD19、CD4、CD7。系列非相关抗原CD34的表达率为67.57%,HLA-DR的表达率为85.41%,CD38的表达率为80.59%,TdT的表达率为62.59%。结论:流式细胞术免疫分型在白血病分型中起重要作用,是FAB分型的补充和修正,提高了儿童急性白血病诊断的准确率有必要进一步加强流式细胞术免疫分型的标准化工作。     

Distinction between HLA class I-positive and -negative cervical tumor subpopulations by multiparameter DNA flow cytometry

BACKGROUND: The study of the molecular-genetic basis of heterogeneity of HLA class I expression in solid tumors is hampered by the lack of reliable rapid cell-by-cell isolation techniques. Hence, we studied the applicability of a flow cytometric approach (Corver et al.: Cytometry 2000;39;96-107). METHODS: Cells were isolated from five fresh cervical tumors and simultaneously stained for CD45 or vimentin (fluorescein isothiocyanate fluorescence), Keratin (R-phycoerythrin fluorescence), HLA class I (APC fluorescence), and DNA (propidium iodide fluorescence). A dual-laser flow cytometer was used for fluorescence analysis. Tissue sections from the corresponding tumors were stained for HLA class I antigens, keratin, vimentin, or CD45. RESULTS: Flow cytometry enabled the simultaneous measurement of normal stromal cells (vimentin positive), inflammatory cells (CD45 positive), epithelial cells (keratin positive), and DNA content readily. Normal stromal/inflammatory cells served as intrinsic HLA class I-positive as well as DNA-diploid references. Good DNA histogram quality was obtained (average coefficient of variation < 4%). Intratumor keratin positive subpopulations differing in HLA class I expression as well as DNA content could be clearly identified. Losses of allele-specific HLA class I expression found by immunohistochemistry were also detected by flow cytometry. CONCLUSIONS: We conclude that multiparameter DNA flow cytometry is a powerful tool to study loss of HLA class I expression in human cervical tumors. The method enables flow-sorting of discrete tumor and normal cell subpopulations for further molecular genetic analysis.     

Identification of cell subpopulations by dual-color surface immunofluorescence using biotinylated and unlabeled monoclonal antibodies

A new method of dual-color immunofluorescence is presented for analysis of surface antigen distribution among heterogeneous cell suspensions. It involves flow cytometric analysis of cells stained with a biotinylated first monoclonal antibody and/or with an unlabeled second monoclonal antibody. After addition of streptavidin-phycoerythrin and/or fluoresceinated goat antimouse immunoglobulin antibody, single-cell fluorescence intensities are measured and biparametric graphic representations are obtained, allowing one to determine the percentage of cells stained by each of the monoclonal antibodies or both. The validity of the method was assessed on human peripheral blood mononuclear cells by using three sets of two monoclonal antibodies: CD8 and CD5, CD3 and CD4, CD11 and HLA-DR. The results showed that dual staining did not induce significant quenching or competition between pairs of antibodies. The procedure is simple and sensitive. It requires only minute amounts of monoclonal antibodies. It is readily applicable to the screening of hybridoma supernatants and to the characterization of new antibodies to cell surface antigens with respect to well-defined markers.     

Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression

Three-color immunofluorescence has been used to determine the co-expression of cell surface antigens on human peripheral blood lymphocytes. Monoclonal antibodies or avidin were coupled to either FITC (green), phycoerythrin (orange), or Texas Red (red) fluorochromes. These three fluorochromes could be independently measured by using a dual laser FACS IV system equipped with an argon ion laser (488 nm) and a dye laser (600 nm). Human peripheral blood lymphocytes were stained with the following combinations of reagents: (1) FITC anti-Leu-11a + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; (2) FITC anti-Leu-11a + PE anti-Leu-3a + TR avidin/biotin anti-Leu-7; (3) FITC anti-Leu-8 + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; and (4) FITC anti-Leu-11a + PE anti-Leu-2 + TR avidin/biotin anti-Leu-8. The light scatter, green fluorescence, orange fluorescence, and red fluorescence signals for each sample were stored by a Consort 40 PDP/11 computer in list mode files. Sequential reanalysis of the data directly demonstrated the existence of several unrecognized subpopulations of lymphocytes. Previously, we reported that the anti-Leu-7 and anti-Leu-11 antibodies can be used to identify discrete subsets of human NK cells with distinct functional capacities. In this report, we show that these subsets can be further subdivided on the basis of Leu-8 and Leu-2 expression. Thus, these studies illustrate how multicolor and multiparameter flow cytometry can further our understanding of cellular heterogeneity within this group of lymphocytes.     

Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies

BACKGROUND: Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards. METHODS: Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors. RESULTS: Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 x 10(4)-1.5 x 10(5); CD8, 5.0 x 10(5)-2.1 x 10(6); CD3, 6.6-7.8 x 10(5). Binding constants were highest for unlabeled CD4 antibody, 2. 7 x 10(10)-2.1 x 10(12) M(-1), and then unlabeled CD3 antibody, 1.1 x 10(10)-1.9 x 10(11) M(-1). FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies. CONCLUSIONS: Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.     

Genetic influence on the levels of circulating CD5 B lymphocytes

By using sensitive two- and three-color immunofluorescence analyses, we readily detect CD5B cells (Leu 1 B cells) in the peripheral blood of normal adults. These circulating CD5 B lymphocytes coexpress B cell differentiation antigens CD20, CD21, CD19, sIgM and sIgD, and HLA-DR. Unlike CD5-negative B cells from most adults, however, these cells co-express CD11, a finding also noted for malignant CD5 B cells from several patients with CLL. Between normal volunteers, there exists heterogeneity in the proportion of PBL that co-express CD5 and B cell surface antigens, such cells representing between 0 and 6% of peripheral lymphocytes. Despite such heterogeneity between unrelated individuals, analyses of repeated blood samples from the same person reveal that the proportions of CD5 B lymphocytes are constant over time. Examination of blood samples from related family members, monozygotic twins, and triplets indicate that the relative proportion of circulating CD5 B cells may be genetically regulated. This is apparent even for monozygotic twins discordant for rheumatoid arthritis. Four sets of such twins are examined, each set having one individual with clinically active, seropositive rheumatoid arthritis and another without detectable rheumatoid factor or clinical pathology. Despite such noted differences, twins from each set share identical proportions of circulating CD5 B cells. In summary, our studies indicate that the level of CD5 B lymphocytes is a rather stable phenotypic trait that is under genetic control.     

Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients

Ten years ago, we made an incidental flow cytometric observation while immunophenotyping biopsy and marrow samples from children suspected to have leukemia/non-Hodgkin's lymphoma, but were subsequently diagnosed with neuroblastoma. The samples contained neoplastic CD45(-) cells that had an extremely bright CD56(+) (beyond the fourth decade on a four-decade scale) population distinguishable from CD45(+)CD56(usual density+) natural killer lymphocytes as well as other CD45(-)CD56(usual density+) nonhematopoietic tumors such as small cell carcinoma or melanoma. Following the "rare event" philosophy of selecting one negative and two positive antigens, we initially tried a "cocktail" of CD45(-)CD56(very bright+) neuron-specific enolase (NSE)(cytoplasmic+). We later modified the procedure to a more clinically applicable "lysed whole blood" CD45(-)CD56(very bright+) ganglioside GD2(+) cocktail to improve turnaround time (eliminating the cell permeabilization step for cytoplasmic NSE analysis), specificity, and sensitivity of the assay. A total of 123 marrow/tissue/fluid samples were analyzed by the various forms of the assay. Clearly interpretable samples had an 83% specificity and a 100% sensitivity. The three-color GD2 assay has successfully detected cells in marrow samples to a level of 0.002% (1 per 10(5) cells) using patient samples (not artificially "spiked" material). We added CD81 expression of the neuroblastoma cells as a fourth color and now use this rare event clinical test to help stage and monitor all patients with neuroblastoma.     

Comparative study of five commercial reagents for preparing normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry.

The flow cytometric analysis of leucocytes in whole blood is usually performed on samples in which the erythrocytes have been lysed and the leucocytes fixed. Because lysis and fixation reagents have the potential to introduce artefacts, several commercially available reagents were used to prepare normal and leukaemic lymphocytes for immunophenotypic analysis by flow cytometry, and the results were compared with those obtained from live whole blood. The reagents tested were the ImmunoPrep system and OptiLyse C (Coulter), LF-1000-Lyse and Flow (Harlan), Uti-Lyse (Dako) and FACS Lysing Solution (Becton Dickinson). The effect of each reagent on the apparent expression of CD3, CD5, CD11b, CD45, FMC7, kappa and lambda antigens was determined on lymphocytes from six normal controls and from six patients with chronic lymphocytic leukaemia (CLL). The following observations were made: (i) the time in minutes for each procedure varied markedly and was 1.5, 15, 20, 30 and 30 for the ImmunoPrep system, OptiLyse C, Uti-Lyse, FACS Lysing Solution, and LF-1000, respectively, but only 0.5 min for live whole blood. (ii) The forward and side scatter characteristics were affected by all of the lysis and fixation procedures, and this was most marked for LF-1000-Lyse and Flow. (iii) OptiLyse C gave preparations with poor forward and side scatter resolution due to the presence of residual red cell fragments. (iv) Lysis and fixation procedures did not affect the apparent expression of the CD3, CD45, or FMC7 antigens on normal or CLL samples, but gave highly variable results for the expression of the CD5, CD11b, kappa, and lambda antigens on the CLL samples. We conclude that lysis and fixation procedures can introduce different artefacts in the analysis of normal and leukaemic samples that are best avoided by analysing live whole blood.     

Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time

Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy.     

Clinical application of four and five-color flow cytometry lymphocyte subset immunophenotyping

A 1995 survey of clinical flow cytometry laboratories in the United States determined that 63% of clinical laboratories used one or two-color, 33% used three-color, 4% used four-color, and none used five-color panels. We show the feasibility and advantages of acquiring routine clinical four-color, six-parameter and five-color, seven-parameter analysis on blood samples derived from a pediatric population. The panels were evaluated by comparing the following cell characteristics: size, internal structure, and up to five distinct fluorochrome-conjugated antibodies to cell surface antigens: CD3, CD16+CD56, CD19, CD8, and CD4. These samples were processed on a commercially available instrument without any special modifications. A comparison of two-color and four-color as well as four-color and five-color panel analysis showed no statistical difference between the groups. We propose that the five-color, single-tube panel will (1) eliminate the need for isotype controls; (2) the relative proportions of lymphocyte subpopulations may be used to validate the operator-defined window, replacing CD45; (3) eliminate the need to run a common factor, in order to establish and maintain a reproducible lymphocyte window between tubes; and (4) create a more complete clinical picture by generating 32 unique, mutually exclusive phenotypes (permutations). Our results show that it is feasible to acquire and integrate seven-parameter data. This may be a powerful tool for immunophenotyping cells in a modern clinical diagnostic cytometry laboratory.     

Endocytosis of the TCR/CD3 complex and the class-I major histocompatibility complex in a human T cell line.

We investigated the expression of the T cell receptor (TCR)/CD3 complex on a CD4-positive human T cell lymphoma cell line treated with phorbol myristate acetate (PMA) and/or CA2+ ionophore using fluorescence flow cytometry and fluorescence microscopic analysis. PMA induced a significant decrease in the expression of the CD3 complex on the cell membranes. Fluorescence microscopy confirmed that the down regulation is due to internalization of the antigens. Ca2+ ionophore treatment had no effect on the internalization of the CD3 complex. Double staining revealed that the vesicles containing the internalized CD3 complex and those containing intra-cytoplasmic class I major histocompatibility complex antigen had similar distribution in the PMA-stimulated cells, implying coexistence of these two antigens in a cytoplasmic perinuclear distribution.     

CD56 identifies monocytes and not natural killer cells in rhesus macaques.

BACKGROUND: CD56 is a lineage-specific marker of human natural killer (NK) cells. There are conflicts in the literature regarding the role of CD56 as a marker of NK cells in non-human primates. In the present study, we examined the role of CD56 in identifying rhesus NK cells. METHODS: The immunophenotype of normal macaque and human NK cells was analyzed by two- and three-color flow cytometry. Flow cytometric cell sorting was subsequently used to deplete or purify NK cells; the resulting cell populations were then used in standard chromium release assays of NK lytic function. RESULTS: In peripheral blood mononuclear cells of the rhesus macaque, CD56 was expressed primarily on cells with the light scatter and immunophenotypic profile of monocytes. Flow cytometric depletion of rhesus CD56(+) monocytic cells did not diminish functional activity against K562 cells, whereas depletion of CD8(+) or CD16(+) lymphocytes completely abrogated functional activity. Three-color flow cytometric analysis of CD8(+), CD16(+) lymphocytes showed that they expressed other markers (CD2, CD7, TIA-1) associated with NK cells, but notably, not CD56. CONCLUSIONS: These studies demonstrate that CD56 is not suitable as a marker of NK cells in the rhesus macaque.     

Profiling CD antigens on leukaemias with an antibody microarray

Cluster of differentiation (CD) antigens are defined when a surface molecule found on some members of a standard panel of human cells reacts with at least one novel antibody, and there is good accompanying molecular data. Monoclonal antibodies to surface CD antigens on leukocytes have been used for flow cytometry, and more recently to construct microarrays that capture live cells. These DotScan^TM microarrays enable the rapid and highly parallel characterization of repertoires of CD antigens whose expression patterns may be correlated with discrete leukaemia subtypes, or used to define biomarker ‘signatures’ for non-hematological diseases. DotScan^TM with fluorescence multiplexing enables profiling of CD antigens for minor subsets of cells, such as colorectal cancer cells and tumour-infiltrating lymphocytes from a surgical sample.     

A flow cytometric screening test for detergent-resistant surface antigens in monocytes.

Rafts resemble cholesterol- and glycosphingolipid-enriched, liquid-ordered plasma membrane microdomains, showing resistance to nonionic detergents, and are involved in various cellular processes. In the present study, we have tested surface antigens on resting and lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes for their detergent resistance (i.e. raft-association), by flow cytometry. Constitutive (CD14, CD32, CD55), or LPS-induced (CD81) raft-association, and detergent solubility (i.e. exclusion of rafts) (CD71) of monocyte antigens in the presence of 0.01% Triton X-100 are clearly demonstrated. Flow cytometric detergent insolubility is a powerful tool for rapid screening the raft-association of monocyte antigens in a whole-blood assay.     

Role of IgE receptors in IgE antibody-dependent cytotoxicity and phagocytosis of ovarian tumor cells by human monocytic cells

Antibodies directed against tumor-associated antigens are emerging as effective treatments for a number of cancers, although the mechanism(s) of action for some are unclear and still under investigation. We have previously examined a chimeric IgE antibody (MOv18 IgE), against the ovarian tumor-specific antigen, folate binding protein (FBP), and showed that it can direct human PBMC to kill ovarian cancer cells. We have developed a three-color flow cytometric assay to investigate the mechanism by which IgE receptors on U937 monocytes target and kill ovarian tumor cells. U937 monocytes express three IgE receptors, the high-affinity receptor, FcεRI, the low-affinity receptor, CD23, and galectin-3, and mediate tumor cell killing in vitro by two mechanisms, cytotoxicity, and phagocytosis. Our results suggest that CD23 mediates phagocytosis, which is enhanced by upregulation of CD23 on U937 cells with IL-4, whereas FcεRI mediates cytotoxicity. We show that effector : tumor cell bridging is associated with both activities. Galectin-3 does not appear to be involved in tumor cell killing. U937 cells and IgE exerted ovarian tumor cell killing in vivo in our xenograft model in nude mice. Harnessing IgE receptors to target tumor cells suggests the potential of tumor-specific IgE antibodies to activate effector cells in immunotherapy of ovarian cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.