of March 25, 2018.This information is current as
Cancer CellsMechanisms of Eradication of OvarianSolid Tumors: Cytotoxic and Phagocytic IgE-Antibody-Dependent Immunotherapy of
Balkwill and Hannah J. GouldBurke, Robert J. Moore, David D. Dombrowicz, Frances R. David J. Fear, Richard G. Thompson, Nicholas East, FrancesNatalie McCloskey, Rebecca L. Beavil, Andrew J. Beavil, Sophia N. Karagiannis, Marguerite G. Bracher, James Hunt,
http://www.jimmunol.org/content/179/5/2832doi: 10.4049/jimmunol.179.5.2832
2007; 179:2832-2843; ;J Immunol
Referenceshttp://www.jimmunol.org/content/179/5/2832.full#ref-list-1
, 27 of which you can access for free at: cites 68 articlesThis article
average*
4 weeks from acceptance to publicationFast Publication! •
Every submission reviewed by practicing scientistsNo Triage! •
from submission to initial decisionRapid Reviews! 30 days* •
Submit online. ?The JIWhy
Subscriptionhttp://jimmunol.org/subscription
is online at: The Journal of ImmunologyInformation about subscribing to
Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:
Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:
Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,
is published twice each month byThe Journal of Immunology
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
IgE-Antibody-Dependent Immunotherapy of Solid Tumors:Cytotoxic and Phagocytic Mechanisms of Eradication ofOvarian Cancer Cells1,2
Sophia N. Karagiannis,3* Marguerite G. Bracher,* James Hunt,* Natalie McCloskey,*Rebecca L. Beavil,* Andrew J. Beavil,* David J. Fear,* Richard G. Thompson,† Nicholas East,†
Frances Burke,† Robert J. Moore,† David D. Dombrowicz,‡ Frances R. Balkwill,†
and Hannah J. Gould*
Abs have a paramount place in the treatment of certain, mainly lymphoid, malignancies, although tumors of nonhemopoieticorigin have proved more refractory ones. We have previously shown that the efficacy of immunotherapy of solid tumors, inparticular ovarian carcinoma, may be improved by the use of IgE Abs in place of the conventional IgG. An IgE Ab (MOv18 IgE)against an ovarian-tumor-specific Ag (folate binding protein), in combination with human PBMC, introduced into ovarian cancerxenograft-bearing mice, greatly exceeded the analogous IgG1 in promoting survival. In this study, we analyzed the mechanismsby which MOv18 IgE may exert its antitumor activities. Monocytes were essential IgE receptor-expressing effector cells thatmediated the enhanced survival of tumor-bearing mice by MOv18 IgE and human PBMC. Monocytes mediated MOv18 IgE-dependent ovarian tumor cell killing in vitro by two distinct pathways, cytotoxicity and phagocytosis, acting respectively throughthe IgE receptors Fc�RI and CD23. We also show that human eosinophils were potent effector cells in MOv18 IgE Ab-dependentovarian tumor cell cytotoxicity in vitro. These results demonstrate that IgE Abs can engage cell surface IgE receptors and activateeffector cells against ovarian tumor cells. Our findings offer a framework for an improved immunotherapeutic strategy forcombating solid tumors. The Journal of Immunology, 2007, 179: 2832–2843.
O varian cancer ranks second among gynecological can-cers in the number of new cases and first in the numberof deaths each year (1, 2). In the early stages, it grows in
and around the ovaries and it metastasizes in the peritoneal cavityand later to the internal organs. It causes no symptoms in the earlystages, and is essentially incurable in the late stages of the disease;around 50–60% of patients die within 5 years (1, 2). Ovariancancer is intractable to current chemotherapies, but immunother-apies using IgG Abs currently in clinical trials are showing somepromising results (3–6).
The current use of the IgG1 Ab isotype in Ab immunotherapystems from Waldmann’s classic work (7, 8) on the efficacy of
Campath-1H (alemtuzumab) IgG Ab subclasses in complement-dependent immunotherapy of non-Hodgkin’s lymphoma. Severalother mechanisms are now known to operate in Ab immunother-apies (3, 9). However, non-IgG isotypes have never been tried incancer patients. IgG Abs are now increasingly being used for thetreatment of blood malignancies, but “solid” tumors are often re-fractory (3–5). Probable reasons are diffusional barrier of tissues,slow recruitment of effector cells, and low affinity of IgG for itsreceptors (8–10).
Abs of the IgE class may offer an alternative to the conventionaltreatments with IgG Abs, particularly for solid tumors such asthose of the ovary. Unlike Abs of the IgG class, IgE binds to itsreceptors with very high affinity. The affinity of IgE for its highaffinity receptor, Fc�RI (Ka � 1011 M�1), is two to five orders ofmagnitude higher than that of IgG for the Fc�Rs (Fc�RI-III) (9–11). Its affinity for the low affinity receptor, CD23 (Ka � 108
M�1), is as high as that of IgG for Fc�RI (11). Furthermore, theperitoneal cavity, where ovarian cancer spreads, is home to IgEreceptor-expressing cells, such as macrophages (12, 13), mast cells(14), and dendritic cells (15). These properties may translate tostrong retention of Abs in tissues and longer antitumor immunesurveillance. IgE may therefore have greater efficacy than IgG intargeting tumors in nonhemopoietic tissues.
A number of epidemiological studies on the relation betweenallergies and the risk of cancer support the idea that IgE mighthave advantages over IgG for the treatment of certain types ofcancer. Mills et al. (16) conducted a prospective study in 34,198Seventh-Day Adventists. They found that the risk of prostate,breast cancer, and lymphatic or hematopoetic cancers actually in-creased with allergy, and further increased with an increasing num-ber of allergies. However, the risk of ovarian cancer was de-creased with allergy and decreased with an increasing number of
*Randall Division of Cell and Molecular Biophysics, New Hunt’s House, King’sCollege London, Guy’s Campus, London, United Kingdom; †Centre for TranslationalOncology, Institute of Cancer and the CR-UK Clinical Centre, Barts and The London,Queen Mary’s School of Medicine and Dentistry, John Vane Science Centre, Char-terhouse Square, London, United Kingdom; and ‡Institut National de la Sante et de laRecherche Medicale, Institut Federatif de Recherche 17, Institut Pasteur de Lille,Unite 547, Lille, France
Received for publication February 15, 2007. Accepted for publication June 28, 2007.
The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by the Association for International Cancer Re-search, U.K.2 The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. section 1734 solely to indicate this fact.3 Address correspondence and reprint requests to Dr. Sophia N. Karagiannis, RandallDivision of Cell and Molecular Biophysics, King’s College London, Room 3.8, NewHunt’s House, Guy’s Campus, St Thomas’s Street, London, U.K. E-mail address:[email protected]
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
www.jimmunol.org
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
allergies. A recent meta-analysis has demonstrated a significantinverse association between a history of both asthma and hay feverand overall cancer mortality and colorectal cancer (17). Recentstudies have also confirmed the increased risk of prostate andbreast cancer (18), and shown an increased risk for lung cancerwith asthma and a decreased risk of pancreatic cancer and glioma(19–24).
Experimental studies have also supported the concept of har-nessing IgE for cancer therapy. Nagy et al. (25) demonstrated thata mouse monoclonal IgE, directed against the murine mammarytumor virus, prevented the growth of the tumor in mice. Kershawet al. (26) showed that a mouse monoclonal IgE, directed againsta human colon carcinoma Ag, conferred a brief survival advantageto mice implanted with colon tumor cells. We have shown that achimeric Ab, MOv18 IgE, directed against an ovarian tumor Ag,folate binding protein, in combination with human PBMC, wasmore active than MOv18 IgG1 in protection of mice from ovariantumor growth in two xenograft models of ovarian carcinoma inscid and nude mice (27, 28).
T cells do not express Fc�RI, and hence treatment with IgE Abswould not exploit their known cytotoxic functions. Kershaw et al.(29) expressed the extracellular portion of the high-affinity IgEreceptor, Fc�RI �-chain, joined to the membrane sequence ofmouse Fc�RII and cytoplasmic sequences of CD28 and the TCR�-chain (CD28-�) in primary human T cells (30). Fc�RI-CD28-� Tcells, together with an IgE Ab directed against CD8, protected scidmice from the growth of a human thymoma tumor. This strategyshould allow any tumor Ag-specific IgE or combination of IgEs tobe used in adoptive cell immunotherapy of any type of cancer.
Another way of exploiting the inherent advantages of IgE overIgG is based on oral vaccination of mice with tumor Ags underalkaline conditions, which favors the production of IgE Abs (31,32). Jensen-Jarolim et al. (33) showed that oral vaccination with abreast tumor “Ag” stimulates the production of IgE Abs that ac-tivate IgE effector cells and mediate tumor cell lysis in vitro. Realiet al. (34) have shown that passive immunization with IgE Abs canalso result in stimulating an active immune response. This reflectsthe sensitization of APCs bearing Fc�RI, which results in uniquelystrong stimulation. It is notable that no adverse effects of IgE Abtreatment in the mouse models of cancer have been observed inany of the above-mentioned studies.
The present study focuses for the first time on the mechanism ofIgE-dependent tumor cell killing. For this, we made use of MOv18IgE developed against the tumor-associated Ag folate binding pro-tein (35, 36), which is overexpressed in 80% of ovarian cancers(37, 38). In a previous study, we observed that monocytes infil-trated human ovarian tumors growing in the nude mice treatedwith MOv18 IgE and human PBMC (28). In the present study, weshow that monocytes are necessary for the protection of the miceby human PBMC.
Human monocytes express the two IgE receptors, the high-af-finity receptor, Fc�RI, and the low-affinity receptor, CD23 (39–41). CD23b expression is induced by IL-4 on a wide range ofhemopoietic cells, including monocytes (42, 43) and has beenshown to act in IgE Ab-dependent phagocytosis (ADCP)4 of hap-ten-coated red cells (44). The nearest IgG receptor homologue toFc�RI is Fc�RIII, which acts in IgG Ab-dependent T cell-, NKcell-, and macrophage-mediated tumor cell cytotoxicity (Ab-de-pendent cell-mediated cytotoxicity, ADCC) (9, 10, 45). In thisstudy, we use a novel three-color cytometric assay (46) to enable
us to distinguish between two modes of IgE-dependent monocyte-mediated tumor cell killing, cytotoxic cell killing (ADCC), andphagocytosis (ADCP), and establish that Fc�RI is responsible forADCC and CD23 for ADCP.
Eosinophils express low levels of Fc�RI, which mediates IgE-dependent stimulation of IL-10 secretion and defense against par-asites (47). Using purified eosinophils from blood, we now showthat they, too, are potent effector cells in MOv18 IgE Ab-depen-dent ovarian tumor cell killing in vitro.
Materials and MethodsAbs and reagents
Chimeric Abs MOv18 IgE against folate binding protein and 4-hydroxy-3-nitro-phenacetyl (NIP) IgE specific for the hapten NIP were prepared asbefore (27, 48). We used goat anti-human IgE-FITC Ab (Vector Labora-tories), anti-CD89-PE (BD Biosciences), anti-CD14-PE, isotype controlmAbs, and anti-mouse IgG (Fab)2-FITC (DakoCytomation). Anti-CD23mAbs IDEC-152 and IDEC-152 Fab (Dr. J. Hopp, Biogen Idec, San Diego,CA) recognize the IgE binding site (49). Anti-Fc�RI mAb 22E7 recognizesan epitope unaffected by IgE occupancy (Hoffmann-La Roche) (50). Thesoluble Fc�RI �-chain (sFc�RI�) was prepared as before (51). HumanrIL-4 (1U � 34.5 pg) was obtained from R&D Systems. Propidium iodide(PI), CFSE dye, tissue culture medium, and reagents were obtained fromInvitrogen Life Technologies.
Flow cytometric evaluation of receptor expression and IgEbinding to monocytes
Monocytes were incubated with 10 �g/ml mAb 22E7 or MHM6, anti-mouse IgG (Fab)2–FITC, followed by anti-CD14-PE. To assess IgE bind-ing, monocytes were given 5 �g/ml MOv18 IgE or no Ab for 30 min at4°C, followed by 10 �g/ml goat anti-IgE-FITC for 30 min at 4°C. To blockIgE binding to cell surface receptors, 5 �g/ml MOv18 IgE were incubatedalone or with 62 �g/ml sFc�RI� for 30 min at 37°C, followed by additionof monocytes and anti-IgE-FITC. Incubations and washing steps were per-formed in FACS buffer (PBS, 5% normal goat serum).
Cell purification, stimulation, and culture
The human ovarian carcinoma IGROV1 cells were grown in RPMI 1640,10% FCS complete medium at 37°C in 5% CO2 (52). Monocytes fromhuman venous blood were isolated to 70–80% purity as described before(28). Cells were incubated overnight at 37°C in AIM-V medium, 5% FCS,in VueLife (FEP) culture bags (American Fluoroseal Corporation) (53).Monocytes were incubated for 20 h with 320 U/ml (10 ng ml�1) humanrIL-4 to stimulate CD23 or 2 �g/ml MOv18 IgE to stimulate Fc�RI. For invivo experiments, PBMCs were isolated from human venous blood as be-fore (27, 28). PBMCs were depleted of monocytes by incubation withanti-CD14-coated immunomagnetic beads to label monocytes, followed bythe removal of labeled monocytes using a VarioMACS immunomagneticdevice (Miltenyi Biotec) according to the manufacturer’s instructions. Eo-sinophils were isolated to �95% purity by Percoll gradient centrifugation(density 1.082 g/ml) (GE Healthcare) followed by immunomagnetic sep-aration with anti-CD16-coated immunomagnetic beads as previously de-scribed (47), and used for assays immediately. All work was performedwith the approval of the Guy’s Research Ethics Committee and with thevolunteers’ written informed consent.
Flow cytometric cytotoxicity/phagocytosis (ADCC/ADCP) assay
Cell treatment. A three-color flow cytometric assay was used to si-multaneously study tumor cell cytotoxicity (ADCC) and phagocytosis(ADCP) of IGROV1 cells by human effector cells as previously de-scribed (46). IGROV1 cells were labeled with 10�5 mM CFSE for 10min at 37°C 1 day before assays. A total of 1.3 � 105 CFSE-labeledIGROV1 cells were mixed with 1.3 � 105 unstained effectors (E:Tratio � 1:1) and 5 �g/ml MOv18, NIP IgE, or no Ab, followed byincubation for 2.5 h at 37°C. In blocking experiments, 25 �g/ml IDEC-152 Fab were added to monocytes for 30 min at 37°C before assays. Inothers, 62 �g/ml sFc�RI� were combined with 5 �g/ml IgEs or withcomplete medium alone for 30 min at 37°C, followed by addition ofcells. All conditions were tested in triplicate. Cells were then incubatedwith 10 �g/ml anti-CD89-PE mAb to label monocytes or anti-CD49d-PE to label eosinophils for 25 min at 4°C, washed, and treatedwith 0.25 �g/ml PI for 15 min at 4oC to identify dead cells. Following
4 Abbreviations used in this paper: ADCP, Ab-dependent cell-mediated phagocytosis;ADCC, Ab-dependent cell-mediated cytotoxicity; NIP, 4-hydroxy-3-nitro-phenac-etyl; sFc�RI�, soluble Fc�RI �-chain; PI, propidium iodide; SL, spontaneous loss.
2833The Journal of Immunology
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
a further wash, cells were mixed thoroughly to interrupt cell-cell con-tact, and 20,000 cellular events were acquired by flow cytometry usinga dual laser FACSCalibur flow cytometer (BD Biosciences).Assay setup and calculations. Acquisition and measurement of singlecell events were monitored by forward scatter vs side scatter dot plots andcompared with control single- and mixed- population samples. CFSE-la-beled IGROV1 were detected in FL1 (530/30 nm band pass filter), PE-labeled effectors in FL2 (582/42 nm band pass filter), and PI� dead cells inFL3 (670 nm LP band pass filter) channels. Appropriate controls were setfor compensation adjustments between fluorochromes and ADCC andADCP (46, 54). To calculate ADCC and ADCP (Fig. 1), two dot plots weregenerated and three regions were identified: 1) R1 (green), total CFSE�
tumor cells � total tumor cells/sample; 2) R2 (orange), CFSE�/PE�
cells � tumor cells phagocytosed by PE� effector cells; and 3) R3 (red),CFSE�/PI� cells � intact dead tumor cells. Deviations between sampleswere accounted for by “R1 Spontaneous Loss (SL) Control” � the averageR1 of three control samples (i.e., effector and target cells without mAb).Below are calculations to determine the proportion of IGROV1 tumor cellskilled by ADCC and ADCP:
R1 SL control – R1 � XADCC � [(X � R3)/R1 SL Control] � 100ADCP � [R2/R1 SL Control] � 100
Immunofluorescence imaging of cells
Monocytes were incubated on glass chamber slides (SLS) with IGROV1and mAbs to assess contact between cells and ADCP as previously de-scribed (28, 46). Following incubations, monocytes were given anti-CD89-PE mAb. In similar assays, anti-CD49d-PE mAb was used to labeleosinophils. Slides were washed, fixed in 1% paraformaldehyde-FACSbuffer, and mounted with fluorescence preserver (DakoCytomation).Slides were observed using an Axioskop 20 upright microscope (CarlZeiss) equipped with a Zeiss A-Plan 40�/0.65 Ph2 lens, an AxioCam14-bit camera, and AxioVision Version 3.0.2 imaging system (ImagingAssociates). Light and fluorescent images were superimposed as de-scribed before (28).
Experiments in the human ovarian carcinoma xenograft model
The human ovarian carcinoma xenograft HUA was established in 8–12-wk-old specific pathogen-free female nude mice and implanted i.p. as de-scribed before (28, 55). Each mouse received an i.p. injection of primarycells with or without Abs 8 h following tumor challenge. The treatmentswere with PBS or 4 � 106 unfractionated PBMC per mouse, 3.2 � 106
PBMC depleted of monocytes using anti-CD14-coated immunomagneticbeads and the MACS system, or 3.2 � 106 PBMC depleted of monocytesand reconstituted with 0.8 � 106 purified monocytes. All cell treatmentswere administered with or without 100 �g MOv18 IgE in a total volume of0.2 ml per mouse. Mice received one further dose of treatments after 14days and were assessed for the duration of survival. All animal work wasconducted according to the guidelines specified by the U.K. Home OfficeAnimals Scientific Procedures Act 1986.
Data handling and statistical analyses
Flow cytometry experiments of receptor expression and blocking of IgEbinding were repeated at least three times, and representative experimentsare illustrated. In vitro ADCC/ADCP assays were performed in triplicate,and data are shown as mean ADCC � SD and ADCP � SD of a numberof independent experiments. Statistical analyses of ADCC/ADCP assaysand of mouse survival were performed by means of the unpaired two-tailed
Student’s t test and significance was accepted at p � 0.05. Changes in IgEreceptor expression and in tumor cell killing by unstimulated and IgE-stimulated monocytes in vitro were studied by means of the paired two-tailed Student’s t test.
ResultsCD23 expression on monocytes and role in IgE-mediated ADCPof tumor cells
In previous studies, we used standard cytotoxicity assays to ex-amine the efficacy of tumor Ag-specific IgE for the immunother-apy of ovarian cancer (27, 28). Standard cytotoxicity assays do notmeasure phagocytosis. Thus, they may underestimate tumor cellkilling and hence the potential of an Ab for immunotherapy ofcancer. To analyze the mechanisms by which IgE effector cellsmediate tumor cell killing, we have developed a three-color cyto-metric assay (Fig. 1) to simultaneously measure cytotoxicity andphagocytosis of tumor cells by effector cells and tumor Ag-specificIgE (46).
Two-color flow cytometric dot plots of CD14� human mono-cytes show that �3% of monocytes cultured without IL-4 stimu-lation express CD23 (Fig. 2A, top). After overnight stimulationwith IL-4, 58% of the cells express CD23. The proportion ofmonocytes expressing Fc�RI (31%) remained unchanged afterIL-4 stimulation (Fig. 2A, middle). Thus, incubation of monocyteswith IL-4 stimulated expression of CD23, but did not affect ex-pression of Fc�RI on the cell surface. With IL-4 stimulation, theproportion of monocytic cells capable of binding IgE increasedfrom 32% in untreated monocytes to 56% in IL-4-treated cells(Fig. 2A, bottom), suggesting that newly expressed CD23 on thesurface of monocytes was capable of binding MOv18 IgE.
We measured ADCC and ADCP of IGROV1 cells cultured for2.5 h with human peripheral blood monocytes and either MOv18IgE, the hapten-specific anti-NIP IgE, or no Ab as controls (TableI; n � 6). Unstimulated monocytes mediated 34.4% ADCC, com-pared with �15% for the NIP IgE and the no Ab controls (Fig. 2B,Table I). No phagocytosis of tumor cells was detected by compar-ison to controls. Following IL-4 stimulation of the monocytes,MOv18 IgE ADCC was 32.5%, compared with �10% for thecontrols, whereas MOv18 IgE-mediated ADCP measured at22.2%, compared with �15% for the control samples.
Thus, IL-4 stimulation did not affect MOv18 IgE-mediatedADCC ( p � 0.77, n � 6), but it significantly enhanced MOv18IgE ADCP ( p � 0.0007, n � 6). Levels of ADCP in controlsamples without Ab or with NIP IgE were also slightly elevatedcompared with those for unstimulated monocytes (Table I). Thissuggests that IL-4 may enhance the innate phagocytic capacity ofmonocytes.
Because IL-4 stimulates the expression of CD23, but not Fc�RI(Fig. 2A), the results of the three-color assay imply that ADCP
FIGURE 1. Setup of the three-color flow cytometricassay. A three-color flow cytometric assay can simulta-neously measure cytotoxicity and phagocytosis of tu-mor cells by effector cells. Dot plots of mixed monocyteeffector and IGROV1 tumor target cells from which cal-culations were made. Region 1 (R1, green) representstotal CFSE� tumor cell targets. Region 2 (R2, orange)depicts the CFSE� tumor cells present within PE-stained monocytes (CFSE�/PE�), depicting phagocyto-sis. Region 3 (R3, red) contains tumor cells killed ex-ternally by effector cells (cytotoxicity) and are thereforeCFSE�/PI�.
2834 MECHANISMS OF IgE ANTIBODY-DEPENDENT TUMOR CELL KILLING
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
FIGURE 2. CD23 up-regulation by IL-4 androle in MOv18 IgE-mediated ADCP of ovariantumor cells. A, Two-color flow cytometric dotplots show IgE receptor expression and bindingof MOv18 IgE in primary monocytes. Primarymonocytes, untreated (left) or incubated withIL-4 (right), were labeled with anti-CD23 mAbIDEC-152 (top) or anti-Fc�RI mAb 22E7 (mid-dle), followed by anti-mouse IgG (Fab)2–FITC(x-axis) and CD14-PE (y-axis). For binding ofMOv18 IgE to untreated (bottom left) and IL-4-stimulated monocytes (bottom right) cells werelabeled with MOv18 IgE, anti-IgE FITC (x-axis), followed by CD14-PE (y-axis). B, MOv18IgE-mediated killing of IGROV1 tumor cells byuntreated (left) and IL-4-stimulated (right)monocytes. C, ADCP killing of tumor cells byIL-4-stimulated primary monocytes (left) wasblocked by anti-CD23 IDEC-152 mAb Fab(right). Cytotoxicity (ADCC), black bars;phagocytosis (ADCP), gray bars. Results shownare means � SD of six independent experi-ments. Significance of values compared withsamples given MOv18 IgE by the Student’s ttest. n/s, p � 0.05; �, p � 0.05; ��, p � 0.005;���, p � 0.0005.
2835The Journal of Immunology
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
may be attributed to CD23. To test this possibility, we preincu-bated monocytes with 20-fold molar excess of the IDEC-152CD23-blocking Ab Fab (Fig. 2C; n � 6). Following IL-4 stimu-lation of monocytes, MOv18 IgE-induced ADCC was 26.2%,compared with �15% for the controls. As before (Fig. 2B), withIL-4 stimulation, MOv18 IgE-induced ADCP measured at 25.8%,
compared with �15% with the controls. Blocking of IgE bindingto CD23 with IDEC-152 Fab resulted in MOv18 IgE ADCC mea-sured at 26.1%, compared with �15% for the controls. Monocytestreated with the CD23 blocking Ab Fab, however, showed lowMOv18 IgE ADCP (13.9%), a value similar to that measured withthe controls.
FIGURE 3. Fc�RI is responsible for monocyte ADCC by tumor Ag-specific IgE. A, Two-color flow cytometric dot plots show Fc�RI expression inprimary monocytes. Monocytes were incubated in medium alone (left) or stimulated with MOv18 IgE for 20 h (middle). Cells were labeled with anti-Fc�RImAb 22E7, anti-mouse IgG (Fab)2–FITC (x-axis), followed by anti-CD14-PE (y-axis). The proportion of monocytes showing cell surface Fc�RI expressionincreased from 31.0 � 9.6% to 37.8 � 10.1% following overnight stimulation with IgE (n � 8). B, Increased Fc�RI cell surface expression correlated withenhanced ADCC of IGROV1 tumor cells by primary monocytes and MOv18 IgE. Primary monocytes were incubated overnight in medium alone orstimulated with medium containing MOv18 IgE, followed by ADCC/ADCP tumor killing assays. Monocytes from eight donors were assessed, and eachset of ADCC measurements represents mean values of triplicate calculations. Tumor cell killing by IgE-stimulated monocytes and MOv18 IgE increasedto 30.7 � 8.1% compared with 24.4 � 7.7% with unstimulated monocytes and MOv18 IgE (n � 8). Phagocytosis was not significantly influenced by IgEstimulation (ADCP: 8.1 � 3.2% with and 6.4 � 1.4% without IgE, n � 8, p � 0.05). Significance of changes in IgE receptor expression and in tumor cellkilling by the paired two-tailed Student’s t test: ��, p � 0.005; ���, p � 0.0005.
Table I. Blocking of IgE-mediated killing of IGROV1 tumor cells by monocytesa
% ADCC � SD % ADCP � SD % ADCC � SD % ADCP � SD
Untreated Monocytes (n � 6) IL-4-Treated Monocytes (n � 6)
No Ab 10.5 � 2.1*** 8.5 � 2.3n/s 9.9 � 1.2** 11.2 � 6.5*NIP IgE 10.3 � 0.8*** 8.9 � 1.5* 8.6 � 1.0*** 11.6 � 6.7*MOv18 IgE 34.4 � 11.2 6.4 � 1.4 32.5 � 11.5 22.2 � 8.0
IL-4-Treated Monocytes (n � 6) IL-4-Treated Monocytes � IDEC-152 Fab (n � 6)
No Ab 10.9 � 3.1** 13.4 � 3.0** 11.8 � 3.0** 14.0 � 4.1n/s
NIP IgE 9.4 � 2.1** 14.2 � 4.3* 10.5 � 2.8** 14.7 � 4.9n/s
MOv18 IgE 26.2 � 8.7 25.8 � 6.9 26.1 � 9.3 13.9 � 4.7
Untreated Monocytes (n � 6) Untreated Monocytes � sFc�RI� (n � 6)
No Ab 8.1 � 3.0*** 4.6 � 0.7n/s 9.7 � 3.1n/s 6.5 � 0.7n/s
NIP IgE 7.7 � 3.5*** 6.4 � 1.3n/s 9.8 � 5.0n/s 7.3 � 0.5*MOv18 IgE 27.6 � 6.2 5.4 � 1.1 13.2 � 4.7 6.5 � 0.5
IL-4-Treated Monocytes (n � 6) IL-4-Treated Monocytes � sFc�RI� (n � 6)
No Ab 7.9 � 3.1*** 7.4 � 2.4** 8.7 � 3.5n/s 6.2 � 1.3n/s
NIP IgE 9.8 � 4.1** 7.5 � 2.0** 9.9 � 2.3n/s 5.5 � 0.6n/s
MOv18 IgE 27.8 � 8.9 16.5 � 4.2 12.0 � 1.6 5.8 � 1.0
a Monocytes were incubated with or without human rIL-4 to stimulate CD23 expression. IGROV1 tumor cells were incubated with monocyte effector cells and MOv18 IgE,NIP IgE, or no Ab, followed by incubation for 2.5 h at 37°C. To block CD23, IDEC-152 Fab was added to monocytes prior to assays. To block binding of IgE to receptors,sFc�RI� was combined with IgE before addition of cells. Significance compared to samples given MOv18 IgE by the Student’s t test: n/s, p � 0.05; �, p � 0.05; ��, p � 0.005;���, p � 0.0005.
2836 MECHANISMS OF IgE ANTIBODY-DEPENDENT TUMOR CELL KILLING
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
Whereas MOv18 IgE ADCC remained unchanged at 26%with or without CD23 blocking ( p � 0.97), MOv18 IgE ADCPwas reduced from 25.8% to 13.9% with blocking of CD23 ( p �0.006). Thus, CD23 mediated the IgE ADCP of ovarian tumorcells effected by monocytes. The observed levels of expressionof Fc�RI and CD23 in unstimulated and IL-4-stimulated mono-cytes (Fig. 2A), together with the activity of anti-CD23, suggestthat Fc�RI mediates the constitutive ADCC and CD23 mediatesthe superimposed ADCP in IL-4-stimulated monocytes (Fig. 2,B and C).
Up-regulation of Fc�RI on monocytes and function in IgEADCC of tumor cells
We established that monocyte-mediated ADCP is due to the actionof CD23 by correlating the increase in ADCP with the up-regula-tion of CD23 by IL-4, and by the inhibitory effect of the anti-CD23-blocking Ab. Similarly, we have been able to attributeADCC to the action of Fc�RI. We up-regulated Fc�RI on mono-cytes by incubation with MOv18 IgE, and examined the effects ofincreased expression on tumor cell killing by IgE. We also testedthe effects of a soluble fragment of the IgE receptor �-chain(sFc�RI�) on ADCC and ADCP.
Primary monocytes were incubated for 20 h with or withoutMOv18 IgE, and then labeled with CD14-PE and the 22E7 anti-Fc�RI Ab to determine the level of receptor expression by flowcytometry. Preincubation with MOv18 IgE led to a modest in-crease, in the proportion of Fc�RI�-expressing CD14� cells
(Fig. 3A, representative dot plots). Without stimulation, 31.0%of CD14� cells expressed Fc�RI and this proportion increasedto 37.8% of the cells in IgE-stimulated monocytes (Fig. 3A,right; n � 8). Increased expression of Fc�RI on monocytesstimulated with MOv18 IgE in culture corresponded to anequivalently modest increase in MOv18 IgE-induced ADCCfrom 24.4% in unstimulated monocytes to 30.7% in IgE-stim-ulated monocytes (Fig. 3B; n � 8). ADCP was measured atbackground levels in unstimulated and IgE-stimulated mono-cytes. These results support a role for Fc�RI in IgE-mediated ADCC.
IgE bound to IL-4-stimulated monocytes was assayed using goatanti-IgE-FITC. We detected endogenous IgE bound to 25% of themonocytes and, after addition of MOv18 IgE, 58% of the mono-cytes bound IgE (Fig. 4A). When 100-fold molar excess ofsFc�RI� was added to MOv18 IgE before incubation with themonocytes, the proportion of IgE� monocytes did not increasewith the addition of MOv18 IgE (Fig. 4A). This demonstrates thatsFc�RI� prevented binding of MOv18 IgE on the surface of mono-cytes. The binding sites of sFc�RI and CD23 on IgE are compet-itive (11, 56, 57), so that sFc�RI� in 100-fold molar excess mustprevent the association of IgE to both receptors.
When a 100-fold molar excess of sFc�RI� was mixed withMOv18 IgE before incubation with monocytes, MOv18 IgEADCC dropped from 27.6 to 13.2% ( p � 0.001, n � 6) (Fig. 4B,Table I). MOv18 IgE-mediated ADCP was low with (6.5%) andwithout (6.2%) treatment with sFc�RI� ( p � 0.052, n � 6). This
FIGURE 4. sFc�RI� blocked bind-ing of IgE to monocytes and IgE-medi-ated tumor killing. A, Flow cytometrichistograms show binding of MOv18IgE to monocytes. IL-4-stimulatedmonocytes bound fresh MOv18 IgE viaboth Fc�RI and CD23 (left). Preincuba-tion of MOv18 IgE with sFc�RI� pre-vented binding of fresh IgE on themonocyte surface (right). (MOv18 IgE,anti-IgE-FITC, black lines; anti-IgE-FITC, gray lines). B, MOv18 IgEmediated killing of IGROV1 tumorcells by unstimulated monocytes (left).sFc�RI� blocked MOv18 IgE-medi-ated IGROV1 tumor killing by un-stimulated monocytes (right). C,MOv18 IgE-mediated ADCC andADCP by IL-4-stimulated monocytes(left). Both ADCC and ADCP wereblocked by incubation of MOv18 IgEwith soluble Fc�RI� (right). Cytotox-icity (ADCC), black bars; phagocyto-sis (ADCP), gray bars. Results aremeans � SD of six independent mea-surements. Significance of valuescompared with samples givenMOv18 IgE by the Student’s t test:n/s, p � 0.05; �, p � 0.05; ��, p �0.005; ���, p � 0.0005.
2837The Journal of Immunology
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
proves that ADCC depends on MOv18 IgE binding to Fc�RI onmonocytes. In the case of IL-4-treated monocytes, sFc�RI� led todecreases in both MOv18 IgE ADCC (from 27.8% to 12.0%; p �0.002, n � 6) and MOv18 IgE ADCP (from 16.5% to 5.8%; p �0.0001, n � 6), approaching the values observed in controls (Fig.4C). This was expected because, as mentioned above, sFc�RI�blocks the binding of IgE to both receptors (11, 56, 57). We con-clude that the binding of MOv18 IgE to both IgE receptors onmonocytes contributed to IgE-mediated tumor cell targeting andkilling.
Visualization of contact between tumor cells and monocytes
To visualize the results, shown quantitatively in the flow cytomet-ric assays described above, we labeled IGROV1 with CFSE beforeincubation with IgE Abs for 2.5 h and then stained monocytes withanti-CD89-PE. Cell interactions were the observed by fluorescencemicroscopy. Incubation with MOv18 IgE led to contact between
monocytes and IGROV1 cells (Fig. 5A), not seen with control AbNIP IgE, or when the MOv18 IgE is preincubated with 100-foldmolar excess of sFc�RI� (Fig. 5A). When the monocytes werestimulated by IL-4, contact between the tumor and effector cellswas enhanced, and phagocytosis was clearly visible in themerged image of the green tumor cells inside the red mono-cytes, resulting in the yellow color (Fig. 5B; arrow); this is notseen with NIP IgE, or when IgE binding to its receptors isblocked by sFc�RI�. Addition of the IDEC-152 anti-CD23blocking Ab Fab to the monocytes cultured with IL-4 substan-tially reduced, but did not eliminate, the contact between mono-cytes and IGROV1 cells. IDEC-152 Fab inhibited phagocytosis,seen by the absence of yellow color inside the monocytes (Fig.5B). In contrast, incubation with sFc�RI� completely inhibitedcontact between monocytes and IGROV1 cells, consistent withIgE binding to both Fc�RI and CD23 and the inhibition of cy-totoxicity as well as phagocytosis (Fig. 4, B and C).
FIGURE 5. Superimposed bright-field and fluorescent images of IGROV1-monocyte interactions. CFSE-stained IGROV1 tumor cells (green) andCD89-PE-labeled monocytes (red) were incubated with IgE Abs for 2.5 h. A, After incubation with MOv18 IgE, unstimulated monocytes were in frequentcontact with IGROV1 cells (left). Rare monocyte-IGROV1 cell contact was seen in samples given control NIP IgE (middle) and when MOv18 IgE wastreated with sFc�RI� before assays (right). B, IL-4-stimulated monocytes show enhanced contact with IGROV1 tumor cells (green) and phagocytosis oftumor cells (green fluorescent CFSE inside monocytes; orange; arrow) after incubation with MOv18 IgE. Little E:T cell contact and phagocytosis are seenwith control Ab NIP IgE, or when MOv18 IgE was preincubated with sFc�RI�. IL-4-treated monocytes given anti-CD23 IDEC-152 blocking mAb Fabbefore MOv18 IgE exhibit reduced effector-tumor cell contact and lack of phagocytosis (absence of CFSE dye inside monocytes; red). Original magni-fication, �400. Scale bars, 20 �m.
2838 MECHANISMS OF IgE ANTIBODY-DEPENDENT TUMOR CELL KILLING
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
Monocytes are necessary for IgE-mediated survival of micebearing ovarian tumor xenografts
We tested the ability of monocytes to promote survival in nudemice bearing the human ovarian carcinoma xenograft HUA, whichexpresses the folic acid receptor at moderate levels (28). HUAcells were derived from a patient with ovarian cancer; they havenot yet been adapted to tissue culture and their characteristics havenot changed in years of passage in mice. The tumors grow andspread in the peritoneal cavity of the mice, as in ovarian cancerpatients, and their histological and cytokine profiles are similar tothe original ascites. This is, therefore, considered a more clinicallyrelevant model than the ovarian carcinoma cell line IGROV1 usedin this study in our in vitro assays and in our previous in vitro andin vivo studies (27, 28, 55). HUA ascites were introduced intonude mice by i.p. injection. Unlike human IgG1, the Fc fragmentof the human IgE (Fc�) is not recognized by the murine Fc�Rs(10). Thus, to study the effect of human IgE and the role of IgEreceptor-bearing effector cells in targeting a human ovarian carci-noma xenograft, we introduced human mononuclear cells fromperipheral blood. Mice were treated with human effector cells, withor without MOv18 IgE, and the length of survival was assessed(Fig. 6).
Mice treated with PBMCs plus MOv18 IgE survived for 39 daysand they had a significant survival advantage compared with mice thatreceived PBMC alone (21 days) or buffer controls (16 days) (TableII). As observed in our previous studies (27, 28), mice given PBMCalone survived longer than buffer controls. Mice given PBMCs de-pleted of monocytes plus MOv18 IgE survived for 21 days, whilethose treated with PBMCs depleted of monocytes without IgE sur-vived for 18 days and their survival was shorter than that of micegiven unfractionated PBMC with MOv18 IgE. Removal of mono-
cytes from the PBMC population thus abolished the survival advan-tage of mice given PBMC and MOv18 IgE, implying that monocytesplay a crucial role in targeting IgE against tumor cells and promotingmouse survival.
The role of monocytes in delaying tumor growth and prolongingmouse survival was further confirmed with reconstitution of mono-cyte-depleted PBMCs with purified monocytes. Adding back pu-rified monocytes to depleted PBMCs at proportions equivalent tothose in unfractionated PBMCs restored the ability of PBMCs andMOv18 IgE to increase mouse survival (44 days) to levels statis-tically equivalent to those seen in mice given whole PBMCs andMOv18 IgE (Fig. 6, Table II). This survival was significantlylonger than monocyte-reconstituted PBMCs alone (25 days) or de-pleted PBMCs with and without MOv18 IgE. Purified monocytes
FIGURE 6. Effect of monocytes and MOv18 IgE onsurvival of human ovarian carcinoma xenograft-bearingmice. Nude mice were transplanted with the HUA hu-man ovarian cancinoma xenografts and given varioustreatments with primary human cells, with or withoutMOv18 IgE. A repeat treatment dose was given 2 wkfollowing tumor challenge. Dot, Survival following tu-mor challenge (days). Horizontal bars, Mean survivalfor each population (days). Significance of survivalcompared by the Student’s t test. n/s, p � 0.05; �, p �0.05; ��, p � 0.005; ���, p � 0.0005.
Table II. Monocytes are crucial effector cells in IgE-mediated survivalof ovarian carcinoma xenograft-bearing micea
Treatment Survival � SD
PBS (n � 21) 15.8 � 2.7***PBMC (n � 21) 20.6 � 5.1**PBMC � MOv18 IgE (n � 21) 38.7 � 23.9Monocyte-depleted PBMC (n � 13) 18.4 � 3.0**Monocyte-depleted PBMC � MOv18 IgE
(n � 13)20.5 � 5.8*
Monocyte-depleted PBMC � Monocytes(n � 13)
24.9 � 9.4*
Monocyte-depleted PBMC � Monocytes �MOv18 IgE (n � 13)
44.4 � 16.4n/s
a Results are mean � SD. Numbers in brackets denote the number of nude miceexamined in each group. Significance levels for comparison to mice treated withPBMC and MOv18 IgE by the Student’s t-test: n/s, p � 0.05; �, p � 0.05; ��, p �0.005; ���, p � 0.0005.
2839The Journal of Immunology
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
were also tested in this model, but they did not confer any survivaladvantage to tumor-bearing mice (results not shown). This may bedue to the requirement of survival factors or trafficking signalsconferred by other cells in the PBMC preparation in these models.
Eosinophils mediate IgE-dependent ADCC
Eosinophils were lost in the purification of PBMCs from blood. Totest their ability to direct IgE Ab-dependent tumor cell killing, wepurified eosinophils and used them as effector cells in the ADCC/ADCP assay. We measured the ADCC and ADCP of IGROV1cells incubated with human eosinophils and MOv18 IgE, NIP IgEor no Ab (Fig. 7A; n � 4). Eosinophils mediated elevated ADCC(32.4%) with MOv18 IgE, compared with controls. No phago-cytosis of tumor cells was detected by comparison to controls(Table III).
We examined the interaction between eosinophils and tumorcells in the presence or absence of MOv18 IgE by fluorescencemicroscopy. We labeled IGROV1 with CFSE before incubation
with IgE Abs for 2.5 h, and then stained eosinophils with anti-CD49d-PE. Cells were then visualized to examine E:T cell inter-actions by fluorescence microscopy (Fig. 7B). Microscopic obser-vations showed that incubation with MOv18 IgE led to contactbetween eosinophils (red) and IGROV1 tumor cells (green), fre-quently accompanied by eosinophil degranulation, loss of tumorcell architecture, and apparent tumor cell death. Less frequent con-tact of tumor cells with eosinophils was seen with NIP IgE or inthe absence of Ab. In the latter specimens, eosinophil degranula-tion was less common and tumor cells appeared more brightlyfluorescent and viable. As previously reported, we found eosino-phils to express low levels of the Fc�RI receptor but not CD23(results not shown) (47). These results support a role for Fc�RI inIgE ADCC of tumor cells by eosinophils as well as monocytes.
DiscussionFollowing our previous demonstration that MOv18 IgE is superiorto MOv18 IgG1, together with human PBMC, in prolonging thesurvival of mice in our xenograft models of ovarian cancer (27,28), we sought to identify the IgE effector cells and the IgE re-ceptors and mechanisms involved in tumor cell killing. The ob-servation of monocytes infiltrating the xenografts in the mice (28)directed our attention to monocytes. We also examined the activityof eosinophils, which are potential IgE effector cells not presentin PBMC.
In earlier work, we observed that Fc�RI, but not CD23, wasconstitutively expressed on primary monocytes, implicating theformer receptor in IGROV1 cell killing (28). In this study, weconfirmed this finding and found that Fc�RI-expressing primarymonocytes exerted primarily ADCC. Upon up-regulation of Fc�RIby preincubation of the monocytes with MOv18 IgE, we observed
FIGURE 7. Assessments of MOv18 IgE-mediated tumor cell interaction and killing by human eosinophils. A, MOv18 IgE mediated IGROV1 killingof tumor cells by primary eosinophils. Cytotoxicity (ADCC), black bars; phagocytosis (ADCP), gray bars. Results are means � SD of four independentexperiments. Significance was compared with samples given MOv18 IgE by the Student’s t test. n/s, p � 0.05; �, p � 0.05; ��, p � 0.005; ���, p � 0.0005.B, Superimposed bright-field and fluorescent images of IGROV1-eosinophil interactions. CFSE-stained IGROV1 tumor cells (green) and CD49d-PE-labeled eosinophils (red) were incubated with IgE Abs for 2.5 h. After incubation with MOv18 IgE, eosinophils were in frequent contact with IGROV1cells and IGROV1 tumor cell destruction was evident (left). Eosinophil-IGROV1 cell contact was observed less frequently in samples given control NIPIgE (middle), or no Ab (right). Original magnification, �400. Scale bars, 20 �m.
Table III. IgE-mediated killing of IGROV1 tumor cells by eosinophilsa
Fresh Eosinophils (n � 4)
% ADCC � SD % ADCP � SD
No Ab 14.6 � 5.4* 6.6 � 3.7n/s
NIP IgE 16.7 � 5.2* 6.8 � 3.9n/s
Mov18 IgE 32.4 � 8.6 5.9 � 3.1
a Human eosinophils freshly isolated from peripheral blood were incubatedwith IGROV1 tumor cells and MOv18 IgE, NIP IgE or no Ab for 2.5 h at 37°C.Significance compared to samples given MOv18 IgE by the Student’s t test: n/s,p � 0.05; �, p � 0.05.
2840 MECHANISMS OF IgE ANTIBODY-DEPENDENT TUMOR CELL KILLING
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
an increase in ADCC, further suggesting that Fc�RI mediates cy-totoxic cell killing. ADCC and the contact between the monocytesand tumor cells, observed by fluorescence microscopy, were in-hibited by sFc�RI�, confirming this mechanism of Fc�RI action.In contrast, up-regulation of CD23 by IL-4, led to MOv18 IgEADCP without affecting the level of ADCC. Moreover, incubationwith the IDEC-152 anti-CD23 Ab Fab inhibited ADCP, but notADCC. Therefore, we were able to attribute IgE-mediated cyto-toxicity of tumor cells to Fc�RI and IgE-mediated phagocytosisto CD23.
Our tumor cell killing assays and microscopic observationssuggest that contact of tumor target and monocytes is necessaryfor IgE-mediated ADCC and ADCP. Addition of tumor Ag-specific IgE was needed for tumor cell death, as the hapten-specific NIP IgE did not mediate tumor killing. This implies therequirement for tumor cell recognition by a tumor Ag-specific Ab.Blocking of MOv18 IgE binding to receptors on monocytes bypreincubation of IgE with sFc�RI� drastically reduced MOv18-IgE-mediated ADCC and ADCP. Therefore, engagement of IgEreceptors by IgE is also required. Finally, microscopic observa-tions show a correlation between E:T cell contact and the presenceof MOv18 IgE. Therefore, bridging tumor cells and IgE receptorson monocytes by IgE is necessary for both mechanisms of IgE-mediated tumor killing, ADCC and ADCP, to occur.
Our observations in human ovarian carcinoma xenograft-bear-ing mice clearly suggest a pivotal role for monocytes in enhancingmouse survival effected by MOv18 IgE. In this in vivo system,monocytes in PBMC, together with MOv18 IgE, are required toenhance mouse survival, compared with controls. This implies thatIgE-receptor-bearing cells like monocytes can act, not only invitro, but also in vivo as effector cells. Monocytes-macrophagesare recruited in the intratumoral environment by tumor-derivedchemoattractant signals. Tumor-associated macrophages can beactivated to promote tumor growth in situ and fail to mount anantitumor response (13, 53, 58). Resident macrophages expressIgE receptors (59) and are capable of mediating NO release (60).Based on our observations on the IgE-mediated antitumor effectsof monocytes in vitro as well as in vivo, we propose that these cellsmay be activated and reprogrammed by a tumor Ag-specific IgE tokill and phagocytose tumor cells, rather than promote their growth.
The PBMC used in our xenograft model contain around 2%human basophils (28), which are known to express Fc�RI. Wehave shown that these cells can be activated to secrete histamine inthe presence of PBMC, tumor cells, and MOv18 IgE (27). It islikely that IgE-dependent mast cell and basophil activation by tu-mor cell Ags would enhance the recruitment of inflammatory cellsto the site of a tumor, but this remains to be tested.
The PBMC used as effector cells lack other IgE receptor-ex-pressing cells, such as eosinophils, tissue mast cells, and macro-phages that might mediate strong antitumor responses. The ab-sence of these potent human effector cells in this system may havecontributed to the observed limited efficacy that MOv18 IgE con-fers in this system. We have previously observed a lack of en-hanced mouse survival using tumor-specific IgE to treat estab-lished tumors; these observations could also be attributed to lack ofpotent human effector cells. Human PBMC have a limited lifespanin the mouse and their antitumor effects may therefore be limitedto a short time following treatments. Cytokines released by humaneffector cells may not act on murine effector cells and, thus, thexenograft model may not reveal indirect mechanisms of tumor cellkilling. Our results may point toward a beneficial role for IgEtherapy following surgical intervention on ovarian cancer patientswith smaller tumor burden or with minimal residual disease.
IgE-Fc�RI complexes on eosinophils are known to exert ADCCagainst parasites (61, 62). In this study, we show for the first timethat a tumor Ag-specific Ab can direct eosinophils to kill tumorcells by ADCC. As observed with monocytes, and consistent withthe expression of low levels of Fc�RI on the surface of eosinophils(47), this IgE receptor on eosinophils appears to mediate MOv18-dependent cytotoxicity of tumor cells. Engagement of IgE Abs onthe surface of eosinophils triggers the release of inflammatory andcytotoxic mediators, such as eosinophil peroxidase and major basicprotein (63), which may contribute to tumor cell death. Our data,therefore, raise the possibility that eosinophils, either resident inintratumoral areas or recruited from the circulation, could contrib-ute to the elimination of tumor cells in vivo. Indeed, eosinophilinfiltrates are associated with host inflammatory responses againsta number of cancers (62, 64).
The human immune system operates with nine Ab classes, butcancer immunotherapy has been attempted only with Abs of theIgG class. It seems unlikely that this would afford optimal efficacyfor all types of tumors, in every anatomical location and in everycancer patient. Other workers have shown IgA ADCC and ADCPof breast tumor cells (54, 65), melanoma cells (66), and non-Hodgkins lymphoma (67) by monocyte-derived macrophages (65)or neutrophils (54, 66, 67). IgA Abs were directed to tumor cellsin the form of bispecific Abs against the IgA receptor, Fc�RI, orCD89, on the effector cells and the specific tumor Ag on thetumor cells. Bispecific Abs were used to compensate for the lowaffinity of IgA for Fc�RI (�106 M�1), which precludes thestable interaction of Ab with the effector cells. Unlike eitherIgG or IgA, IgE binds to Fc�RI with sufficiently high affinity(�1011 M�1) to be effective as a monospecific Ab. Consistentwith the stability of the IgE-Fc�RI interaction, circulatingmonocytes and eosinophils are able to transport IgE into tissueswhere this IgE may engage effector cells in tumor surveillanceand killing, as we have shown (11, 13, 59, 61, 62). This prop-erty is also likely to enhance Ag presentation by Fc�RI APCs(e.g., dendritic cells) leading to active immunity.
There is an increasing call for rational therapies for the treat-ment of cancer, requiring knowledge of mechanisms of drug action(68). There are no previous reports that analyze the mechanisms ofIgE-dependent tumor cell killing and the role of IgE receptor-ex-pressing effector cells. Our findings offer a framework for an im-proved immunotherapeutic strategy for combating solid tumors.
AcknowledgmentsThis study was supported by the Association for International Cancer Re-search. We thank Kate Kirwan for expert assistance with the figures. Weare grateful to Dr. T. Hagemann for technical help and practical advice forthe growth of primary monocytes and to Dr. Silvana Canevari, Dr. Marie-Helene Jouvin, and Dr. J. Hopp, for the generous provision of materials andadvice.
DisclosuresThe authors have no financial conflict of interest.
References1. Bray, F., A. H. Loos, S. Tognazzo, and C. La Vecchia. 2005. Ovarian cancer in
Europe: cross-sectional trends in incidence and mortality in 28 countries, 1953–2000. Int. J. Cancer 113: 977–990.
2. Quirk, J. T., and N. Natarajan. 2005. Ovarian cancer incidence in the UnitedStates, 1992–1999. Gynecol. Oncol. 97: 519–523.
3. Glennie, M. J., and J. G. J. van de Winkel. 2003. Renaissance of cancer thera-peutic antibodies. Drug Discov. Today 8: 503–510.
4. Luborsky, J. L., A. Barua, S. V. Shatavi, T. Kebede, J. Abramowicz, andJ. Rotmensch. 2005. Anti-tumor antibodies in ovarian cancer. Am. J. Reprod.Immunol. 54: 55–62.
5. Nicodemus, C. F., and J. S. Berek. 2005. Monoclonal antibody therapy of ovariancancer. Expert Rev. Anticancer Ther. 5: 87–96.
2841The Journal of Immunology
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
6. Weiner, L. M., and H. Borghaei. 2006. Targeted therapies in solid tumors: mono-clonal antibodies and small molecules. Hum. Antibodies 15: 103–111.
7. Bruggemann, M., G. T. Williams, C. I. Bindon, M. R. Clark, M. R. Walker,R. Jefferis, H. Waldmann, and M. S. Neuberger. 1987. Comparison of the effectorfunctions of human immunoglobulins using a matched set of chimeric antibodies.J. Exp. Med. 166: 1351–1361.
8. Dyer, M. J. S., G. Hale, F. G. J. Hayhoe, and H. Waldmann. 1989. Effects in vivoin patients with lymphoid malignancies; influence of antibody isotype. Blood 73:1431–1439.
9. Clynes, R. A., T. L. Towers, L. G. Presta, and J. V. Ravetch. 2000. Inhibitory Fcreceptors modulate in vivo cytoxicity against tumor targets. Nat. Med. 6:443–446.
10. Ravetch, J. V., and J. P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9:457–492.
11. Gould, H. J., B. J. Sutton, A. J. Beavil, R. L. Beavil, N. McCloskey, H. A. Coker,D. Fear, and L. Smurthwaite. 2003. The biology of IgE and the basis of allergicdisease. Annu. Rev. Immunol. 21: 579–628.
12. Schoppmann, S. F., P. Birner, J. Stockl, R. Kalt, R. Ullrich, C. Caucig,E. Kriehuber, K. Nagy, K. Alitalo, and D. Kerjaschki. 2002. Tumor-associatedmacrophages express lymphatic endothelial growth factors and are related toperitumoral lymphangiogenesis. Am. J. Pathol. 161: 947–956.
13. Gordon, I. O., and R. S. Freedman. 2006. Defective antitumor function of mono-cyte-derived macrophages from epithelial ovarian cancer patients. Clin. CancerRes. 12: 1515–1524.
14. Chan, J. K., A. Magistris, V. Loizzi, F. Lin, J. Rutgers, K. Osann, P. J. DiSaia,and M. Samoszuk. 2005. Mast cell density, angiogenesis, blood clotting, andprognosis in women with advanced ovarian cancer. Gynecol. Oncol. 99: 20–25.
15. Wei, S., I. Kryczek, L. Zou, B. Daniel, P. Cheng, P. Mottram, T. Curiel,A. Lange, and W. Zou. 2005. Plasmacytoid dendritic cells induce CD8� regu-latory T cells in human ovarian carcinoma. Cancer Res. 65: 5020–5026.
16. Mills, P. K., W. L. Beeson, G. E. Fraser, and R. L. Phillips. 1992. Allergy andcancer: organ site-specific results from the Adventist Health Study.Am. J. Epidemiol. 136: 287–295.
17. Turner, M. C., Y. Chen, D. Krewski, P. Ghadirian, M. J. Thun, and E. E. Calle.2005. Cancer mortality among U.S. men and women with asthma and hay fever.Am. J. Epidemiol. 162: 212–221.
18. Wiemels, J. L., J. K. Wiencke, J. Patoka, M. Moghadassi, T. Chew, A. McMillan,R. Miike, G. Barger, and M. Wrensch. 2004. Reduced immunoglobulin E andallergy among adults with glioma compared with controls. Cancer Res. 64:8468–8473.
19. Brenner, A. V., M. S. Linet, H. A. Fine, W. R. Shapiro, R. G. Selker, P. M. Black,and P. D. Inskip. 2002. History of allergies and autoimmune diseases and risk ofbrain tumors in adults. Int. J. Cancer 99: 252–259.
20. Wiemels, J. L., J. K. Wiencke, J. D. Sison, R. Miike, A. McMillan, andM. Wrensch. 2002. History of allergies among adults with glioma and controls.Int. J. Cancer 98: 609–615.
21. Wang, H., and T. L. Diepgen. 2005. Is atopy a protective or a risk factor forcancer? A review of epidemiological studies. Allergy 60: 1098–1111.
22. Turner, M. C., Y. Chen, D. Krewski, and P. Ghadirian. 2006. An overview of theassociation between allergy and cancer. Int. J. Cancer 118: 3124–3132.
23. Wang, H., and T. L. Diepgen. 2006. Atopic dermatitis and cancer risk.Br. J. Dermatol. 154: 205–210.
24. Wang, H., D. Rothenbacher, M. Low, C. Stegmaier, H. Brenner, andT. L. Diepgen. 2006. Atopic diseases, immunoglobulin E and risk of cancer of theprostate, breast, lung and colorectum. Int. J. Cancer 119: 695–701.
25. Nagy, E., I. Berczi, and A. H. Sehon. 1991. Growth inhibition of murine mam-mary carcinoma by monoclonal IgE antibodies specific for the mammary tumorvirus. Cancer Immunol. Immunother. 34: 63–69.
26. Kershaw, M. H., P. K. Darcy, J. A. Trapani, D. MacGregor, and M. J. Smyth.1998. Tumor-specific IgE-mediated inhibition of human colorectal carcinomaxenograft growth. Oncol. Res. 10: 133–142.
27. Gould, H. J., G. A. Mackay, S. N. Karagiannis, C. M. O’Toole, P. J. Marsh,B. E. Daniel, L. R. Coney, V. R. Zurawski, Jr., M. Joseph, M. Capron, et al. 1999.Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in aSCID mouse xenograft model of ovarian carcinoma. Eur. J. Immunol. 29:3527–3537.
28. Karagiannis, S. N., Q. Wang, N. East, F. Burke, S. Riffard, M. G. Bracher,R. G. Thompson, S. R. Durham, L. B. Schwartz, F. R. Balkwill, and H. J. Gould.2003. Activity of human monocytes in IgE antibody-dependent surveillance andkilling of ovarian tumor cells. Eur. J. Immunol. 33: 1030–1040.
29. Kershaw, M. H., P. K. Darcy, J. A. Trapani, and M. J. Smyth. 1996. The use ofchimeric human Fc� receptor I to redirect cytotoxic T lymphocytes to tumors.J. Leukocyte Biol. 60: 721–728.
30. Teng, M. W., M. H. Kershaw, J. T. Jackson, M. J. Smyth, and P. K. Darcy. 2006.Adoptive transfer of chimeric Fc�RI gene-modified human T cells for cancerimmunotherapy. Hum. Gene Ther. 17: 1134–1143.
31. Untersmayr, E., I. Scholl, I. Swoboda, W. J. Beil, E. Forster-Waldl, F. Walter,A. Riemer, G. Kraml, T. Kinaciyan, S. Spitzauer, et al. 2003. Antacid medicationinhibits digestion of dietary proteins and causes food allergy: a fish allergy modelin BALB/c mice. J. Allergy Clin. Immunol. 112: 616–623.
32. Scholl, I., E. Untersmayr, N. Bakos, F. Roth-Walter, A. Gleiss, G. Boltz-Nitulescu,O. Scheiner, and E. Jensen-Jarolim. 2005. Antiulcer drugs promote oral sensitizationand hypersensitivity to hazelnut allergens in BALB/c mice and humans. Am. J. Clin.Nutr. 81: 154–160.
33. Riemer, A. B., E. Untersmayr, R. Knittelfelder, A. Duschl, H. Pehamberger,C. C. Zielinski, O. Scheiner, and E. Jensen-Jarolim. 2007. Active induction of
tumor-specific IgE antibodies by oral mimotope vaccination. Cancer Res. 67:3406–3411.
34. Reali, E., J. W. Greiner, A. Corti, H. J. Gould, F. Bottazzoli, G. Paganelli,J. Schlom, and A. Siccardi. 2001. IgEs targeted on tumor cells: therapeutic ac-tivity and potential in the design of tumour vaccines. Cancer Res. 61: 5517–5522.
35. Campbell, I. G., T. A. Jones, W. D. Foulkes, and J. Trowsdale. 1991. Folatebinding protein is a marker for ovarian cancer. Cancer Res. 51: 5329–5338.
36. Coney, L. R., A. Tomassetti, L. Carayannopoulos, V. Frasca, B. A. Kamen,M. I. Colnaghi, and V. R. Zurawski, Jr. 1991. Cloning of a tumor-associatedantigen: MOv18 and MOv19 antibodies recognize a folate-binding protein. Can-cer Res. 51: 6125–6132.
37. Coney, L. R., D. Mezzanzanica, D. Sanborn, P. Casalini, M. I. Colnaghi, andV. R. Zurawski, Jr. 1994. Chimeric murine-human antibodies directed againstfolate binding receptor are efficient mediators of ovarian carcinoma cell killing.Cancer Res. 54: 2448–2455.
38. Toffoli, G., C. Cernigoi, A. Russo, A. Gallo, M. Bagnoli, and M. Boiocchi. 1997.Overexpression of folate binding protein in ovarian cancers. Int. J. Cancer 74:193–198.
39. Williams, J., S. Johnson, J. J. Mascali, H. Smith, L. J. Rosenwasser, andL. Borish. 1992. Regulation of low affinity IgE receptor (CD23) expression onmononuclear phagocytes in normal and asthmatic subjects. J. Immunol. 149:2823–2829.
40. Wong, H. L., M. T. Lotze, L. M. Wahl, and S. M. Wahl. 1992. Administration ofrecombinant IL-4 to humans regulates gene expression, phenotype, and functionin circulating monocytes. J. Immunol. 148: 2118–2125.
41. Boltz-Nitulescu, G., M. Willheim, A. Spittler, F. Leutmezer, C. Tempfer, andS. Winkler. 1995. Modulation of IgA, IgE, and IgG Fc receptor expression onhuman mononuclear phagocytes by 1 �,25-dihydroxyvitamin D3 and cytokines.J. Leukocyte Biol. 58: 256–262.
42. Mossalayi, M. D., N. Paul-Eugene, F. Ouaaz, M. Arock, J. P. Kolb, E. Kilchherr,P. Debre, and B. Dugas. 1994. Involvement of Fc�RII/CD23 and L-arginine-dependent pathway in IgE-mediated stimulation of human monocyte functions.Int. Immunol. 6: 931–938.
43. Paul-Eugene, N., D. Mossalayi, M. Sarfati, K. Yamaoka, J. P. Aubry,J. Y. Bonnefoy, B. Dugas, and J. P. Kolb. 1995. Evidence for a role of Fc�RII/CD23 in the IL-4-induced nitric oxide production by normal human mononuclearphagocytes. Cell. Immunol. 163: 314–318.
44. Yokota, A., K. Yukawa, A. Yamamoto, K. Sugiyama, M. Suemura, Y. Tashiro,T. Kishimoto, and H. Kikutani. 1992. Two forms of the low-affinity Fc receptorfor IgE differentially mediate endocytosis and phagocytosis: identification of thecritical cytoplasmic domains. Proc. Natl. Acad. Sci. USA 89: 5030–5034.
45. Dyall, R., L. V. Vasovic, R. A. Clynes, and J. Nikolic-Zugic. 1999. Cellularrequirements for the monoclonal antibody-mediated eradication of an establishedsolid tumor. Eur. J. Immunol. 29: 30–37.
46. Bracher, M. G., H. J. Gould, B. J. Sutton, D. Dombrowitz, and S. N. Karagiannis.2007. Three-colour flow cytometric method to measure antibody-dependent tu-mour cell killing by cytotoxicity and phagocytosis. J. Immunol. Methods 323:160–171.
47. Kayaba, H., D. Dombrowicz, G. Woerly, J. P. Papin, S. Loiseau, and M. Capron.2001. Human eosinophils and human high affinity IgE receptor transgenic mouseeosinophils express low levels of high affinity IgE receptor, but release IL-10upon receptor activation. J. Immunol. 167: 995–1003.
48. Neuberger, M. S., G. T. Williams, E. B. Mitchell, S. S. Jouhal, J. G. Flanagan,and T. H. Rabbitts. 1985. A hapten-specific chimaeric IgE antibody with humanphysiological effector function. Nature 314: 268–270.
49. Wakai, M., P. Pasley, Z. M. Sthoeger, D. N. Posnett, R. Brooks, S. Hashimoto,and N. Chiorazzi. 1993. Anti-CD23 monoclonal antibodies: comparisons ofepitope specificities and modulating capacities for IgE binding and production.Hybridoma 12: 25–43.
50. Riske, F., J. Hakimi, M. Mallamaci, M. Griffin, B. Pilson, N. Tobkes, P. Lin,W. Danho, J. Kochan, and R. Chizzonite. 1991. High affinity human IgE receptor(Fc�RI): analysis of functional domains of the �-subunit with monoclonal anti-bodies. J. Biol. Chem. 266: 11245–11251.
51. Cook, J. P., A. J. Henry, J. M. McDonnell, R. J. Owens, B. J. Sutton, andH. J. Gould. 1997. Identification of contact residues in the IgE binding site ofhuman Fc�RI�. Biochemistry 36: 15579–15588.
52. Benard, J., J. Da Silva, M. C. De Blois, P. Boyer, P. Duvillard, E. Chiric, andG. Riou. 1985. Characterization of a human ovarian adenocarcinoma line,IGROV1, in tissue culture and in nude mice. Cancer Res. 45: 4970–4979.
53. Hagemann, T., J. Wilson, F. Burke, H. Kulbe, N. F. Li, A. Pluddemann,K. Charles, S. Gordon, and F. R. Balkwill. 2006. Ovarian cancer cells polarizemacrophages toward a tumor-associated phenotype. J. Immunol. 176: 5023–5032.
54. Otten, M. A., E. Rudolph, M. Dechant, C. W. Tuk, R. M. Reijmers, R. H. Beelen,J. G. van de Winkel, and M. van Egmond. 2005. Immature neutrophils mediatetumor cell killing via IgA but not IgG Fc receptors. J. Immunol. 174: 5472–5480.
55. Balkwill, F. R., A. Lee, G. Aldam, E. Moodie, J. A. Thomas, J. Tavernier, andW. Fiers. 1986. Human tumor xenografts treated with recombinant human tumornecrosis factor alone or in combination with interferons. Cancer Res. 46:3990–3993.
56. Vercelli, D., B. Helm, P. Marsh, E. Padlan, R. S. Geha, and H. Gould. 1989. TheB-cell binding site on human immunoglobulin E. Nature 338: 649–651.
57. Keegan, A. D., C. Fratazzi, B. Shopes, B. Baird, and D. H. Conrad. 1991. Char-acterization of new rat anti-mouse IgE monoclonals and their use along withchimeric IgE to further define the site that interacts with Fc�RII and Fc�RI. Mol.Immunol. 28: 1149–1154.
58. Schoppmann, S. F., P. Birner, J. Stockl, R. Kalt, R. Ullrich, C. Caucig,E. Kriehuber, K. Nagy, K. Alitalo, and D. Kerjaschki. 2002. Tumor-associated
2842 MECHANISMS OF IgE ANTIBODY-DEPENDENT TUMOR CELL KILLING
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
macrophages express lymphatic endothelial growth factors and are related toperitumoral lymphangiogenesis. Am. J. Pathol. 161: 947–956.
59. Leung, D. Y., E. E. Schneeberger, R. P. Siraganian, R. S. Geha, and A. K. Bhan.1987. The presence of IgE on macrophages and dendritic cells infiltrating into theskin lesion of atopic dermatitis. Clin. Immunol. Immunopathol. 42: 328–337.
60. Thomsen, L. L., and D. W. Miles. 1988. Role of nitric oxide in tumor progres-sion: lessons from human tumors. Cancer Metastasis Rev. 17: 107–118.
61. Gounni, A. S., B. Lamkhioued, K. Ochiai, Y. Tanaka, E. Delaporte, A. Capron,J. P. Kinet, and M. Capron. 1994. High-affinity IgE receptor on eosinophils isinvolved in defence against parasites. Nature 367: 183–186.
62. Cormier, S. A., A. G. Taranova, C. Bedient, T. Nguyen, C. Protheroe, R. Pero,D. Dimina, S. I. Ochkur, K. O’Neill, D. Colbert, et al. 2006. Pivotal advance:eosinophil infiltration of solid tumors is an early and persistent inflammatory hostresponse. J. Leukocyte Biol. 79: 1131–1139.
63. Munitz, A., and F. Levi-Schaffer. 2004. Eosinophils: ‘new’ roles for ‘old’ cells.Allergy 59: 268–275.
64. Lotfi, R., J. J. Lee, and M. T. Lotze. 2007. Eosinophilic granulocytes and damage-associated molecular pattern molecules (DAMPs): role in the inflammatory re-sponse within tumors. J. Immunother. 30: 16–28.
65. Keler, T., P. K. Wallace, L. A. Vitale, C. Russoniello, K. Sundarapandiyan,R. F. Graziano, and Y. M. Deo. 2000. Differential effect of cytokine treatment onFc� receptor I- and Fc� receptor I-mediated tumor cytotoxicity by monocyte-derived macrophages. J. Immunol. 164: 5746–5752.
66. van Spriel, A. B., H. H. van Ojik, A. Bakker, M. J. Jansen, andJ. G. J. van de Winkel. 2003. Mac-1 (CD11b/CD18) is crucial for effective Fcreceptor-mediated immunity to melanoma. Blood 101: 253–258.
67. Stockmeyer, B., M. Dechant, M. van Egmond, A. L. Tutt, K. Sundarapandiyan,R. F. Graziano, R. Repp, J. R. Kalden, M. Gramatzki, M. J. Glennie, et al. 2000.Triggering Fc�receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy. J. Immunol. 165: 5954–5961.
68. Park, J. W., R. S. Kerbel, G. J. Kelloff, J. C. Barrett, B. A. Chabner,D. R. Parkinson, J. Peck, R. W. Ruddon, C. C. Sigman, and D. J. Slamon. 2004.Rationale for biomarkers and surrogate end points in mechanism-driven oncologydrug development. Clin. Cancer Res. 10: 3885–3896.
2843The Journal of Immunology
by guest on March 25, 2018
http://ww
w.jim
munol.org/
Dow
nloaded from
