Effects of irradiation on the expression of surface antigens in human ovarian cancer.

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The physiologic and cellular changes associated with high INTRODUCTION doses of irradiation have been well documented [for review, see 9].However, it has only recently been shown that after Cell surface antigens are important for both recognition irradiation an increased synthesis of specific proteins, includand destruction of tumor cells by the host immune system.ing MHC Class I antigens, has been observed.Hauser et For a specific immune response to ensue, T cells must recogal.[10] reported an increased expression of MHC Class I nize a target peptide in the context of a major histocompatimolecules after fractionated doses of irradiation on a murine bility complex (MHC) 3 molecule.However, additional comelanoma cell line in vitro and Hareyama et al. [11] demonstrated an increased expression of MHC Class I and carci- 1 This work was supported in part by grants from Memorial Health Sernoembryonic antigen after lethal irradiation of a human gasvices of Long Beach Memorial Hospital (Long Beach, CA), Oncotech, tric adenocarcinoma cell line.
(Irvine, CA), and the Camillo Golgi Foundation (Brescia, Italy).stimulate systemic immunity against parental tumor cells in complete media (CM) containing RPMI 1640 (Gibco Life Technologies, Grand Island, NY) 10% fetal bovine serum many murine models has stimulated clinical investigators to design human trials in an attempt to translate such results (FBS, Gemini Bioproducts, Calabasas, CA).for human cancer immunotherapy.However, allogeneic or High-dose gamma irradiation.Tumor cell lines were autologous human tumor vaccines require methods such as irradiated with 5000 and 10,000 cGy in 15-ml conical tubes irradiation, to inhibit cell replication, so as to prevent possiin CM at room temperature with gamma rays (Cesium 137) ble tumor formation at the immunization site.Moreover, at a dose rate of 200 cGy/min.Immediately after irradiation, such methods should not compromise the immunogenicity cells were seeded into T 75 tissue culture flasks (Corning, of the tumor vaccine; rather, they should maintain or when Corning, NY) in CM and cultured at 37ЊC in a 5% CO 2 possible enhance its intrinsic immunogenic potential.We atmosphere.Spent media were changed every other day.recently observed upregulation of the expression of MHC Irradiated cells were harvested at Days 2 and 6 for analysis Class I molecules and tumor-associated antigens (TAA) after of surface antigen expression by FACS analysis.irradiation of an IL-4 transduced human ovarian carcinoma Indirect immunofluorescence and flow cytometry.Cells cell line produced to be used in a phase I immunotherapy were harvested with 0.25% trypsin in HBSS (Gibco) and trial [12].This observation has directed our interest in studywashed once in CM.Cell suspensions were counted and ing the effects of high doses of gamma irradiation on the distributed into 12 1 75-mm tubes at 5 1 10 5 cells/tube.expression of MHC and other surface antigens in several Mouse monoclonal antibodies [anti-HLA Class I (mAb W6/ ovarian carcinoma cell lines.

cells (GATC) secreting immuno-enhancing cytokines to
32; Accurate Chemical and Scientific Corp., Westbury, NY); Our studies show that high doses of gamma irradiation anti-HLA Class II (mAb CR3-43; Accurate Chemical and induce a significant and long-lasting upregulation of all sur-Scientific Corp.); anti-ICAM-1 (mAb LB-2; Becton-Dickinface antigens expressed on the ovarian carcinoma cell lines son); anti-CA 125 (mAb OC125; Signet Laboratories, Dedprior to irradiation.The possible implications of such findham, MA); anti-HER-2/neu p185 (mAb TA-1; Oncogene ings in light of the recent proposed use of genetically modi-Science, Uniondale, NY)] were diluted in cold assay buffer fied tumor cells as vaccines for the treatment of women with (PBS, pH 7.2, supplemented with 0.1% FBS) and added in advanced ovarian cancer will be discussed.
a 50-ml volume.A mouse IgG preparation (mAb IgG 2 a; Becton-Dickinson) was used as a negative control.Tubes MATERIAL AND METHODS were incubated for 30 min followed by two washes with assay buffer.Phycoerythrin-conjugated goat anti-mouse IgG Tumor cell lines.Four human epithelial ovarian carci-F(ab) 2 (H&L) (Leinco Technologies, St. Louis, MO) diluted noma cell lines (UCI-101, UCI-107, SKOV-3, and T-222) 1:100 in assay buffer was added to the cells and the tubes were used for this study.UCI-101 and UCI-107 have been were incubated for 30 min.Following two washings in assay previously characterized [13, 14] and were kindly provided buffer, the final cell pellet was resuspended in 500 ml assay by Dr. Alberto Manetta, University of California, Irvine.
buffer for subsequent analysis.Cells were analyzed with a SKOV-3 was purchased from American Type Culture Colfluorescence-activated cell sorter (FACS; Becton-Dickinlection (ATCC), while T-222 was kindly provided by Dr.
son) [15] with a 15-mW argon laser with an excitation of Benjamin Bonavida, University of California, Los Angeles.All tumor cell lines were maintained at 37ЊC, 5% CO 2 in 488 nm.Fluorescent signals were gated on the basis of for-ward and right-angle light scattering to eliminate dead cells and aggregates from analysis.Gated signals (10 4 ) were detected at 585-bp filter and analyzed using Lysis II software.
Statistical analysis.Significance analysis was performed using a paired Student's t test.

RESULTS
Expression of surface antigens on ovarian carcinoma tumor cell lines.Flow cytometric analysis of MHC Class I and Class II antigens, ICAM-1, and the TAA CA 125 and Her2-neu antigens was performed on four continuous ovarian cell lines.The results shown in Table 1 indicate that MHC Class I antigens were expressed at variable levels (range of MFI from 41.8 to 834.3) on all four cell lines.Her2-neu antigen was expressed also in all four cell lines (range of MFI from 21.5 to 224.3).In contrast, Class II antigens and CA 125 were not expressed in any cell line.ICAM-1 was expressed in only two cell lines (i.e., UCI-101 and T-222) (range of MFI from 43.8 to 92.2).
Expression of surface antigens after high-dose gamma irradiation.Cell surface antigen expression on the four established cell lines was evaluated 2 days after irradiation and the results were compared to the of expression on unirradiated control cells.As shown in Figs.1-3, irradiation with 10,000 cGy caused a significant upregulation of all the surface antigens expressed prior to irradiation.The induction indices for MHC Class I ranged from 1.42 to 2.62, for ICAM-1 from 1.74 to 2.20, and for Her2-neu from 1.22 to 1.71, respectively.It is interesting that irradiation did not induce neoexpression of antigens previously not present on these cells, such as MHC Class II.In one cell line (i.e., T-222), irradiation-induced upregulation was also noted at 5000 cGy and was essentially identical to that found at 10,000 cGy.However, because 5000 cGy was not a sufficient dose to totally block cell replication in some cell lines, we performed the majority of our studies using 10,000 cGy, a dose which consistently blocked cell replication in all cell lines.
Kinetics of expression of surface antigens after high-dose gamma irradiation.Kinetics studies were performed to de-

FIG. 1. Expression of surface antigens on UCI-101 ovarian cancer cells
termine whether the radiation induced of surface antigens prior to and after 10,000 cGy of gamma irradiation.1, unirradiated cells; was persistent or was a transient response by the tumor cells.2, irradiated cells.Thus, tumor cells from the T-222 cell line were irradiated with 10,000 cGy and analyzed for antigen expression 2 and studies showed that the enhancement of antigen expression 6 days after irradiation.As seen in Fig. 4, irradiation of these was persistent until all cells died (data not shown).cells caused increased expression of all the surface antigens at Day 2 as well as Day 6 when compared to the levels expressed by the unirradiated parental cells.Viability studies DISCUSSION (trypan blue exclusion) showed that by Day 2 the viability of the irradiated cells was approximately 90% and this The importance of surface antigen expression and immune recognition has been underscored by studies on human tumor dropped to about 30% by Day 6 after 10,000 cGy.Additional Irradiation, together with surgery and chemotherapy, represents one of the most commonly used, standardized, and effective modalities for contemporary cancer therapy.However, in a significant number of cases where whole-body irradiation with fractionated low doses is used (i.e., malignant lymphoma) the efficacy of the therapy cannot be explained merely by the lethal effects of radiation on the tumor cells [17].There may be additional reasons to explain these findings.Reports dating more than two decades ago note an increased infiltration of lymphocytes surrounding tumor cells following radiation therapy, suggesting that such cells may have become more immunogenic [18,19].Most reports evaluating the phenotype of such tumor-infiltrating cells have shown that the majority of these cells are CD 4 / helper inducer T cells [20].Cameron et al. [21] reported that local tumor irradiation synergizes with tumor-infiltrating lymphocytes (TIL) and IL-2 administration in mediating the regression of established macrometastases.Such results support the possibility that the synergy observed between local irradiation and the systemic administration of IL-2 and TIL cells may be due in part to the upregulated expression of cell surface antigens on the irradiated tumor cells.Indeed, such cells exposed to TNF-a plus 5,16].Such studies have shown that the induction of cytotoxic effector cells requires a higher level of antigen expression than cytotoxic interaction.In fact, tumors not previously considered to be immunogenic but so induced to express higher levels of MHC antigens appear to recruit high-avidity T cells, which can then recognize target cells which express virtually undetectable levels of these surface antigens [4,5].Moreover, in studies on melanoma [5, 6] the expression of ICAM-1 antigens in different melanoma clones correlated with the susceptibility of the tumor cells to lysis by specific and nonspecific T-cell clones and, more importantly, upregulation of these antigens, by treatment with interferon-g in low expressor clones, could boost tumor lysability only by specific, TCR-dependent and HLA-restricted effector cells.In such studies, the relative surface expression of ICAM-1 was a key factor for the outcome of the interaction with T cells and only those clones that expressed these molecules at lev-  a phenomenon would render these cells more recognizable animals against a subsequent challenge of viable parental cells, even though the unirradiated tumor cells were consid-by the IL-2-activated, MHC Class I-restricted cytotoxic T lymphocytes.However, adequate documentation of the ef-ered to be poorly immunogenic.Such data support the concept that following irradiation important cell surface changes fects of irradiation on surface antigen expression is lacking.
Further studies documenting the effects of irradiation on occur which alter the intrinsic immunogenicity of the tumor cells.In addition to these findings, the necessity of blocking tumor immunogenicity were recently reported by Dranoff et al. and Huang et al. [7,8] who showed that immunogenicity cell replication in any human clinical immunotherapy trial using whole-cell tumor vaccines has directed our interest of irradiated tumor cells was increased and would protect

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To whom correspondence should be addressed at UCI Medical Center, Building 23, Room 314, University of California, Irvine, 101 The City few years due to advances in genetics, immunology, and Drive, Orange, CA 92668-3298.Fax: 714-456-5039.molecular biology that have provided high efficiency gene 3 Abbreviations used: PPC, percentage of positive cells; MFI, mean chantransfer systems for vaccine approaches for the treatment of nel fluorescence intensity; TNF-a, tumor necrosis factor-a; IFN-g, intercancer.The success in the use of genetically altered tumor feron-g MHC, major histocompatibility complex; ICAM-1, intercellular adhesion molecule-1.

FIG. 2 .
FIG. 2. Expression of surface antigens on UCI-107 ovarian cancer cellsprior to and after 10,000 cGy of gamma irradiation.1, unirradiated cells; 2, irradiated cells.Note that UCI-107 cells do not express MHC Class II, ICAM-1, or Ca 125 surface antigens and these were not induced after irradiation.

FIG. 3 .
FIG. 3. Expression of surface antigens on SKOV-3 ovarian cancer cellsels high enough for an efficient interaction with antigenprior to and after 10,000 cGy of gamma irradiation.1, unirradiated cells; 2, irradiated cells.

FIG. 4 .
FIG. 4. Kinetics of expression of surface antigens following exposure to high-dose gamma irradiation.T-222 cells were irradiated with 10,000 cGy and analyzed for surface antigen expression 2 and 6 days after treatment.1, unirradiated cells; 2, irradiated cells.

TABLE 1 Expression of Surface Antigens on Four Established Ovarian Cell Lines
PPC, percentage of positive cells (values are expressed as means { standard deviation).b MFI, mean channel fluorescence intensity (values are expressed as means { standard deviation). a