Experiments were carried out in triplicate. of the tissue of origin are prerequisite to validate new potentially therapeutic tools. Uterine cervical cancer is a good model to evaluate immunotherapy protocols, because the etiological agent of this tumor, the human papillomavirus (HPV), has been well-defined.7,8 This cancer is preceded by well-characterized preneoplastic stages designated as squamous intraepithelial lesions. Moreover, several HPV proteins induce overexpression of EGFR by different mechanisms9,10 and this overexpression is associated with poor prognosis in cervical carcinomas.11 The organotypic (raft) culture system has been increasingly used to examine the effects of viral or biochemical therapeutic agents on a variety of malignant keratinocytes.12-15 The raft technique permits cell proliferation and differentiation at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, whereas HPV-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to high-grade lesions seen Cell Death Detection Kit; Roche, Germany). Briefly, slides were fixed in cold acetone for 3 minutes, washed twice with PBS, and 50 l of TUNEL reaction mixture was added. After incubation in a humid chamber for 1 hour at 37C, slides were washed three times with PBS, mounted, and examined by fluorescence microscopy (Olympus IX50, Micro Image software). Nuclei of all cells were revealed with DAPI staining (4, 6-diamidine-2-phenylindole dihydrochloride; Roche). Enzyme-Linked Immunosorbent Assay (ELISA) Levels of interferon- (IFN-) and tumor necrosis factor- (TNF-) in the cultures were measured using specific ELISA assays (Biosource, Nivelles, Belgium). Recombinant human IFN- and TNF- were used as reference standards. Statistical Analysis The nonparametric Mann-Whitney test was applied using Instat Mac 2.01 software (GraphPad Software, San Diego, CA). Differences were considered significant at 0.05. Results Neoplastic HPV+ Keratinocytes Overexpress EGFR Fluorescence-activated cell sorting (FACS) analysis of EGFR on cell surface revealed high expression levels of EGFR on all HPV+ keratinocytes (HPV-transformed keratinocytes KT1 and KT2 cells and tumor-derived SiHa and CasKi cells) whereas HPV? tumor cell line C33 showed expression level as low as that of normal keratinocytes (Figure 1A)? . EGFR was differentially expressed in the epithelium of the uterine cervix and, interestingly, this differential expression was also found in organotypic cultures as indicated by immunohistochemistry staining. Indeed, staining was evident only in basal layers of normal exocervix biopsies (Figure 1B)? and of normal keratinocyte organotypic cultures (Figure 1C)? , whereas all cells were strongly stained in high-grade cervical lesions (Figure 1D)? and in organotypic cultures of HPV+ cell lines CasKi, KT1 (Figure 1, E and F)? , KT2, and SiHa (data not shown). Open in a separate window Figure 1. EGFR expression on normal and HPV+ keratinocytes. A: EGFR expression by FACS analysis. Fluorescence index represents the total fluorescence intensity in the presence of mAb MINT5 and FITC-labeled secondary antibodies/background level in the presence of the FITC-labeled R306465 secondary antibody alone. Values are means (SD) of five independent experiments. B: R306465 Immunohistochemical staining with mAb MINT5 on biopsy specimens (original magnification, 20) of normal exocervix and high-grade squamous intraepithelial lesions (D), organotypic culture sections of normal keratinocytes (C), CasKi cells (E), and KT1 cells (F). Allogeneic Lymphocytes Retargeted by BimAb Kill HPV+ Keratinocytes Rabbit Polyclonal to Cytochrome P450 3A7 in Monolayer Cultures To evaluate the efficacy of bimAb against HPV+ R306465 keratinocytes, cytotoxicity assay of lymphocytes retargeted by the anti-CD3/anti-EGFR bimAb M26.1 was performed using normal and transfected cervical keratinocytes or cervical carcinoma cell lines in monolayer cultures as targets and lymphocytes from healthy donors as effectors. Cytotoxic assay revealed highly increased 51Cr release in wells with activated T lymphocytes M26. 1-retargeted and EGFR+ target cells KT2, SiHa (Figure 2, A and B)? , and CasKi (data not shown) as compared to activated lymphocytes incubated in absence of bimAb, which exerted a low level of natural killer-like cytotoxic activity, particularly evident at the higher E:T ratios. Parental antibodies either alone or in combination failed to trigger cytolytic activity against SiHa cells (Figure 2B)? or against the other targets (data not shown). A low but detectable cytolytic activity was also observed against normal keratinocytes (Figure 2C)? , which, as shown.