After 6h of incubation, radioactivity of the supernatant (100��l) was measured with a ��-counter. The percentage of specific lysis=100 �� (experimental c.p.m.?spontaneous c.p.m.)/(maximum c.p.m.?spontaneous c.p.m.). Flow cytometry For the expression of CD247 (transducing �� molecule) on NK cells, PBMCs were selleck Ruxolitinib stained with CD56-FITC (DAKO, Glostrup, Denmark) and CD3-APC (DAKO). Thereafter, the stained cells were fixed with 0.25% formaldehyde in PBS for 10min, followed by permeabilisation with digitonin (Invitrogen, Carlsbad, CA, USA; 10��gml?1) for 15min on ice, and stained with anti-CD247 mAbs conjugated with PE (IMMUNOTEC, Marseille, France) or with IgG1 isotype control mAbs for 10min on ice. The mean fluorescence intensity for CD247 gated on CD56(+)CD3(?) cells was assessed by flow cytometric analysis.

For the expression of perforin and granzyme-B on NK cells, PBMCs were stained with CD56-FITC and CD3-APC. Thereafter, the stained cells were fixed and permeabilised using the Cytoperm/Cytofix kit (BD Bioscience, San Diego, CA, USA), followed by staining with mouse anti-human perforin mAbs conjugated with FITC (BD Pharmingen, San Diego, CA, USA) or mouse anti-human Granzyme-B mAbs conjugated with FITC (BD Pharmingen). The mean fluorescence intensity for perforin or granzyme-B gated on CD56(+)CD3(?) cells was assessed by flow cytometric analysis. For the expression of IL-21 receptor (IL-21R) on NK cells, PBMCs were stained with CD56-FITC, CD3-APC, and PE-labelled anti-human IL-21R mAb (R&D Systems, Minneapolis, MN, USA).

Statistical analysis Paired and non-paired Student’s t-tests were used to examine AV-951 the differences between groups. Findings were considered significant when P-values were <0.05. Results Trastuzumab- and Cetuximab-mediated ADCC was impaired in patients with ESCC We examined Trastuzumab- and Cetuximab-mediated ADCC of PBMCs derived from patients and healthy donors. High EGFR-expressing KYSE30 and low EGFR-expressing KYSE110 were killed by Cetuximab-mediated ADCC, and the ADCC activity reflected the degree of EGFR expression, as shown in Figure 1A (high EGFR-expressing KYSE30 vs low EGFR-expressing KYSE110). It is noteworthy that the levels of Cetuximab-mediated ADCC in patients with ESCC were significantly impaired in comparison with those in healthy donors (Figure 1A), in line with our previous reports (Kawaguchi et al, 2007a). Figure 1 Trastuzumab- and Cetuximab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) of peripheral blood mononuclear cells (PBMCs) from patients and healthy donors. (A) Cetuximab-mediated ADCC of PBMCs from patients (oesophageal squamous cell carcinoma …