Quantifying T-Cell Populations in Tumor Samples

Part of the selected n = 188 tumor samples could not be analyzed due to insufficient tumor material to obtain at least one microscopic image (n = 26). Triple immunofluorescent staining for CD3 (ab828, Abcam, Cambride, UK), FoxP3 (ab20034, Abcam) and IL-17 (AF-317-NA, R&D Systems, Abingdon, UK) was performed on 162 tumor samples as described before [29 (link)]. These comprised 86 p16 positive and 76 p16 negative tumors. Images were obtained using an LSM700 confocal laser scanning microscope containing an LCI Plan-Neofluar 25×/0.8 Imm Korr DIC M27 objective (Zeiss, Göttingen, Germany). One to four random images sampled a total vital tumor (epithelium + stroma) area of up to 1.0 mm². Total tumor epithelium and stroma surface area and double or triple positivity of cells were determined in each image using LSM Image Browser (version 4.2.0.121, Zeiss). Single-, double- and triple-positive cells were scored separately in the tumor epithelial and stromal areas using ImageJ version 1.47 (http://rsb.info.nih.gov/ij). Cells within blood vessels and largely autofluorescent areas were not scored.