Cells were analyzed by a FACScan equipped with Cell Quest software (Becton Dickinson,

Mountain View, CA, USA). CXCL10, CCL2, and CXCL8 were measured with OptEIA™ kits (BD Pharmingen), LY294002 nmr in cell-free supernatants [sups] from resting or stimulated keratinocyte cultures, according to the manufacturer’ protocols. The plates were analyzed in an ELISA reader mod. 3550 UV Bio-Rad. Results are given as ng/106 cells ± SD. Skin biopsies were minced with a scalpel and placed in culture in complete medium plus 60 U/mL IL-2. After 2–5 days, T cells emigrated from tissue samples were collected for phenotypic and functional characterization and T-cell cloning by limiting dilution (0.6 cells per well), in the presence of irradiated allogeneic feeder cells plus 1% PHA. Subconfluent keratinocytes seeded in culture slides (BD Biosciences) were pretreated with the indicated concentrations of peptides and, then, stimulated with IFN-γ. After 24 h, cells were cocultured with 5 × 105 CFSE-stained autologous T cells clones. In blocking experiments, keratinocytes were incubated for 1 h with 10 μg/mL anti-CD54 prior to cocultures with effector T cells. After 6 h, cocultures were extensively washed in PBS, fixed in 4% paraformaldehyde, and counterstained with hematoxylin. T cells

that adhered to keratinocytes were counted in 20 casual fields for each Poziotinib in vitro condition, as fluorescent dots using a fluorescent microscope (Zeiss, Oberkochen, Germany), and average T-cell number per square millimeter ± SD was calculated. Complete RPMI with 0.5% BSA alone or supernatants (sups) from untreated or 24 h IFN-γ-stimulated keratinocyte cultures

(0.6 mL total amount) were added to the bottom chamber of 24-well Transwell chambers with uncoated 5 mm pore polycarbonate filters (Corning Janus kinase (JAK) Costar, Cambridge, MA). T autologous cells were resuspended in complete RPMI with 0.5% BSA, and 0.1 mL cell suspension (106 cells/mL) was added to the top chamber. Transwells were then incubated for 1 h at 37°C with 5% CO2. The number of cells transmigrated to the lower chamber relative to the input was measured with a FACScant by 60 s acquisition at a flow rate of 100 mL/min. The experiments were carried out in triplicate for each condition and the results are given as ng/106 cells ± SD. Five-millimeter punches of normal human skin from three healthy donors undergoing to plastic surgery. Biopsies were taken after informed consent and the study was approved by the Ethical Committee of the Istituto Dermopatico dell’Immacolata (IDI)–IRCCS (Rome, Italy). Biopsies were placed in Keratinocye Basal Medium with 0.1% normal human serum in a humidified incubator at 37°C, with enough medium to just cover the explants.