After one wash with PBS, slides were analyzed by fluorescent microscopy using a Nikon eclipse E400 microscope (Nikon, Tokyo, Japan) with a ×20 or ×60 plan objective. For flow cytometry evaluation, staining was performed

without DAPI. Cells were analyzed GSI-IX using BD FACSCalibur software (Becton Dickinson, San Jose, CA, USA). A plasmid containing the human NF-κB promoter upstream to the luciferase reporter gene, kindly provided by Y. Ben-Neriah (The Hebrew University), was purified using the Qiagen EndoFree Plasmid Kit (Qiagen, Düsseldorf, Germany) according to the manufacturer’s instructions. Highly purified plasmid DNA, 3 μg, was used to electroporate 0.5–2×106 DC, which were introduced with the Human Dendritic Cell Nucleofector Kit (Amaxa Biosystems, Cologne, Germany). Cells then were incubated

for varying times, under varying conditions, as indicated. The iDC were then harvested, washed, and lysed. Luciferase activity was measured by the Floustar luminometer, using the Luciferase Assay Kit (Promega, Madison, WI, USA). Statistical significance was assessed using the JNK signaling pathway inhibitors Student’s t-test for unpaired data comparisons unless indicated otherwise. Kolmogorov−Smirnov analysis was used for flow cytometry analysis. The authors wish to thank Shifra Fraifeld for her editorial assistance with the preparation of this article. Conflict of interest: The authors declare no financial or commercial conflict of interest. “
“MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell Y-27632 molecular weight receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism

as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues.