Target cells were labeled with Na251CrO4 (Hartmann,
Analytik, Braunschweig, Germany) for 1.5 h at 37°C, washed, and added at a concentration of 1×105 cells/well resulting in the indicated effector/target ratios. To study the underlying mechanisms of NK cell induced tumor cell death, neutralizing anti-FasL (BD Pharmingen), anti-TRAIL (BioVender), or isotype control antibody was added to the co-culture system. To inhibit perforin-mediated cytolysis, CMA (Sigma-Aldrich, Taufkirchen, Germany) was added to the NK cells 2 h prior to co-culture with target cells. The radioactive content of the supernatant was measured in a gamma counter (Berthold, Wildbad, Germany). Specific lysis was determined according to the following formula: specific lysis (%)=100×(Exp−Spo)/(Max−Spo), where Exp is the experimental release, Spo is the spontaneous release, and Max is the maximum release. Assays were Nivolumab in vivo performed as triplicates/quadruplicates, and data are depicted as means±standard deviation (SD). The experimental design of the Treg cell-NK co-culture experiments is illustrated in the Supporting Information Fig. S1. Student’s t-test for means (two-tailed, paired samples) from at least three individual experiments was used to calculate significance, and p-values equal or below 0.05 were considered as significant. We thank Kirsten Bruderek for her excellent
technical assistance. We also thank Johannes Schulte for his help with the chromium release assays. Antibodies directed against ULBP1, ULBP2, ULBP3, MICA, and MICB were a kind gift from Annette Tyrosine Kinase Inhibitor Library mouse Paschen (UK Essen). Research described in this article was supported in part by the IFORES program
of the Medical Faculty, University Duisburg-Essen (to S. B.) and the Deutsche Forschungsgemeinschaft (DFG 4190/1-1 to C. B.). Conflict of interest: The authors have declared no conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. Celecoxib They are made available as submitted by the authors. “
“Cyclooxygenase-2 is a promising target for cancer immunotherapy. Here, we designed the analogues p321-9L and p321-1Y9L (YLIGETIKL) from cyclooxygenase-2-derived native peptide p321. Then, we tested the binding affinity and stability of the analogues and their ability to elicit specific immune response both in vitro (from PBMCs of HLA-A*02+ healthy donors) and in vivo (from HLA-A2.1/Kb transgenic mice). Our results indicated that the activity of cytotoxic T lymphocytes induced by p321-9L and p321-1Y9L was more potent than that of p321. In conclusion, the epitope analogue, especially p321-1Y9L, may be a good candidate which could be used to the immunotherapy of patients with tumours expressing cyclooxygenase-2. Cytotoxic T lymphocytes (CTLs) specific for various tumour antigens play an important role in elimination of tumour cells [1, 2].