Anti il22 apc

Cryopreserved PBMC and freshly isolated MMC were rested over night at 37°C before stimulation for 5 hours with 40 ng/mL of Phorbolmyristate acetate (PMA) and 1 µM Ionomycin (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 1 µl/mL of Brefeldin A (BD Bioscience, San Jose, CA, USA) to prevent cytokine release. As negative control cells remained unstimulated with 1 µl/mL Brefeldin A added for 5 hours of incubation. After two washes with RPMI medium containing 10% heat-inactivated HAB serum, cells were fixed and permeabilized according to the BD Cytofix/Cytoperm protocol. Subsequently cells were stained with the following antibodies: anti-CD3 PE-Cy7 (Invitrogen, Eugene, Oregon, USA), anti-CD4 ECD (Beckman Coulter, Brea, CA, USA), anti-IL-2 FITC, anti-CD8 PerCP-Cy5.5 (BD Bioscience, San Jose, CA, USA), anti-IL17A PE (BD Pharmingen, San Diego, CA, USA), anti-IL22 APC (R&D systems, Minneapolis, MN, USA) and IFNγ eFlour450 (eBioscience, San Diego, CA, USA) for 20 min at 4°C. Samples were acquired and analyzed as described above. Positive responses shown were calculated after unstimulated background was subtracted. Frequencies of IFNγ, IL-2 and IL-17 and/or IL-22 expressing cells is based on the population of CD4+T cells, while the frequency of triple-cytokine producing cells (IFNγ, IL-2 and IL-22) is based on the population of CD4+IL-17+T cells.