Th1 th2 th17 cytokine bead array

Rag1-deficient mice were injected i.p. with approximately 3x10⁵ wild type or Tpl2^−/− naïve T cells (CD4⁺CD25⁻CD45RB^hi). Mice were weighed prior to injection and weekly thereafter. Blood was collected at 3, 6 and 8 weeks from the tail vein or by terminal cardiac puncture, and serum cytokines were quantified by Th1/Th2/Th17 cytokine bead array (BD Biosciences). Spleen and mesenteric lymph nodes were isolated and counted. Cells were restimulated ex vivo for 4 hours with PMA, ionomycin, and Golgi Plug (BD Biosciences) at a concentration of 1–2x10⁶ cells/ml and stained similarly to in vitro cultures.

Peripheral blood lymphocytes (PBL) were isolated after informed consent from PBMC of healthy donors, by depleting CD14⁺ cells using CD14 Microbeads (Miltenyi Biotec). PBL were stored in liquid nitrogen until further use. PBL were labeled with 1 µM CFSE (Invitrogen) and plated in a 96-well plate at 1 × 10⁵ per well. Mature bleb-loaded MoDC were added to the wells at DC/PBL ratios of 1:5, 1:10, or 1:20, and CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell proliferations were analyzed using flow cytometry after 6 days. The day 6 supernatant was analyzed for T cell cytokines, using a TH1/TH2/TH17 cytokine bead array (BD Biosciences, Breda, the Netherlands).