Human il 17 duoset elisa

PBMCs from healthy donors were enriched by performing ficoll gradient centrifugation and then labeled with anti-CD8a (PRA-T8; BioLegend), anti-CD4 (OKT-4; BioLegend) and anti-CD45RA (HI100; BioLegend). CD8⁺CD45RA⁻ cells were sorted using a FACSAria™ III. Tc17 cells were stimulated with anti-CD3 (2 μg ml⁻¹, clone TR66) and anti-CD28 (2 μg ml⁻¹ CD28.2; BD Biosciences) in the presence of rh IL-1β 20 ng ml⁻¹, rh TGF-β 20 ng ml⁻¹, rh IL-6 10 ng ml⁻¹, rh IL-2 50 U ml⁻¹. Tc17 cells were treated with 0.1% DMSO as a control, or with 10 μM DMF alone or in combination with 50 μM GSH. Living cells were analyzed by flow cytometry for IL-17A Pacific Blue, anti-IL-17A (BL168; Biolegend) and IFN-γ APC-Cy7 anti-IFN-γ (4 S.B3; BioLegend) production after 4 days of culture and restimulation with PMA/ionomycin for 2.5 h and brefeldin A for further 2.5 h. Supernatants were collected after 4 days and IL-17 was measured with the Human IL-17 DuoSet ELISA (R&D, DY317).

Autologous DC of patients V-A-01 and V-A-08 were matured for 48 h with the conventional cytokine cocktail (cDC), the complete prophylactic vaccine cocktail (VAC-DC), or the separate prophylactic vaccines BCG, Typhim, or Act-HIB. cDC was loaded with KLH, gp100 peptides (10 μM gp100:280–288 + 10 μM gp100:154–162), or tyrosinase peptide (10 μM tyrosinase:369–377). 1 × 10⁴ DC were co-cultured with 5 × 10⁴ autologous cells obtained from a bronchoalveolar lavage in RPMI + 7 % human serum. Cytokine production was measured in the supernatant after 24 h by cytometric bead array (human Th1/Th2 11 plex kit, eBioscience) or standard sandwich ELISA (human IL-17 DuoSet ELISA, R&D Systems). To study T cell proliferation, cells were pulsed after 4 days with 1 μCi/well tritiated thymidine for 8 h, and incorporation of tritiated thymidine was measured with a beta-counter.