Golgiblocker

T cells were activated and grown in X-VIVO 15 Medium supplemented with 1% Penicillin-Streptomycin (both from Lonza), 5 μg/ml plate-bound α-CD3, 1 μg/ml soluble α-CD28 (both from Biolegend), 300 U/ml IL-2 (Preprotech) and 6% CO₂ at 37 °C. Naïve T cells were differentiated towards the Th17 lineage by culturing them with 10 ng/ml TGF-β, 10 ng/ml IL-1β, 25 ng/ml IL-6 and 10 ng/ml IL-23 (all from Peprotech) for 6 days. Four hours prior to harvesting, cells were restimulated with 50 ng/ml PMA (Sigma) and 1 μg/ml ionomycin (Life Technologies) and treated with GolgiBlocker (BD Biosciences).

DCs were enriched by lineage (CD3, CD14, CD16, CD19) depletion, then lineage negative cells (2 × 10⁵ in 200 μL culture medium) were stimulated with TLR ligands or viruses for 4 h followed by another 2 h in the presence of Golgi Blocker (BD Biosciences, Franklin Lakes, NJ, USA). Cells were stained with surface markers and permeablized and stained with antibodies against various cytokines. TLR ligands were purchased from Invivogen (San Diego, CA, USA) and used at following concentrations: LPS (1 μg/mL), CpG 2216 (2 μg/mL), R848 (2 μg/mL). Heat inactivated influenza virus A/PR8/34 was used at 10 MOI for stimulation.

Infiltrating lymphocytes in islets were isolated as described [24 (link)]. Briefly, pancreatic tissues were collected and digested with Liberase RI (Roche Applied Science) at 37°C for 15–20 min. Dissociated islets were isolated under a dissecting microscope. To release intra-islet lymphocytes, islets were digested with Trypsin-EDTA for 10 min, followed by treatment with cell dissociation buffer (GIBCO/BRL) for 15 min at 37°C. After overnight incubation at 37°C, 4-5x10⁴ cells (70–80% viable) were recovered from pooled pancreata from 5 mice. Lymphocytes were then fixed and externalization of CD107a (1D4B, Biolegend) was determined by flow cytometry. To detect IFN-γ production, lymphocytes were re-stimulated with 1μM OVA peptide (SIINFEKL) in the presence of Golgi blocker (BDbiosciences) for 5 hours. Cells were fixed and permeabilized according to the manufacturer's instructions. The level of intracellular IFN-γ was detected by APC labeled anti-IFN-γ (XMG1.2, Biolegend). To detect T-bet and pStat5, cells were fixed using BD Phosflow Lyse/Fix Buffer and stained with PE labeled anti-T-bet (4B10, eBioscience) and anti-pSTAT5 (pY649, BDbiosciences), respectively.