M 2 me

DC3.2 is a J2 virus-immortalized dendritic cell line (13 (link)). A particular DC3.2 clone (with Renilla luciferase) was used for all experiments in this study as this clone has very strong cross-presentation and MHC class II presentation, as compared to other clones. RF33.70 is a T cell hybridoma that recognizes the ovalbumin (OVA) peptide OVA_257–264 in the context of H2-K^b (14 (link)). MF2.2D9 is a T cell hybridoma that recognizes OVA_258–276 in the context of I-A^b (13 (link)). RF33.70 and MF2.2D9 were transduced with lentivirus containing NFAT-luciferase. NIH-3T3 cells were stably transfected with the mouse H2-K^b molecule. A549 and MCF7 were kindly provided by Leslie Shaw (UMass), and the D53m and H50m mouse MCA-induced sarcoma lines were kindly provided by Robert Schreiber (Wash U, St. Louis). The MCA-induced sarcoma lines were grown in R10 media and all other cell lines were grown in RPMI 1640 (Gibco) supplemented with 10% FBS (Hyclone), 1% NEAA (Gibco), 1% HEPES (Gibco), 1% Antibiotic-Antimycotic (Gibco), and 5 × 10⁻⁵ M 2-ME (Sigma). The MCF7 growth media was also supplemented with 10μg/mL insulin. Antibiotic selection for CRISPR-Cas9 knockout cells was done for two weeks in media containing 5μg/mL blasticidin (Invivogen). All cells were grown in a 10% CO₂ atmosphere at 37°C.

Mice were immunized sub-cutaneously (s.c.) with 20 nmol hen egg-white lysozyme (HEL) emulsified in complete Freund’s adjuvant (CFA) (Difco). After 7 days, draining lymph nodes cells were harvested, and 0.5-1 × 10⁶ cells were plated in triplicate with medium only, varying concentrations of HEL, or purified protein derivative (PPD) of Mycobacterium tuberculosis for 4 days in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum (PAA), 5×10⁻⁵ M 2-ME (Sigma-Aldrich), 2mM GlutaMAX (Life Technologies), 1mM sodium pyruvate (Cellgro), 0.1mM non-essential amino acids (Lonza), 25mM HEPES buffer (Cellgro), and 50μg/mL gentamicin (Life Technologies), with 0.4μCi tritiated thymidine ([³H]TdR) added for the last 24 h of culture. Assays were harvested on a Skatron cell harvester and counted using a LKB (Pharmacia/LKB) β plate counter.

Peripheral venous blood (20 mL) was collected into a heparinized tube before and 1 week after infliximab infusion in BD patients with or without recurrent uveitis during at least 1 year of infliximab therapy, and from healthy controls at any time. PBMCs were isolated immediately by density gradient centrifugation (Ficoll-Hypaque; Pharmacia Biotech, Shanghai, China) and suspended at 2 × 10⁶ cells/mL in RPMI 1640 medium supplemented with 10 mM HEPES, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin (all from BioWhittaker, Walkersville, MD), 1 × 10⁻⁵ M 2-ME (Sigma Chemical Co., St Louis, MO), and 10% fetal calf serum (Sigma Chemical Co.).