Complete rpmi medium

LMP1/CD40-expressing mice have been already described [5 (link)]. Animals were housed at 21–23 °C with a 12-h light/dark cycle. All procedures were conducted under an approved protocol according to European guidelines for animal experimentation (French national authorization number: 87–022 and French ethics committee registration number “CREEAL”: 09-07-2012). For in vivo PD-L1 treatment, LMP1/CD40-expressing mice were injected intraperitoneally every 4 days for 3 weeks with 200 μg anti-PD-L1 antibody (clone 10F.9G2; Bio X cell; US). For ex vivo treatments, splenocytes were cultured for 48 h in complete RPMI medium (Eurobio) supplemented with 10% of FBS, 2 mM of L-Glutamine, 1% of Na pyruvate, 100 U/ml of penicillin and 100 μg/ml of streptomycin (ThermoFisher Scientific) and with the following treatments: either 10 μM of PHA-408 or 1.5 μM of ruxolitinib or 1 μM of ibrutinib.

The MH-S mouse alveolar macrophage cell line (American Type Culture Collection, Bethesda, MD) was grown in complete RPMI medium (Eurobio, Courtaboeuf, France).
3X10⁵ MH-S cells were resuspended in 20μl Nucleofector solution (Cell Line 96-well Nucleofector Kit SF; Lonza, Levallois, France) per one well and transfection was performed with Nucleofector device according to manufacturer protocol (Lonza).
To normalize for transfection efficiency, pGL4.10[luc2] plasmids bearing either the wild-type mCxcr1 promoter or the NOD sequence were cotransfected with the pGL4-74[hRluc/TK] plasmid, which encodes Renilla luciferase. Negative and positive controls were respectively obtained with empty pGL4.10[luc2] vector and pGL3[luc/SV40] vector (Firefly luciferase under SV40 large T antigen promoter). Following nucleofection and a 16h-incubation, cells were lysed and luciferase assays were performed using the Dual-Glo Luciferase Assay system (Promega). Luminescence was measured on a Wallac MicroBeta TriLux (Perkin Elmer, Courtaboeuf, France). Values of firefly luciferase activity were normalized to the ones of Renilla luciferase. Each plasmid of interest was transfected into three wells per experiment, and three independent experiments were performed.

Peripheral blood mononuclear cells from healthy donors were separated by Ficoll-Hypaque density-gradient centrifugation. PBMC were cultured in complete RPMI medium (Eurobio) supplemented with 10% AB-human serum (Invitrogen, Saint Aubin, France), 2% Penicillin–Streptomycin (Eurobio), and 1% l-glutamine (Eurobio). Cocultures were performed in flat-bottomed 96-well culture plates in which 10⁴ synoviocytes/well were seeded and allowed to adhere to the plate for several hours. PBMC (5 × 10⁴) were then added to the synoviocytes in the presence or not of 5 ug/mL phytohemagglutinin (PHA, Sigma Aldrich, Saint-Quentin Fallavier, France). After 24 h, supernatants were collected and cells were washed with PBS, followed by partial separation of PBMC from synoviocytes by incubating the cocultures with a 1 mM EDTA–PBS solution at 37°C for a brief time. PBMC present in the EDTA wash and synoviocytes still attached on the plate layer were then lysed for the 24 h timepoint. complete RPMI medium in the presence or not of PHA was then added to the cells for 24 or 48 h more before collection of the supernatants and lysis of the cells. For each condition, twelve wells were combined in order to obtain sufficient material to perform gene expression quantifications.