Jes3 19f1

Ko143 and LPS (Eschericia coli strain O111:B4) were obtained from Sigma Chemical Co (St. Louis, MO). Isotype control antibodies (Abs) (IgG1, IgG2a and IgG2b), anti-ABCG2-FITC (IgG2b, 5D3), anti-CD1c-APCcy7 (IgG1, L161), anti-CD4-PerCP/Cy5.5 (IgG2b, OKT4), anti-CD8-Pacific Blue (IgG1, HIT8a), anti-CD11c-APC (IgG1, 3.9), anti-CD123-PEcy7 (IgG1, 6H6), anti-CD83-FITC (IgG1, HB15e), anti-CD25-APC and -PEcy7 (IgG1, M-A251), anti-FOXP3-Pacific Blue and -Alexa Flour-488 (IgG1, 206D), anti-IL-10-PE (IgG2a, JES3-19F1) and anti-IFN-γ-Alexa Flour-488 (IgG1, 4S.B3) Abs were obtained from Biolegend; and neutralizing Ab against human IL-10 (IgG2a, JES3-19F1) was purchased from Biolegend. Abs against phosphorylated form of p38, AKT, ERK and phospho-IKK were obtained from Cell Signaling Technology (Beverly, MA).

Unsorted cell suspensions or B cell populations sorted by FACS were suspended in RPMI-1640 containing 10% FBS, 100 U/ml penicillin/ 100 μg/ml streptomycin and seeded into 96 well plates prior to stimulation with 250 ng/ml ionomycin, 50 ng/ml PMA (both Sigma) and GolgiStop (1:1,000; BD Biosciences) for 4 h. Cells were washed and resuspended with PBS prior to live dead staining with Zombie Aqua. Subsequently, cells were washed and fixed with 2% paraformaldehyde, prior to treatment with permeabilization buffer (both eBioscience/Thermo Fisher Scientific). Cells were stained with anti-TNFα (Mab11, BioLegend, 1:50) and anti-IL10 (JES3-19F1, BioLegend, 1:20) and data acquired using a FACS Canto II instrument (BD Biosciences). See Supplemental Figure 4 for gating strategies for analysis of cytokine production by whole CD19⁺ populations from GALT and blood and Supplemental Figure 3 for sorting CD27⁺IgD⁻, CD27⁻IgD⁻, CD27⁺IgD⁺, and CD27⁻IgD⁺ populations and for analysis of cytokine production by cultured sorted cells.

Bulk CD4⁺ T cells were cultured and stimulated as described above for 7 days in the absence or presence of adalimumab (1 µg/mL), with supplementation of 20 U/mL (Peprotech) of IL‐2 on day 4. Cells were washed and rested for 24 h at 37°C. Fibroblasts generated from RA patients' knee replacement synovial tissue were seeded in 96‐well flat bottom plates at a density of 10 000/well in DMEM, supplemented with 10% FCS, 1% penicillin/streptomycin, 2% l‐glutamine (all from GIBCO), and 1 µg/mL Amphotericin B/Fungizone (GIBCO) and allowed to adhere for 24 h. Twenty‐five thousand pre‐cultured CD4⁺ T cells were added to the fibroblasts and cultured in DMEM for 3 days with soluble aCD3 mAb (100 ng/mL) with or without 10 µg/mL of anti‐IL‐10 (JES3‐19F1, Rat IgG2a, Biolegend) and anti‐IL‐10R (3F9, Rat IgG2a, Biolegend) blocking antibodies or matching isotype control Ab (RTK2758, Rat IgG2a, Biolegend). Supernatants were collected post co‐culture.