Recombinant il 2

Peripheral blood mononuclear cells were isolated from healthy volunteers, and the naïve B cells were purified by negative selection using magnetic cell separation by using the Naive B Cell Isolation Kit II (Miltenyi Biotec) with anti-CD10 antibodies (Miltenyi Biotec). The purity of the isolated CD19+ CD27− naïve B cell population was routinely >95%. The cells were labeled with 1 μM CFSE (Invitrogen) in serum-free medium at 37 °C for 10 min and washed in complete medium so as to monitor cellular division. Purified naive B cells were cultured at 7.5 × 10⁵ cells/mL in 24-well plates and stimulated during 4 days with 2.6 μg/ml F(ab’)² fragment goat anti-human IgA+IgG+IgM (H+L) (Jackson ImmunoResearch Laboratories), 100 ng/ml recombinant human soluble CD40L (Millipore), 1.0 mg/ml CpG oligodeoxynucleotide 2006 (Invivogen), and 50 U/ml recombinant IL-2 (R&D Systems). Day 4-activated B cells were washed and cultured at 4 × 10⁵ cells/ml for up to 3 days with 50 U/ml IL-2, 50 ng/ml IL-6, 50 ng/ml IL-10, and 2 ng/ml IL-12 (R&D Systems). At day 7 of culture, cells were washed and cultured with 50 ng/ml IL-6, 10 ng/ml IL-15 and 500U/ml IFN-α for 3 days. The cells were subjected to flow cytometry to analyze cell surface CD38, CD138. QRT-PCR was used to examine the expression of BLIMP1 mRNA and miR-26a.

96 well round bottom plates were coated with 1 μg/ml CD3 (145–2C11, BD Biosciences) and 0.5 μg/ml CD28 (37.5, BD Biosciences). Macrophages were M1 (250 ng/ml LPS) or M2 (20 ng/ml IL-4) polarized prior to the experiment for 16 h. CD4⁺CD25⁻ T cells were stained with CFSE proliferation dye (Thermo) and co-cultured with macrophages at a 1:2 ratio for 4 days in RPMI supplemented with 20 ng/ml recombinant IL-2 (R&D Systems). Upon collection, samples were stained with LIVE/DEAD™ Fixable Near-IR Stain Dye (Thermo) and analyzed by flow cytometry. Proliferation modeling was performed using Flow-Jo 10.1 and results normalized to the T cell only control group.