Cd11c positive isolation kit

CD4⁺ T cells were purified by a CD4⁺ Naive T cell Negative Isolation Kit (Stem Cell). CD11c⁺ DCs were purified by a CD11c Positive Isolation Kit (Miltenyi Biotec) (>95% purity). The method for generating bone-marrow dendritic cells (BMDCs) has been described previously (42 (link)). For in vitro differentiation, CD4⁺ Naive T cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (GIBCO) and stimulated with 5 µg/ml plate-bound anti-CD3 and 1 µg/ml soluble anti-CD28 under the appropriate conditions to obtain either Th0 (50 U/ml IL-2, 10 µg/ml anti-IL-12 and 10 µg/ml anti-IL-4), Th1 (10 ng/ml IL-12, 10 µg/ml anti-IL-4 and 50 U/ml IL-2) or Th17 (3 ng/ml TGF-β1, 20 ng/ml IL-6, 10 ng/ml IL-1β, 10 ng/ml IL-23, 10 µg/ml anti-IFN-γ, and 10 µg/ml anti-IL-4) cells in the presence or absence of ECM1 protein or human IgG control.

Splenic DCs were sorted to more than 95% purity using the CD11c Positive Isolation Kit (Miltenyi Biotec). Naive 2D2 T cells were isolated from the lymph nodes and spleens of 2D2 TCR transgenic mice using the CD4⁺CD62L⁺ T-cell isolation kit (Miltenyi Biotec). Co-culture of CD4⁺ T cells with splenic DCs has been described previously (29 (link), 30 (link)). Briefly, purified naive 2D2 CD4⁺ T cells were cultured with splenic DCs at a ratio of 5:1 in the presence of the MOG_35–55 peptide (10 µg/ml), IL-6 (50 ng/ml; Peprotech), IL-1β (10 ng/ml; Peprotech), FICZ (6-formylindolo [3,2-b] carbazole; 300 nM; Biomol) and TGF-β (1 ng/ml; R&D Systems) or latent-TGF-β (50 ng/ml). For RGD blockade experiments, cRGD peptide was added at 4 µg/ml. For the co-culture assay, co-cultured cells were maintained in X-VIVO™ 15 Chemically Defined, Serum-free Hematopoietic Cell Medium (Lonza). The 2D2 T cells were analyzed by flow cytometry after 3 days of culture. Cytokines in the culture supernatants were measured using an IL-17A ELISA kit (R&D, USA). The activation of TGF-β was measured with the Plasminogen Activator Inhibitor-1 Promoter Luciferase Assay (details described below).