Plate coated anti cd3

Naïve CD4⁺ T cells were sorted from spleen and lymph nodes using the MACS CD4⁺CD62L⁺ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in supplemented IMDM medium in the presence of 5 µg/ml plate-coated anti-CD3 (produced in house) and 1 µg/ml soluble anti-CD28 antibodies (BD Bioscience, San Jose, California USA).The following conditions were used: Th0 – no further cytokines or antibodies; Th17 -TGF-β [5 ng/ml], IL-6 [20 ng/ml], IL-23 [10 ng/ml], neutralizing anti-IL-4 [2 µg/ml] and anti-IFN-γ [2 µg/ml] antibodies; Th1 - IL-12 [10 ng/ml], neutralizing anti-IL-4 antibody [5 µg/ml] (eBioscience, San Diego, California, USA). Where indicated, concentration of secreted cytokines in culture supernatants was measured by Bioplex System (Bio-Rad, Hercules, California, USA), according to the manufacturer's instructions.

CD4⁺ or CD8⁺ T Cells were sort-purified and labeled with CellTrace Violet (CTV) according to the manufacturer’s protocol (Invitrogen). 0.1 million CTV labeled T cells were cultured with plate-coated anti-CD3 (2.5 μg/ml; BD Biosciences) and soluble anti-CD28 (2 μg/ml; BD Biosciences) antibodies, in certain groups of cells were cultured in the present of Dleu2-17aa (10 μM) or PBS control the phenotype of proliferation was determined by flow cytometry.

Naive splenic CD4⁺ T-cells were isolated using anti-CD4-conjugated magnetic beads (Miltenyi Biotec, San Diego CA, USA) with an autoMACS cell separator. CD4⁺ splenic T-cells were differentiated under T_H1 polarizing conditions. Briefly, 2.0–2.5 × 10⁶/ml cells were activated with 1.5 µg/ml plate-coated anti-CD3 (BD Pharmingen) and 1.5 µg/ml soluble anti-CD28 antibodies (BD Pharmingen) in addition to 10 µg/ml anti-IL-4 blocking antibody, 50 U/ml IL-2 and 20 ng/ml IL-12. Cells were cultured for 3–5 days, then harvested and washed for intracellular staining of IFN-γ.