Ciclosporin a

Splenic T cells of WT mice were cultured to examine the major stimuli and their signaling pathways to induce LSP1 expression. Briefly, T cells were isolated from the spleens and prepared as single-cell suspensions. CD4⁺ T cells or CD8⁺ T cells were purified by magnetic separation using anti-CD4 beads or anti-CD8 beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Purified CD4⁺ T cells or CD8⁺ T cells were stimulated with recombinant IFN-γ (10 ng/mL, R&D Systems), transforming growth factor-β (TGF-β, 2 ng/mL, R&D Systems), IL-10 (10 ng/mL, R&D Systems) or antimouse CD3ε Ab (1 µg/mL, 145-2 C11, Invitrogen) plus antimouse CD28 Ab (1 µg/mL, 37.51, Invitrogen) in complete media for 72 hours. In some experiments, ciclosporin A (Sigma), tacrolimus (FK506, Sigma) and rapamycin (Sigma) were treated to the T cells stimulated with anti-CD3/anti-CD28 Abs for 72 hours to determine whether the calcineurin pathway is involved in LSP1 expression. The cultured cells were harvested and stained to detect intracellular LSP1 expression by flow cytometry and/or western blot analysis.

OE-19 cell line was purchased from ECACC, tested for Mycoplasma once a month and cultured in complete RPMI 1640 medium supplemented with 10% fetal calf serum and 100 μg/ml streptomycin. Counting was performed using Cellometer Auto T4 (Nexcelom Bioscience LLC). Cells were maintained at 37°C and 5% CO2 at all times. OE-19 cell line was exposed to increasing concentrations of T-DM1 for 6 months in the absence or presence of 1 μg/ml ciclosporin A (C3662; Sigma-Aldrich) to obtain resistance models.