Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples of SLE patients and healthy controls by Ficoll density gradient centrifugation (STEMCELL Technologies, Vancouver, CA). Human CD4+ or naïve CD4+ T cells were purified from fresh PBMCs by using the EasySep Human CD4+ or Naïve CD4+ T Cell Isolation kits (STEMCELL Technologies), stimulated with anti-CD3/CD28 beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, CD3/CD28 beads-stimulated naïve CD4+ T cells were treated with tofacitinib, STAT3 inhibitor (NSC74859), AKT inhibitor (GSK690693), or p38 MAPK inhibitor (SB203580) for 24 h. NSC74859, GSK690693, and SB203580 were purchased from ApexBio (Houston, TX, USA). Tofacitinib was added at the determined concentrations (0.1 µM, 1 µM, and 10 µM) for 24 h. In some experiments, CD4+ T cells were stimulated with anti-CD3/CD28 beads in the presence of recombinant cytokines, such as TGF-β (5 ng/ml; PeproTech, Rocky Hill, NJ) and IL-6 (100 ng/ml; PeproTech).
Spleens were isolated, and the erythrocytes in splenocyte suspensions were lysed with red cell lysate. Mouse CD4+ T cells were purified from splenocytes by using the EasySep Mouse CD4+ T Cell Isolation kit (STEMCELL Technologies) and processed for real-time PCR or Western blotting analysis.
Spleens were isolated, and the erythrocytes in splenocyte suspensions were lysed with red cell lysate. Mouse CD4+ T cells were purified from splenocytes by using the EasySep Mouse CD4+ T Cell Isolation kit (STEMCELL Technologies) and processed for real-time PCR or Western blotting analysis.