Anti il 4

Human peripheral blood CD4⁺ T cells (Lonza, Walkersville, MD) were cultured with X-VIVO medium (Lonza) containing 2% FBS. Th1 cells were differentiated with anti-CD3-coated plates (5 μg/ml, BD Biosciences), anti-CD28 antibodies (2 μg/ml, BD Biosciences) plus IL-12 (10 ng/ml, R&D Systems, United States) and anti-IL-4 neutralizing antibodies (2.5 μg/ml, BD Biosciences) for 6 days. Th17 cells were differentiated with anti-CD3-coated plates (5 μg/ml) plus anti-CD28 (2 μg/ml), in the presence of IL-6 (50 ng/ml, R&D Systems), TGF-β1 (5 ng/ml, BioVision, United States) and anti-IFN-γ (2.5 μg/ml, BD Biosciences) and anti-IL-4 (2.5 μg/ml, BD Biosciences) neutralizing antibodies for 6 days. Treg cells were differentiated with anti-CD3-coated plates (5 μg/ml) and anti-CD28 (2 μg/ml), in presence of TGF-β1 (10 ng/ml, BioVision, United States) and anti-IFN-γ (2.5 μg/ml, BD Biosciences) and anti-IL-4 (2.5 μg/ml, BD Biosciences) neutralizing antibodies for 6 days. The effect of hHF-MSCs on Th1, Th17 and Treg differentiation was tested by co-cultivating cells at a ratio of 10:1 (T cells/hHF-MSCs) after 3 days of Th1, Th17 or Treg polarizing conditions (day 3). After 3-day co-culture of T cells and hHF-MSCs, flow cytometry was performed in order to detect IFN-γ+CD4⁺ Th1 cells, IL-17 + CD4⁺ Th17 cells, and CD4⁺CD25 + Foxp3+ Treg cells.

Naïve CD4⁺ T cells (5 × 10⁵ cells per well) were grown in a 96-well plate containing T cell expansion medium (Thermo-Fisher) at 37 °C and 5% CO₂, and then transfected with miRNA-374b-5p inhibitor (200 nmol/mL; Qiagen) and miRNA-374b-5p mimic (50 nmol/mL; Qiagen) using the Hiperfect Transfection Reagent (Qiagen). MiScript NC siRNA (Qiagen) was employed as a control. After 4 h, T cell differentiation was performed in using 48-well plates different cytokine regimens according to a previous method [23 (link)]. For Th1, the transfected cells were incubated with complete RPMI, plate-bound 1 μg/mL anti-CD3 and 1 μg/mL soluble CD28 antibodies, 20 ng/mL IL-2, 10 ng/mL anti-IL-4 and 50 ng/mL IL-12 antibodies (BD Biosciences) for 96 h. For Th2, the cells were exposed to 1 μg/mL plate-bound anti-CD3 and 1 μg/mL anti-CD28 antibodies, 10 ng/mL IL-4, 20 ng/mL IL-2 and 10 ng/mL anti-IFN-γ antibodies (BD Biosciences) for 96 h. For Th17 cell differentiation, the transfected cells were incubated with complete RPMI, 1 μg/mL plate-bound anti-CD3 and 0.2 μg/mL soluble CD28 antibodies, 5 ng/mL TGF-β, 100 ng/mL IL-6, 50 ng/mL IL-23, 10 ng/mL anti-IL-4, and 10 ng/mL anti-IFN-γ antibodies (BD Biosciences) for 96 h.

CD4⁺ T cells were freshly isolated from spleen of C57BL/6 mice by negative selection with Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen, Thermo Fisher, USA) according to manufacturer’s instructions. Once purified, they were labeled with CellTrace Violet (CTV) (Life-Technology, Thermo Fisher, USA) and activated with CD3/CD28 beads (Invitrogen, Thermo Fisher, USA). Lymphocytes were cultured in mixed lymphocyte reaction (MLR) media, containing 10% Fetal Bovine Serum, 1% Pen/Strep, 1% sodium pyruvate, 1% non-essential amino acids, 1% glutamine and 25 µM β-mercaptoethanol (Gibco, Thermo Fisher, USA), in Iscove's Modified Dulbecco's Media (IMDM) (Gibco, Thermo Fisher, USA). To differentiate towards Th1 subtype, purified CD4⁺ T cells were stimulated with 10 ng/ml of IL12 (R&D Systems, USA) and 2.5 μg/ml of anti-IL4 (BD Pharmingen, BD Biosciences, USA). Similarly, Th17 phenotype was induced with 50 ng/ml of IL6 (R&D Systems, USA), 2.5 ng/ml of TGBβ1 (R&D Systems, USA), 2.5 μg/ml of anti-IFNγ (BD Pharmingen, USA) and 2.5 μg/ml of anti-IL4 (BD Pharmingen, USA).
To assess immunosuppressive properties of murine MSCs, CD4⁺ T cells were cultured alone or in the presence of MSCs (control vs pretreated) at a cell ratio of 1 MSC per 10 lymphocytes in MLR media. After 72 h, proliferation and CD4⁺ T cell differentiation was quantified by flow cytometry.

Naive CD4⁺T cells from normal BALB/c mice were purified using CD4⁺ naive T cell isolation kit (STEMCELL Technologies) according to the manufacturer’s instruction. Purified cells were activated by plate-coating anti-CD3 (10 μg/ml; BD Pharmingen) plus anti-CD28 (2 μg/ml; BD Pharmingen) for 5 days under the following polarizing conditions: TGF-β (3 ng/ml, Peprotech), IL-6 (30 ng/ml; eBioscience), IL-23 (20 ng/ml; R&D), anti-IFN-γ (10 μg/ml, BD Pharmingen), anti-IL-4 (10 μg/ml, BD Pharmingen) for Th17 polarization, and TGF-β1 (5 ng/ml, Peprotech), anti-IFN-γ (10 μg/ml, BD Pharmingen), anti-IL-4 (10 μg/ml, BD Pharmingen) for Treg polarization. VPA or vehicle solution was added on day 0.

Naïve CD4⁺ T cells were purified from mouse spleen and MLNs using Naive CD4⁺ T Cell Isolation Kit (Miltenyi Biotec). Purified naïve CD4⁺ T cells were plated at a density of 2.5 × 10⁵ cells/well at 37 °C in 48-well plates pre-coated with anti-CD3e (Clone: 145-2C11, 1 μg/ml, BD Biosciences) and anti-CD28 (Clone: 37.51, 1 μg/ml, BD Biosciences) antibodies. Activated cells were polarized under Th0 (no supplement), Th1 [IL-12 (5 ng/ml, R&D Systems), IL-2 (10 ng/ml, R&D Systems) and anti-IL-4 antibody (5 μg/ml, BD Biosciences)], Treg [TGF-β (5 ng/ml, R&D Systems), IL-2 (10 ng/ml, R&D Systems), anti-IFN-γ (5 μg/ml, BD Biosciences), anti-IL-12 p40/p70 (5 μg/ml, BD Biosciences) and anti-IL-4 (5 μg/ml, BD Biosciences) antibodies] differentiation conditions in complete RPMI medium or polarized in splenic DC-conditioned medium (overnight culture supernatant of CpG-ODN-stimulated splenic DCs).