Anti il 4 neutralizing antibodies

For Th1 polarization, purified CD4⁺ Th0 cells were cultured in expansion media supplemented with IL-12 (10 ng/mL) (Peprotech) and anti-IL-4 neutralizing antibodies (5 μg/mL) (BD Bioscience, San Jose, CA, USA) for 5 days. For Th2 induction, purified CD4⁺ Th0 cells were cultured in expansion media with IL-4 (20 ng/mL) (Peprotech) and anti-IFN-γ neutralizing antibodies (5 μg/mL) (BioxCell, West Lebanon, NH, USA) for 5 days. To evaluate the spontaneous induction ratio of Treg cells, purified CD4⁺ Th0 cells were cultured in expansion media for 5 days without any lineage-specific stimulants. To assess the Ech A impact on Th cell polarization, Ech A or vehicle was also treated during the culture period.

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.

Purified CD4⁺ T cells were co-cultured with hUCB-MSCs or exosome from hUCB-MSCs. Cells were plated at a 1:10 ratio (CD4⁺ T cells: hUCB-MSCs or CD4⁺ T cells: Exosome from hUCB-MSCs) in 24-well culture plates. Th1 cells were polarized in ImmunoCult -XF T Cell Expansion Medium supplemented with ImmunoCult Human CD3/CD28/CD2 T Cell Activator, 10 ng/mL recombinant human interleukin-12 (IL-12; Peprotech), and 5 μg/mL anti-IL-4 neutralizing antibodies (BD Bioscience) for 5 days. Th2 cells were polarized in ImmunoCult-XF T Cell Expansion Medium supplemented with ImmunoCult Human CD3/CD28/CD2 T Cell Activator, 20 ng/mL recombinant human interleukin-4 (IL-4; Peprotech), and 5 μg/mL anti-IFNγ neutralizing antibodies (BioxCell, Lebanon, NH, USA) for 5 days. Th1 and Th2 were treated with 20 ng/mL IL-2 on day 3 of polarization. Treg cells were polarized in RPMI 1640 supplemented with 10% FBS, 2 mM glutamax (Gibco), 50 μM β-mercaptoethanol, 2 ng/mL transforming growth factor beta 1 (TGF-β1; Peprotech), and 10 ng/mL IL-2 for 5 days.