Figure 5. Effect of rshCD5 on in vitro T_reg and T_H1 polarization.

Sorted naïve T cells CD4⁺CD25^-CD62L^hiCD44^lo cells (1 × 10⁵) from C57BL/6 mice were activated for 96 h in triplicate with plate-bound α-CD3 (2 μg/mL) and soluble α-CD28 mAb (0.5 μg/mL) under (A) T_reg polarization (α-IL-4 mAb, 1 μg/mL; TGF-β, 2 ng/mL; IL-2, 5 ng/mL; and of α-IFN-γ, 1 μg/mL) or (B) T_H1 polarization (α-IL-4 mAb, 10 μg/mL; IL-2, 5 ng/mL; and IL-12, 10 ng/mL) conditions in the presence of different amounts of rshCD5 (0-10 μg/mL). Then cells were (A) stained for surface CD4, CD25 and intracellular FoxP3 expression or (B) re-stimulated for 5 h with PMA (80 nM) and Ionomycin (1 μg/mL) in the presence of 2 μM Monensin followed by surface CD4 and intracellular IFN-γ staining, for further flow cytometry analyses. Data represent the mean percentage of double-positive cells (mean ± SD) from three experiments (A) or one representative experiment of two (B) performed. ^*, p<0.05; ^**, p<0.01 (Unpaired t-test).