Anti cd3 anti cd28 mab coated microbeads

MMC were stimulated in vitro with medium as a control, with anti-CD3/anti-CD28 mAb-coated microbeads in a 1:10 bead-to-cell ratio (Invitrogen, Bleiswijk, The Netherlands) alone, rhIL-15 (100 ng/ml; Gibco Life Technologies) alone, and beads together with rhIL-15 in 96-well U-bottom plates. The rationale for using IL-15 is because IL-2 is hardly detected in decidua and after implantation, endometrial NK cells start to differentiate as a result of local IL-15 production^{9 (link)}. After 5 days of culture at 37 °C in a humidified 5% CO₂ incubator, cells were harvested and the presence of T cell subsets and differentiation was measured with flow cytometry.

Treg were stimulated with rhIL-2 (25 U/mL) alone as control, or together with either soluble CD28 agonist mAb (1 μg/mL, Clone L293, Cat# 348040; BD Bioscience), plate-bound CD3 mAb (5 μg/mL, Clone UCHT1, BD Bioscience), plate-bound CD3 (5 μg/mL) plus soluble CD28 mAb (1μg/mL), CD28-superagonist ANC28.1 (1 μg/mL, Clone ANC28.1/5D10, Cat# 177–820, preservative free; Ancell, Bayport, USA), or anti-CD3/anti-CD28 mAb-coated microbeads (Invitrogen by Life technologies, Bleiswijk, The Netherlands) used in a 1:2 bead-to-cell ratio. Titration of soluble CD28-agonist and CD28-superagonist antibodies at a concentration range of 1–10 μg/mL led to similar FOXP3 expression levels as determined by flow cytometry. Cells were cultured in 96-well round bottom plates in RPMI 1640 (Invitrogen) as described previously44 (link). In selected experiments immunomodulating agents were analyzed; Treg were pretreated with wortmannin (5 μM), rapamycin (200 nM) (Sigma-Aldrich, St. Louis, USA)15 (link), or vehicle control for 30 min at 37 °C. Thereafter, stimulators as indicated were added to the culture mixture.