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. Author manuscript; available in PMC: 2015 Mar 1.
Published in final edited form as: Stroke. 2014 Feb 4;45(3):857–864. doi: 10.1161/STROKEAHA.113.004100

Figure 1. Tregs inhibit neutrophil-derived MMP-9 through PD-L1 in in vitro cultures.

Figure 1

(A) Isolated Tregs were stimulated with anti-CD3Ab and anti-CD28 Abs for 3 days and pretreated with various neutralizing Abs (anti-PD-L1, -TGF-β or -CTLA-4; 20 μg/mL) or isotype control IgG (20 μg/mL) for 1 h. Stimulated Tregs were co-cultured with neutrophils and then exposed to TNF-α (100 ng/mL) for 24 h. MMP-9 in the culture medium was measured by ELISA. (T) indicates that neutrophils were cultured in transwells. (B) Neutrophils were incubated with PD-L1 protein at indicated concentrations. Cells were then exposed to TNF-α (100 ng/mL) for 24 h. MMP-9 in the culture medium was measured by ELISA. n=6/group in 3 independent experiments. *P<0.05, **P<0.01. (C) Immunocytochemical staining of PD-L1 on Tregs. Red: PD-L1. Blue: DAPI.