Figure 2.

Administration of Tregs dramatically reduces tPA-induced intracerebral haemorrhage in a murine suture model of stroke. (A) Scheme for experimental design in a suture model of MCAO. tPA (10 mg/kg) was continuously infused into the femoral vein over 20 min at 2 h after MCAO. Tregs were isolated from pooled spleens and lymph nodes of donors and transferred (2 × 10⁶ cells/animal) to ischaemic recipients immediately after tPA delivery through the femoral vein. The timeline for outcome measurements is also illustrated. (B) The percentages of Tregs in CD3⁺CD4⁺ T cells in the blood collected from sham, MCAO, and MCAO+tPA groups. (C) Representative flow cytometry plots of CD25⁺Foxp3⁺ cells in CD4⁺CD25⁺ Tregs and CD4⁺CD25⁻ T effective cells (Teff) after double selection. (D) T cell suppression test. Graded numbers of Tregs were added to the Teff cells in the presence of anti-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml) stimulation. The proliferation of Teff was measured 24 h later by BrdU incorporation test. (E and F) Regional cerebral blood flow (CBF) was monitored using 2D laser speckle imaging. (E) Representative images of cerebral blood flow before MCAO, during MCAO, and at 10 min after reperfusion for each group. (F) Quantification of cerebral blood flow. Results are expressed as percent change from baseline (pre-MCAO). n = 4–5 per group. (G) Representative images of the dorsal (top) and ventral (middle) surfaces of the brain and a coronal section (bottom) showing the location of the intracerebral haemorrhage 1 day after stroke in mice treated with PBS, PBS+Treg, tPA+PBS, or tPA+Treg. (H) Quantification of cerebral haemorrhage after different doses of Treg treatment by spectrophotometric haemoglobin assay 1 day after stroke. (I) Exogenous Tregs reduced tPA-induced brain haemorrhage in the absence of endogenous Tregs. Mice were injected intraperitoneally with either PBS (control) or 300 mg of CD25-specific antibody (CD25 Ab) 2 days prior to MCAO. tPA (10 mg/kg) was given at 2 h after MCAO. Brain haemorrhage was measured 1 day after MCAO. (n = 4–6/group). (J) Tregs did not directly inhibit tPA activity. 20 µg tPA was incubated with or without 1.6 × 10⁵ Tregs in 160 µl media for 30 min or 1 h. The activity of tPA was determined by zymography (top) and tPA chromogenic activity assay (bottom). Data are mean ± SE. ^*P ≤ 0.05; ^**P ≤ 0.01.