Fig 4. Role of TGFβ and IL-27 in rat cardiac allograft tolerance.

A) Recipients that have been transferred with 20×10⁶ of spleen CD4⁺T cells from long-term tolerant recipients (Transf.) were treated with neutralizing mouse anti-rat TGFβ mAb (clone 2G7, IgG2b) injected i.p (5 mg/kg) twice a week beginning the day of transplantation and until rejection (Transf.+2G7) n≥4, **p<0.01.

B) Representative pictures of immuno-fluorescence merged staining for IL-27 p28 (red), GFP (green) and DAPI (blue) of untransfected (UT) or GFP or IL-27 AAV transfected HEK 293 cells as described in Material and Methods. Original magnification: ×600.

C) IL-27 level was assessed by ELISA in the sera from GFP or IL-27 AAV transfected allograft recipients harvested at days −14, −4, 0, 17, 40, and 300 before or after transplantation. Results are expressed in fold change compared to the IL-27 basal level assessed from each recipient.

D) Rat cardiac allograft recipients were treated with GFP or IL-27 AAV (4.5×10¹¹ vg) injected i.v. at recipients 3 weeks before transplantation. Alternatively, sub-optimal dose of rapamycin (Rapa) was given orally from day 0 to day 10 (0.4 mg/kg), n≥4, **p<0.01.

E) Assessment of anti-donor-specific IgG antibodies in the sera of IL-27 AAV tolerant recipient or GFP AAV treated recipient and harvested at days 0, 17, 25, 71 and 300 after transplantation. Data are expressed in Geometric Mean Fluorescence.

F) T cells from GFP or tolerant IL-27 AAV treated recipients (day 300 after transplantation) were stimulated by allogeneic (LEW.1W) APC for four days (MLR) and T cell proliferation was assessed in triplicates by ³H incorporation. Data are expressed in cpm ± SEM.

G) Ratio of IL-10/INFγ expression assessed by ELISA in the supernatant of MLR from stimulated CD4⁺ T cells from GFP or tolerant IL-27 AAV treated recipients. Data are expressed in arbitrary unit (AU).