Immunoregulatory function of IL-27 and TGF-β1 in cardiac allograft transplantation

Abstract

Background: Deciphering the mechanisms of tolerance represents a crucial aim of research in transplantation. We previously identified by DNA chip interleukin (IL)-27 p28 and transforming growth factor (TGF)-β1 as overexpressed in a model of rat cardiac allograft tolerance mediated by regulatory CD4CD25 T cells. The role of these two molecules on the control of the inflammatory response remains controversial. However, both are involved in the regulation of the T helper 17/Treg axis, suggesting their involvement in tolerance.

Methods: We analyzed regulation of IL-27 and TGF-β1 expression in allograft response and their role in tolerance by using blocking anti-TGF-β antibody and by generating an adeno-associated virus encoding IL-27.

Results: Here, we confirmed the overexpression of IL-27 and TGF-β1 in tolerated cardiac allografts in two different rodent models. We observed that their expression correlates with inhibition of T helper 17 differentiation and with expansion of regulatory CD4CD25 T cells. We showed in a rat model that anti-TGF-β treatment abrogates infectious tolerance mediated by the transfer of regulatory CD4CD25 T cells. Moreover, overexpression of IL-27 by adeno-associated virus administration in combination with a short-term immunosuppression allows prolongation of cardiac allograft survival and one tolerant recipient. We found that IL-27 overexpression did not induce Foxp3CD4CD25 T-cell expansion but rather IL-10-expressing CD4 T cells in the tolerant recipient.

Conclusions: Taken together, these data suggest that both TGF-β1 and IL-27 play a role in the mechanisms of tolerance. However, in contrast to TGF-β1, IL-27 seems not to be involved in regulatory CD4CD25 T-cell expansion but rather in their mode of action.

Fig 1. Expression of IL-27 p28, EBI3, IL-12 p35, TGFβ1, IL-17 and IL-6 in rat cardiac allograft model

(A): mRNA expression of a) IL-27 p28, b) EBI3, c) IL-12 p35, d) TGFβ1, e) IL-17 and f) IL-6 was analyzed by quantitative RT-PCR in cardiac syngeneic grafts (Syng), acutely rejected allografts (ARej), chronically rejected allografts (CRej) or tolerated allografts (Tol) harvested at day 5 (d5) or 100 (d100) after transplantation (n=4). Results are expressed in AU of the gene/HPRT transcript ratio ± SEM and expressed as relative expression compared to the reference syngeneic grafts at day 5 (value=1), * p<0.05, ** p<0.01, ***p<0.001. (B): Representative pictures of immuno-fluorescence merged staining by histology for DAPI (blue) and for: IL-27 p28 (green) or EBI3 (green) and CD172a (red) in respectively graft and spleen a), b) and c) d). IL-27 p28 (green) or EBI3 (green) and TCR (red) in respectively graft and spleen e), f) and g) h). EBI3 (green) and IL-27 p28 (red) in graft i). TGFβ (LAP complex) (green) and TCR (red) in respectively graft and spleen j) and h). Original magnification: ×600. Data are representative of three independent experiments on different rat tolerant recipients. (C): Expression of IL-27 p28 protein was assessed by histology by counting the number of positive cells per field in chronically rejected (CRej) and tolerated allografts (Tol). Results are expressed in number of cells per field ± SEM, n=4,* p<0.05.

Fig 2. mRNA expression of EBI3, IL-27 p28, IL-12 p35 and TGFβ1 in rat CD4⁺CD25⁺ T cells

mRNA expression of (A) EBI3, (B) IL-27 p28, (C) IL-12 p35 and (D) TGFβ1 was analyzed by quantitative RT-PCR in purified CD4⁺CD25⁺, CD4⁺CD25⁻ and CD8⁺ T cells from naive rats (Naive) or from long-term tolerant recipients (Tol). Results are expressed in AU of the gene/HPRT transcript ratio ± SEM, n=4 * p<0.05.

Fig 3. mRNA expression of IL-27 p28, TGFβ1, EBI3, CD3 and Foxp3 in cardiac allografts in mice

mRNA expression of (A) IL-27 p28, (B) TGFβ1, (C) EBI3, (D) CD3 and (E) Foxp3 were analyzed by quantitative RT-PCR in cardiac untreated rejected allografts (Rejected), treated tolerant allografts (Tolerant) and allografts from RAG ⁻/⁻ recipients harvested at day 2, 5, 7, 8 or 10 after transplantation (n≥2). Results are expressed in mRNA Units/HPRT ratio ± SEM * p<0.05, ** p<0.01.

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).