Tim-1-Fc suppresses chronic cardiac allograft rejection and vasculopathy by reducing IL-17 production

Abstract

Previously, we demonstrated that Tim-1-Fc prevents acute cardiac graft rejection by inhibiting Th1 response. In the present report, we tackled the impact of Tim-1-Fc on Th17 cells in a model of cardiac chronic rejection. Administration of Tim-1-Fc did not result in a detectable impact on innate immunity and regulatory T cells, while it provided protection for Bm12-derive cardiac grafts against chronic rejection in B6 recipients, as manifested by the reduction of inflammatory infiltration along with less severity of vasculopathy. Studies in T-bet(-/-) recipients by implanting Bm12-derived cardiac grafts further revealed that Tim-1-Fc significantly protected cardiac grafts from chronic rejection along with attenuated production of IL-17 producing T cells. Depletion of CD4 and CD8 T cells or blockade of IL-17 in T-bet(-/-) recipients demonstrated that Tim-1-Fc selectively suppresses Th17 differentiation along with attenuated IL-17 secretion. Together, our data suggest that Tim-1-Fc protects cardiac grafts from chronic rejection by suppressing CD4 Th17 development and functionality. Therefore, Tim-1-Fc might be a potential immunosuppressive agent in the setting of cardiac transplantation.

Keywords: Th17; Tim-1; vasculopathy.

Figure 1

Tim-1-Fc attenuates chronic cardiac rejection in MHC II mismatched cardiac grafts. A: Survival rate of Bm12-derived cardiac grafts in B6 recipients treated with either Tim-1-Fc or control IgG. Loss of graft function was defined as cessation of a palpable impulse. B: Hematoxylin and eosin (H&E) staining of cardiac graft sections harvested after day 35 of transplantation. C: Scores for the severity of vasculopathy in cardiac grafts after day 35 of transplantation. D: Intragraft expression of IL-2, IL4, IFN-γ, IL-17 and IL-6. The relative expression levels of cytokines within the grafts were assessed by real-time PCR. E: Administration of recombinant IL-17 abolished the protective effect conferred by Tim-1-Fc. Recombinant IL-17 was administrated along with Tim-1-Fc or control IgG after transplantation every other day until day 15. Histological data and real-time PCR data were obtained from studies of 3 mice.

Figure 2

Tim-1-Fc protects Bm12-derived cardiac grafts from rejection in T-bet deficient recipients. A: Survival rate of Bm12-derived cardiac grafts in T-bet^-/- recipients after treating with Tim-1-Fc or control IgG (n=5 for each study group). B: Results for H&E staining and vasculopathy scores of cardiac grafts after day 14 of transplantation. C: Relative expression levels for IL-2, IL-4, IFN-γ, IL-17, CD11b and CD3 in the grafts after 14 days of transplantation. D: Serum levels for cytokines IL4, IFN-γ and IL-17 in the recipient mice. All data are presented as means ± SD, and 3 replications were included for each assay.

Figure 3

Tim-1-Fc inhibits the number of effector T cells. A: Flow cytometry analysis of lymphocytes in the periphery blood of T-bet^-/- recipients. Tim-1-Fc treatment significantly reduced the number of CD44^hiCD62^low effecter T cells. B: Flow cytometry data for regulatory T cells. Addition of Tim-1-Fc did not affect the number of regulatory T cells. All experiments were conducted with 3 replications. *, P<0.01.

Figure 4

The impact of Tim-1-Fc administration on DC number and maturation. A: Tim-1-Fc treatment did not result in a significant change for the number of splenic DCs and MHC II expressions. B: Flow cytometry data for CD80 and CD86 expressions. Data are a representative of 3 independent experiments conducted.

Figure 5

Tim-1-Fc suppresses Th17 cell differentiation. Bm12-derived cardiac grafts were implanted into T-bet^-/- recipients, and Tim-1-Fc or control IgG were administered as described earlier. The recipient mice were sacrificed 14 day after transplantation, and splenic T cells were prepared for flow cytometry analysis of Th17 production. A: Results for IL-17 producing Th17 cells. Splenic T cells were stained for CD4 and then co-stained for IL-17, IL-4 and IFN-γ, respectively. A significant reduction for CD4 Th17 cells was noted in Tim-1-Fc treated recipient mice. B: Results for flow cytometry analysis of IL-17 producing CD8 T cells. The above prepared splenic T cells were first stained for CD8 and then co-stained for IL-17. Staining of CD4 Th17 cells was used as a control. No detectable change was noted for IL-17 producing CD8 T cells. C: Tim-1-Fc dose-dependently suppressed the production of CD4 Th17 cells. CD4 naive T cells were cultured under Th17 condition in the presence of different doses of Tim-1-Fc or control IgG for 3 days, and then subjected to flow cytometry analysis of Th17 production. D: Tim-1-Fc attenuated DC induced Th17 differentiation. CD4 naive T cells were induced for Th17 differentiation with DCs in the presence of Tim-1-Fc or control IgG as described. All data are shown as means ± SD of 3 independent experiments conducted.

Figure 6

Tim-1-Fc confers protection against cardiac rejection relying on its effect on CD4 Th17 cells. A: Depletion of CD4 T cells diminished the protection conferred by Tim-1-Fc treatment. T-bet^-/- recipients were depleted for CD4 T cells and then implanted with Bm12-derived cardiac grafts along with administration of Tim-1-Fc or control IgG as described (n=5 for each study group). B: Administration of Tim-1-Fc provided protection for cardiac grafts against rejection in CD8 depleted T-bet^-/- recipients (n=5 for each study group). C: Results for intragraft cytokine expressions by real-time PCR. Data presented here are means ± SD of 3 independent experiments. D: Neutralization of IL-17 completely abolished the protection conferred by Tim-1-Fc (n=5 for each study group). IL-17 was neutralized with a mAb in T-bet^-/- recipients after receiving Bm12-derived cardiac grafts along with treatment of Tim-1-Fc or control IgG as described.