A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy

Xueli Yuan, Jesus Paez-Cortez, Isabela Schmitt-Knosalla, Francesca D'Addio, Bechara Mfarrej, Michela Donnarumma, Antje Habicht, Michael R Clarkson, John Iacomini, Laurie H Glimcher, Mohamed H Sayegh, M Javeed Ansari, Xueli Yuan, Jesus Paez-Cortez, Isabela Schmitt-Knosalla, Francesca D'Addio, Bechara Mfarrej, Michela Donnarumma, Antje Habicht, Michael R Clarkson, John Iacomini, Laurie H Glimcher, Mohamed H Sayegh, M Javeed Ansari

Abstract

T-bet plays a crucial role in Th1 development. We investigated the role of T-bet in the development of allograft rejection in an established MHC class II-mismatched (bm12 into B6) model of chronic allograft vasculopathy (CAV). Intriguingly, and in contrast to IFN-gamma(-/-) mice that are protected from CAV, T-bet(-/-) recipients develop markedly accelerated allograft rejection accompanied by early severe vascular inflammation and vasculopathy, and infiltration by predominantly IL-17-producing CD4 T cells. Concurrently, T-bet(-/-) mice exhibit a T helper type 1 (Th1)-deficient environment characterized by profound IFN-gamma deficiency, a Th2 switch characterized by increased production of interleukin (IL) 4, IL-5, IL-10, and IL-13 cytokines, as well as increased production of the proinflammatory cytokines IL-6, IL-12p40, and IL-17. Neutralization of IL-17 inhibits accelerated allograft rejection and vasculopathy in T-bet(-/-) mice. Interestingly, CD4 but not CD8 T cell deficiency in T-bet(-/-) mice affords dramatic protection from vasculopathy and facilitates long-term graft acceptance. This is the first study establishing that in the absence of Th1-mediated alloimmune responses, CD4 Th17 cells mediate an aggressive proinflammatory response culminating in severe accelerated allograft rejection and vasculopathy. These results have important implications for the development of novel therapies to target this intractable problem in clinical solid organ transplantation.

Figures

Figure 1.
Figure 1.
T-bet deficiency accelerates cardiac allograft rejection characterized by severe vasculopathic changes and PMN infiltration. (A) MHC class II–mismatched cardiac allograft survival (bm12 into C57BL/6) in WT, T-bet−/−, IFN-γ−/−, and T-bet/Ig DKO recipients (n = 8 in WT, and n = 6 in T-bet−/−, IFN-γ−/−, and T-bet/Ig DKO groups). Survival data are presented as a Kaplan-Meier plot. (B) Pathology (top, H&E staining; bottom, elastin staining) of bm12 cardiac allografts from WT, T-bet−/−, and IFN-γ−/− recipients at 2 and 8 wk after transplantation. Note the severe vascular inflammation in the T-bet−/− recipient and the normal vascular integrity in the IFN-γ−/− recipient in contrast to that of the WT recipient even at 8 wk after engraftment. Bars, 20 μm. (C) Pathology (H&E staining) of bm12 cardiac allografts from WT, T-bet−/−, and IFN-γ−/− recipients at 2 wk after transplantation (top, low magnification; bottom, high magnification) showing severe PMN infiltration (both neutrophils and eosinophils) in the T-bet−/− recipients and minimal PMN (predominantly eosinophilic) infiltration in IFN-γ−/− recipients, in contrast to modest mononuclear infiltration in WT recipients. Insets show infiltrating cell morphology. Histology results presented are from one experiment and are representative of three independent experiments. Bars, 20 μm.
Figure 2.
Figure 2.
IL-17–expressing graft-infiltrating lymphocytes in T-bet−/− recipients. Immunofluorescence staining and confocal microscopy of heart allografts harvested 2 wk after transplantation for IL-17 expression by CD4 and CD8 T cells. IL-17 expression by most of the CD4 (top) and some of the CD8 (bottom) graft-infiltrating T cells in the T-bet−/− recipients is seen in contrast to WT recipients (not depicted), where only a few of the infiltrating T cells express IL-17 and no IL-17–expressing cells were seen in grafts from IFN-γ−/− recipients (not depicted). Results presented are from one experiment and are representative of three independent experiments. Bars, 20 μm.
Figure 3.
Figure 3.
T-bet deficiency promotes CD4 and CD8 T cell effector/memory differentiation and proliferation in MHC class II–mismatched cardiac allograft recipients. (A) FACS analysis of lymphocytes in the peripheral blood of WT, IFN-γ−/−, and T-bet−/− recipients bearing a CD44hiCD62Llow effector/memory phenotype gated on either CD4 (top) or CD8 (bottom) T cell populations. Percentages of cells are shown. (B) CD25+/Foxp3+ regulatory CD4 T cells in the MHC class II–mismatched cardiac allograft recipients. Gates were set on the CD4+ population, and percentages of cells are shown. Bar graphs show means ± SD. All results are representative of at least three different sets of experiments.
Figure 4.
Figure 4.
Up-regulation of Th2 and Th17 proinflammatory cytokine production by T-bet−/− allograft recipients of bm12 hearts. Th1, Th2, and Th17 proinflammatory cytokine production by the splenocytes of WT, IFN-γ−/−, and T-bet−/− recipients of bm12 heart grafts assessed by the Luminex assay. IL-12 data are presented separately because of the need to use a different scale. Results presented are means ± SD and are representative of at least three independent experiments.
Figure 5.
Figure 5.
T-bet−/− CD4 T cells produce proinflammatory cytokines and mediate accelerated cardiac allograft rejection in the MHC class II–mismatched cardiac allograft model of CAV. (A) Survival of allogeneic bm12 cardiac graft in CD4- or CD8-depleted (left) and CD4/T-bet or CD8/T-bet DKO (right) recipients (n = 4 in each group). Survival data are presented as Kaplan-Meier plots. (B) Pathology (elastin staining) of bm12 cardiac allografts from CD4- or CD8-depleted T-bet−/− recipients at 8 and 3 wk, respectively, after transplantation. Note the significant vasculopathy in the CD8-depleted T-bet−/− recipient and, in contrast, the relatively normal vascular integrity in the CD4-depleted T-bet−/− recipient even at 8 wk after engraftment. Bars, 20 μm. (C) IL-6 and IL-17 proinflammatory cytokine production by splenocytes of T-bet−/−, CD4/T-bet DKO, and CD8/T-bet DKO recipients of bm12 heart grafts assessed by the Luminex assay. Bar graphs show means ± SD. Results presented are from one experiment and are representative of three independent experiments.
Figure 6.
Figure 6.
IL-17 neutralization inhibits accelerated cardiac allograft rejection in a model of CAV in T-bet−/− mice. (A) MHC class II–mismatched cardiac allograft survival (bm12 into C57BL/6) in T-bet−/− recipients treated with anti–IL-17 mAb (n = 4) or control IgG (n = 4). Survival data are presented as a Kaplan-Meier plot. (B) Th1, Th2, and Th17 proinflammatory cytokine production, assessed by the Luminex assay at days 14 or 40 after transplantation, by the splenocytes of T-bet−/− recipients of bm12 heart grafts treated with control Ig or anti–IL-17 mAb. IL-12 data are presented separately because of the need to use a different scale. Results presented are means ± SD and are representative of three independent experiments. (C) Pathology of bm12 cardiac allografts from anti–IL-17 mAb–treated T-bet−/− recipients at 14 d after transplantation (left and middle, H&E staining; inset, infiltrating cell morphology), showing a decrease in PMN infiltration (particularly neutrophils), and at 40 d after transplantation (right, elastin staining), showing minimal CAV. Bars, 20 μm.

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