Role of T cell TGFbeta signaling and IL-17 in allograft acceptance and fibrosis associated with chronic rejection

Abstract

Chronic allograft rejection (CR) is the main barrier to long-term transplant survival. CR is a progressive disease defined by interstitial fibrosis, vascular neointimal development, and graft dysfunction. The underlying mechanisms responsible for CR remain poorly defined. TGFbeta has been implicated in promoting fibrotic diseases including CR, but is beneficial in the transplant setting due to its immunosuppressive activity. To assess the requirement for T cell TGFbeta signaling in allograft acceptance and the progression of CR, we used mice with abrogated T cell TGFbeta signaling as allograft recipients. We compared responses from recipients that were transiently depleted of CD4(+) cells (that develop CR and express intragraft TGFbeta) with responses from mice that received anti-CD40L mAb therapy (that do not develop CR and do not express intragraft TGFbeta). Allograft acceptance and suppression of graft-reactive T and B cells were independent of T cell TGFbeta signaling in mice treated with anti-CD40L mAb. In recipients transiently depleted of CD4(+) T cells, T cell TGFbeta signaling was required for the development of fibrosis associated with CR, long-term graft acceptance, and suppression of graft-reactive T and B cell responses. Furthermore, IL-17 was identified as a critical element in TGFbeta-driven allograft fibrosis. Thus, IL-17 may provide a therapeutic target for preventing graft fibrosis, a measure of CR, while sparing the immunosuppressive activity of TGFbeta.

Figure 1. TGFβ dependent and independent mechanisms of allograft acceptance

WT (squares and triangles) and CD4-DNR (circles and diamonds) mice were transplanted with allogeneic or syngeneic cardiac grafts and were either left untreated (A), given inductive anti-CD40L mAb (B), or transiently depleted of CD4+ cells (C). Graft function was monitored by palpation and recipients were recovered either at the time of rejection or 50 days post-transplant. (D) Flow cytometric analysis of CD4+ splenocytes that were harvested from WT or CD4-DNR allograft recipients on day 20 and day 40 post-transplant. Allograft recipients were treated inductively with anti-CD4 mAb.

Figure 2. Donor-reactive Th1, Th2 and Th17 responses in WT and CD4-DNR recipients

WT (open bars) or CD4-DNR (shaded bars) allograft recipients were left untreated (A), treated with inductive anti-CD40L mAb (B), or treated with inductive anti-CD4 mAb (C). Graft function was monitored by palpation and recipients were recovered either at the time of rejection, 50 days post-transplant (for anti-CD40L mAb treated WT and CD4-DNR recipients), or 40 days post-transplant (for anti-CD4 mAb treated WT recipients in order to directly compare to CD4-DNR recipients, which reject between days 35-40). At the termination of the experiment, splenocytes were harvested and processed for ELISPOT assays to quantify primed, donor-reactive IFN-γ, IL-4, or IL-17-producing cells. Bars represent the mean number of cytokine producing cells (+/- S.E.M.) from at least six recipients per group.

Figure 3. Allograft fibrosis in WT versus CD4-DNR recipients treated with anti-CD4 mAb

(A) Sections of grafts from recipients treated with inductive anti-CD40L (day 50 post-transplant) or anti-CD4 mAb (between days 35-40 post-transplant due to rejection in CD4-DNR recipients) were stained with Masson's trichrome, which stains fibrotic tissue blue. Frames are of grafts from WT and CD4-DNR recipients and are representative of at least 6 anti-CD40L mAb treated mice and 6-8 mice transiently depleted of CD4+ T cells. 200× magnification. (B) Quantification of mean fibrotic area by morphometric analysis in inductive anti-CD40L and (C) anti-CD4 mAb treated WT and CD4-DNR recipients. Bars represent the average percentage (+/- S.E.M.) of graft area positive for fibrosis in 5 anti-CD40L treated recipients and 6 anti-CD4 treated recipients. WT (open bars) and CD4-DNR (shaded bars).

Figure 4. Effect of TGFβ unresponsiveness on donor-reactive alloantibody levels

Sera were obtained from WT (open bars) or CD4-DNR (shaded bars) allograft recipients that were left untreated (A), treated with inductive anti-CD40L mAb (B), or treated with inductive anti-CD4 mAb (C). Sera were obtained at the time of rejection for unmodified recipients), 50 days post-transplant (for anti-CD40L mAb treated WT and CD4-DNR recipients), or 40 days post-transplant (for anti-CD4 mAb treated WT recipients). P815 (H-2^d) cells were incubated with 1:50 dilution of sera and bound donor-reactive Ab were detected by incubation with FITC-tagged anti-IgG, anti-IgG1 or anti-IgG2a Abs. The mean channel fluorescence is indicative of the relative amount of donor-reactive antibodies. Bars represent the average mean channel fluorescence of at least 6 WT and 6 CD4-DNR recipient samples (+/- S.E.M.). IgG1 and IgG2a donor-reactive antibody levels were analyzed in anti-CD4 mAb treated WT and CD4-DNR recipients (D).

Figure 5. IgG, C3d and C4d deposition in allografts of WT and CD4-DNR recipients

WT (left column) or CD4-DNR (right column) allograft recipients were treated with inductive anti-CD4 mAb therapy. Grafts were recovered at either the time of rejection or 40 days after transplantation. Graft sections were fixed and incubated with goat anti-mouse IgG (A) or the goat anti-mouse C3d or C4d (B) followed by development with 3-amino-9-ethylcarbazole or DAB to visualize mouse Ab and complement deposition. Results are representative of grafts from 6-10 recipients. Magnification, 400× (A) and 200× (B).

Figure 6. Reduction of TGFβ-dependent intragraft gene expression from CD4-DNR recipients transiently depleted of CD4+ T cells

RNA was harvested from allografts of WT and CD4-DNR recipients transiently depleted of CD4+ T cells. Intragraft transcript levels of FoxP3 and IL-17 were assessed by real-time RT-PCR. Allografts were recovered between days 35-40 post-transplant. Bars depict the means of RNA expression from 6 WT and 9 CD4-DNR grafts.

Figure 7. In recipients that fail to produce IL-17, allografts are protected from fibrosis

(A) WT (squares) and IL-17-/- (circles) mice were transplanted with BALB/c cardiac allografts and were either left untreated (closed symbols) or transiently depleted of CD4+ cells (open symbols). Graft function was monitored by palpation. Unmodified recipients were harvested at time of rejection, while inductive anti-CD4 mAb treated recipients were harvested at day 50 post-transplantation. Numbers in parentheses represent the number of recipients in each group. (B) Sections of grafts from recipients transiently depleted of CD4+ T cells (day 50 post-transplant) were stained with Masson's trichrome stain. Frames are of grafts from WT and IL-17-/- recipients and are representative of at least 6 WT and 9 IL-17-/- recipient allografts. 200× magnification. (C) Quantification of fibrosis by morphometric analysis. Bars represent the average percentage (+/- S.E.M.) of graft area positive for fibrosis in at least 5 WT and IL-17-/- recipients treated with anti-CD4 mAb. WT (open bars) and IL-17-/- (closed bars). (D) On day 50 post-transplant, RNA was harvested from allografts of WT and IL-17-/- recipients transiently depleted of CD4+ T cells. Intragraft transcript levels of FoxP3 and IL-17 were assessed by real-time RT-PCR. Bars depict the means of RNA from 5 WT and 9 IL-17-/- grafts.