Indirect recognition of allopeptides promotes the development of cardiac allograft vasculopathy

Abstract

Graft loss from chronic rejection has become the major obstacle to the long-term success of whole organ transplantation. In cardiac allografts, chronic rejection is manifested as a diffuse and accelerated form of arteriosclerosis, termed cardiac allograft vasculopathy. It has been suggested that T-cell recognition of processed alloantigens (allopeptides) presented by recipient antigen-presenting cells through the indirect pathway of allorecognition plays a critical role in the development and progression of chronic rejection. However, definitive preclinical evidence to support this hypothesis is lacking. To examine the role of indirect allorecognition in a clinically relevant large animal model of cardiac allograft vasculopathy, we immunized MHC inbred miniature swine with synthetic polymorphic peptides spanning the alpha(1) domain of an allogeneic donor-derived swine leukocyte antigen class I gene. Pigs immunized with swine leukocyte antigen class I allopeptides showed in vitro proliferative responses and in vivo delayed-type hypersensitivity responses to the allogeneic peptides. Donor MHC class I disparate hearts transplanted into peptide-immunized cyclosporine-treated pigs not only rejected faster than unimmunized cyclosporine-treated controls (mean survival time = 5.5 +/-1.7 vs. 54.7 +/-3.8 days, P < 0.001), but they also developed obstructive fibroproliferative coronary artery lesions much earlier than unimmunized controls (<9 vs. >30 days). These results definitively link indirect allorecognition and cardiac allograft vasculopathy.

Figure 1

Histological analysis of cardiac allografts. (a) Voerhoeff elastin stain of the rejected cardiac allograft from recipient no. 13511 on POD 5 (×100). Asterisk indicates internal elastic lamina. (b) Trichrome stain of the rejected cardiac allograft from recipient no. 13692 on POD 5 (×100). Blue staining indicates the presence of collagen within occluding neointima, as indicated by transparent arrow. (c) α-Actin staining of the rejected cardiac allograft from recipient no. 13692 on POD 5 showing smooth muscle cell accumulation within the intima, indicated by filled arrow (×100). (d) Voerhoff elastin stain of cardiac allograft from DRβ control pig no. 13914 at POD 15 showing no intimal thickening (×100). (e) Immunofluorescent staining for IgM on the rejected cardiac allograft from recipient no. 13692 on POD 5 showing antibody deposition along the arteriolar endothelium (×250). (f) Immunofluorescent staining for IgG on the rejected cardiac allograft from recipient no. 13692 on POD 5 showing antibody deposition along arteriolar endothelium (×250). Naïve control hearts did not stain for antibody.

Figure 2

ELISPOT detection of swine reactivity to allogeneic PC14 peptide 3. Representative IFN-γ ELISPOT wells by using 3 × 10⁵ pig responder splenocytes per well plus PC14 peptide 3 (50 μg/ml) are shown. Responder splenocytes were harvested from peptide-immunized CyA-treated rejecter pig no. 14071 (Top), unimmunized acute rejecter pig no. 13384 (Middle), and a naïve pig (Bottom).

Figure 3

Immunization with allogeneic donor class I^c peptides accelerated the generation of anti-donor IgM in host sera. Flow cytometric analysis was performed to evaluate the levels of antidonor IgM in sera from unimmunized acute rejecters (nos. 13384 and 13896), unimmunized, CyA-treated pigs (nos. 13495 and 13262), PC14-peptide-immunized, CyA-treated pigs (nos. 13692, 13511, 14071, and 14311), and DRβ-peptide-immunized, CyA-treated pig (nos. 13914 and 14146).