Louis C. Benjamin

Modeling chronic lung allograft rejection in miniature swine.

BACKGROUND The success of lung transplantation has been limited by the perplexing problem of chronic rejection. The development of a large-animal model for the systematic study of the mechanisms underlying chronic lung rejection has been problematic. We have developed a new preclinical model of chronic lung rejection using MHC-inbred miniature swine. METHODS Using standard operative techniques, four orthotopic left lung allografts were performed using MHC-matched, minor-antigen-mismatched donors. Recipient animals received a 12-day course of postoperative cyclosporine. Grafts were followed with open biopsies and high-resolution computed tomography. Cellular immune responses were monitored by mixed lymphocyte reaction, cytometric analysis of graft-infiltrating lymphocytes, and skin grafting. RESULTS All grafts survived > or = 5 months and developed manifestations of chronic rejection, including obliterative bronchiolitis, interstitial fibrosis, and occlusive vasculopathy. A mononuclear infiltrate was also present in all grafts by the fourth posttransplant month. High-resolution computed tomography demonstrated several cardinal radiographic findings known to correlate with chronic rejection. Cytometric analysis of graft-infiltrating lymphocytes showed a predominance of CD8+ cells. The development of alloreactivity in the host was confirmed by mixed lymphocyte reaction and skin grafting. CONCLUSIONS We report a reproducible, whole-lung, large-animal model of chronic lung rejection. In this immunogenetically defined construct, we have observed a full spectrum of histopathologic lesions that reproduce with fidelity those lesions observed in human lung transplant recipients suffering from chronic rejection. We anticipate that this preclinical model will facilitate further study of the pathogenesis and therapy of chronic lung rejection.

Induction of tolerance to heart transplants by simultaneous cotransplantation of donor kidneys may depend on a radiation-sensitive renal-cell population.

Background. To determine the mechanism by which cotransplantation of a donor kidney and heart allograft induces tolerance to both organs in miniature swine, we examined the renal elements responsible for tolerance induction. Methods. Recipients received 12 days of cyclosporine, and transplants were performed across a major histocompatibility complex (MHC) class I mismatch. Group 1 animals received heart transplants (n=5); group 2 animals received heart and kidney allografts with no other manipulation (n=4); group 3 animals received heart transplants and donor-specific renal parenchymal cells (n=4); group 4 animals received heart and kidney allografts from lethally irradiated donors (n=7); group 5 animals received irradiated hearts and nonirradiated kidneys (n=2); group 6 animals received nonirradiated hearts and peripheral blood leukocytes from swine MHC matched to recipients and becoming tolerant to donor antigen (n=2); group 7 animals received nonirradiated hearts and donor-specific peripheral blood monocyte cells (PBMC) (n=2). Results. Animals in group 1 developed vasculopathy and fulminant rejection by day 55. Animals in group 2 never developed vascular lesions. Parenchymal kidney cell infusion (group 3) did not prolong cardiac survival. Animals in group 4 developed arteriopathy by postoperative day (POD) 28. Group 5 recipients accepted allografts without vascular lesions. Adoptive transfer of leukocytes from tolerant swine (group 6) prolonged cardiac graft survival as much as 123 days, whereas donor PBMC infusion (group 7) did not affect cardiac survival or development of arteriopathy. Conclusions. Radiosensitive elements in kidney allograft may be responsible for tolerance induction and prevention of chronic vascular lesions in recipients of simultaneous heart and kidney allografts.

Indirect recognition of MHC class I allopeptides accelerates lung allograft rejection in miniature swine.

The role of indirect allorecognition in graft rejection is examined in two experiments using a swine lung transplantation model. First, two swine received class I mismatched grafts without immunosuppression; another two recipients were treated postoperatively with cyclosporine (CsA). These swine exhibited acute and chronic rejection, respectively. All four recipients developed T‐cell reactivity to donor‐derived class I major histocompatibility complex (MHC) peptides. Second, six swine were immunized with synthetic donor‐derived class I allopeptides prior to transplantation. Control groups consisted of nonimmunized recipients (n = 6) and recipients immunized with an irrelevant peptide (n = 3). These recipients all received a 12‐day course of post‐operative CsA. Swine immunized with allopeptides exhibited accelerated graft rejection, as compared to both control groups (p < 0.01 and p = 0.03, respectively). Within the experimental group, the dominant histologic finding was acute rejection (AR). Obliterative bronchiolitis (OB) was seen in the graft with the longest survival. Both control groups showed a lesser degree of AR, with four out of six nonimmunized swine ultimately developing OB. These studies suggest that indirect allorecognition is operative during lung allograft rejection, and that pre‐transplant sensitization to donor‐derived MHC allopeptides can accelerate graft rejection.

Recipients of MHC class I disparate lung grafts develop T cell reactivity to donor MHC class I allopeptides in an orthotopic swine model

logical reactivity to donor antigens, followed by vascularized heterotopic heart transplantation to determine if CAV still develops in the tolerant recipient. Procedures: In mice, specific immunological nonreactivity was achieved in recipients by (1) neonatal administration of allogeneic spleen cells from F1 donors between class I mismatched donor and recipient strains, (2) acheivement of mixed chimerism by bone marrow infusion of irradiated adult recipients, (3) use of genetically immunodeficient recipients (RAG1-/or SCID). In swine, reduced reactivity was achieved through the induction of mixed chimerism. After nonreactivity was induced and verified through extended persistence of skin grafts, hearts from donors were heterotopically transplanted into the recipients. Results: Most hearts transplanted into tolerant or immunodeficeint mice developed striking vasculopathy in their coronary arteries (neonatal tolerance: 12 of 15, mixed chimeras: 15 of 23, immunodeficient recipients: 16 of 31). In contrast, isotransplants were virtually free of CAV. Three of the four transplanted swine hearts showed neointimal proliferation characteristic of CAV in 1%-6% of donor coronary arteries. Control hearts transplanted into untreated recipients across similar minor antigen barriers were acutely rejected within 44 days whereas control isografts survived indefinitely without evidence of CAV. Conclusions: Elimination of T and B cell immune responses was not sufficient to prevent the development of CAV. Other immune effectors, perhaps NK cells, could play a role in the development of CAV in this immunodeficient state. Additional results on these alternate effectors will be presented.

Intracoronary interferon-γ accellerates cardiac allograft rejection in miniature swine

Purpose: The effect of interferon(IFN) on allograft rejection remains controversial. Some have proposed that IFNhas a protective effect on the microcirculation of allografts, while others have shown IFNto be deleterious. We investigated the effects of intracoronary IFNon cardiac rejection in a preclinical large animal model. Methods: MHC class I disparate hearts were heterotopically transplanted into swine treated with a 12-day course of cyclosporine (10-14mg/kg/day) (n 3). An Alzet osmotic mini-pump delivered IFNinto the coronary vasculature at a rate of 100ng/day. Control recipients were treated with a similar course of cyclosporine but without IFN(n 3). Results: Swine treated with IFNdeveloped an earlier onset of high grade acute rejection as compared to controls, and rejected their allografts in 19 days vs. 46 days for controls (p 0.02). Elastin staining of the cytokine treated allografts showed severe endothelialitis and intimal thickening as early as POD 13. Conclusion: The delivery of IFNinto the coronary circulation of cardiac allografts appears to accelerate both acute and chronic rejection. Targeting IFNin antirejection regimens may ameliorate both of these processes.