M.E. Cochrane

Repetitive Gastric Aspiration Leads to Augmented Indirect Allorecognition after Lung Transplantation in Miniature Swine

Introduction. Lung transplant recipients with documented gastroesophageal reflux disease (GERD) are at increased risk for graft dysfunction. Here, we present the first large-animal model of gastric aspiration after allogeneic lung transplantation and some preliminary data demonstrating the effect of chronic aspiration on the direct and indirect pathways of allorecognition. Methods. Left orthotopic lung transplants (n=3) were performed in miniature swine across a major histocompatibility complex class I disparity, followed by 12 days of high-dose cyclosporine A. At the time of transplantation, a transtracheal catheter was placed at the carina, above the bronchial anastomosis. A gastrostomy tube was placed for daily aspiration of gastric contents. Subsequently, graft lungs were instilled with gastric aspirate daily (3 mL/hr×8 hr/day) for 50 days. Recipients were followed up with daily complete blood count, scheduled chest radiographs, and biopsies. In vitro studies, including cell-mediated lympholysis, mixed lymphocyte reactions, and peptide proliferation assays, were performed. Results from these three recipients were compared with those of historical controls (n=6) who were treated identically, except for the tracheal cannulation and simulated gastric aspiration. Results. Two of the experimental animals were euthanized with nonviable lungs soon after the postoperative day 50 biopsy. In both cases the native lung was normal. The third animal survived over 180 days without the evidence of chronic rejection. After immunosuppressive treatment, all animals demonstrated donor-specific hyporesponsiveness by assays of direct alloresponse (cell-mediated lympholysis, mixed lymphocyte reaction). A significant response to synthetic donor-derived class I peptide, however, was seen in all animals. A more pronounced and diffuse response was seen in the animals rejecting their grafts. The historical controls showed medium-term graft survival with evidence of chronic rejection in the majority of animals, as previously reported. Conclusion. In a model of GERD after lung transplantation, a spectrum of clinical outcomes was observed. The in vitro data suggest that acid reflux enhances the indirect alloresponse to processed donor class I antigen, giving mechanistic insight into the manner in which GERD may be deleterious to the transplanted lung.

The Indirect Alloresponse Impairs the Induction but not Maintenance of Tolerance to MHC Class I-Disparate Allografts

We studied the effects of indirect allorecognition on the induction and maintenance phases of tolerance in miniature swine cotransplanted with heart and kidney allografts. MHC class I‐mismatched heart and kidney grafts were cotransplanted in recipients receiving CyA for 12 days. Recipients were unimmunized or immunized with a set of donor‐derived or control third‐party MHC class I peptides either 21 days prior to transplantation or over 100 days after transplantation. T‐cell proliferation, delayed type hypersensitivity reaction (DTH) and antibody production were assessed. All animals injected with donor MHC class I peptides developed potent indirect alloresponses specific to the immunizing peptides. While untreated recipients developed stable tolerance, all animals preimmunized with donor allopeptides rejected kidney–heart transplants acutely. In contrast, when peptide immunization was delayed until over 100 days after kidney–heart transplantation, no effects were observed on graft function or in vitro measures of alloimmunity. Donor peptide immunization prevented tolerance when administered to recipients pre transplantation but did not abrogate tolerance when administered to long‐term survivors post transplantation. This suggests that the presence of T cells activated via indirect allorecognition represent a barrier to the induction but not the maintenance of tolerance.

Donor Brain Death Inhibits Tolerance Induction in Miniature Swine Recipients of Fully MHC‐Disparate Pulmonary Allografts

We have previously shown that a short course of high‐dose tacrolimus induces long‐term tolerance to fully mismatched lung allografts procured from healthy MHC‐inbred miniature swine. Here, we investigate whether donor brain death affects tolerance induction. Four recipient swine were transplanted with fully mismatched lung grafts from donors that were rendered brain dead and mechanically ventilated for 4 h before procurement (Group 1). These recipients were compared to two control groups (Group 2: 4 h of donor ventilation without brain death [n = 5]; and Group 3: no donor brain death with <1 h of ventilation [n = 6]). All recipients were treated with a 12‐day course of tacrolimus. In contrast to both groups of control animals, the swine transplanted with lung allografts from brain dead donors all rejected their grafts by postoperative day 45 and showed persistent responsiveness to donor antigen by MLR. Several additional swine underwent brain death induction and/or mechanical ventilation alone to determine the effects of these procedures on the expression of proinflammatory molecules. Significant increases in serum concentrations of IL‐1, TNF‐α and IL‐10 were seen after brain death. Upregulation of IL‐1 and IL‐6 gene expression was also observed.