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Background. Cytotoxic T lymphocyte (CTL)-mediated destruction of allogeneic vascular endothelium is important in the pathogenesis of both acute and chronic allograft rejection. Despite the importance of this phenomenon, the effector mechanisms responsible for endothelial cell killing are not well defined, and conflicting conclusions have been reached based on variation in experimental methodology. Methods. We used a recently described method for isolating mouse vascular endothelium to evaluate endothelial cell lysis by CTLs. Endothelial cell destruction was assessed in vitro both by 51 Cr release and DNA fragmentation using wild-type and lpr (Fas deficient) endothelium of C3H/HeJ (H2 k ) mice by MHC alloantigen-specific T cells of wild-type, gld (Fas ligand deficient), and perforin-deficient mice on a C57BL/6 (H2 b ) background. Results. Although maximal lysis of 56.6±0.8% was seen when using wild-type targets and effectors, only a moderate decrease in apoptosis to 37.6±4.0% was detected when the Fas/Fas ligand death receptor pathway was eliminated. This decrease in cytotoxicity occurred despite the preserved functional capacity of this pathway. Alternatively, a significant decrease in cytotoxicity to 17.4±4.7% was seen when the perforin/ granzyme exocytosis pathway was eliminated. Conclusions. These data indicate that CTLs destroy vascular endothelium primarily by the perforin/granzyme exocytosis pathway with only a minor contribution to apoptosis by the Fas/Fas ligand death receptor pathway. These data are critical for the proper interpretation of studies evaluating acute and chronic allograft rejection and for the design of rational strategies to ameliorate vascular injury concomitant to the rejection process.

BACKGROUND Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts. METHODS The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI-->LEW and the acceptor combination PVG-->DA. RESULTS We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI-->LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for >100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG). CONCLUSIONS Our findings demonstrate that long-term acceptance of liver allografts and tolerance induction is not dependent on the presence of donor-type passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or third-party passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI-->LEW strain combination.

Background. We have described that chimeric rat hearts bearing recipient-type antigen-presenting cells (APCs) do not reject acutely, but develop cardiac allograft vasculopathy (CAV) in untreated recipients. This suggests that CAV is triggered either by CD8 + direct allorecognition or by CD4 + indirect allorecognition. To determine the allorecognition pathway responsible for CAV in this model, recipients of chimeric hearts underwent either CD8 + or CD4 + T cell depletion. Methods. Chimeric hearts were created via bone marrow transplantation in two fully major histocompatibility-mismatched rat strain combinations. DA recipients were thymectomized and treated with Ox8 and Ox38 murine monoclonal antibodies, which deplete CD8 + and CD4 + T cells, respectively. Chimeric PVG hearts bearing DA APCs, abbreviated PVG(DA), were heterotopically transplanted into recipients undergoing thymectomy alone or recipients undergoing thymectomy plus either CD4 + or CD8 + T cell depletion. Results. PVG(DA) allografts survived 100 days, but developed CAV in thymectomized recipients and in those permanently depleted of CD8 + T cells. In contrast, chimeric hearts transplanted into permanently CD4 + T cell-depleted recipients survived 100 days and demonstrated no evidence of CAV. Conclusions. In this specific strain combination, recipient CD8 + T cells are neither necessary nor sufficient for the development of CAV, whereas recipient CD4 + T cells are required for the development of CAV. These findings suggest that CAV is dependent on CD4 + indirect allorecognition and that CD8 + direct allorecognition stimulated by nonprofessional APCs plays a minor role.

A 50-year-old woman underwent single lung transplantation for advanced chronic obstructive pulmonary disease. Shortly after the procedure, it was discovered that the donor suffered from both a renal cell carcinoma and a spindle-cell sarcoma of the ascending aorta, which had metastasized to the spleen. The patient was emergently listed for a retransplantation and underwent bilateral lung transplantation after a new donor became available 4 days after the initial transplantation procedure. After 24 months, the patient is without evidence of malignancy. This case illustrates the role of immediate retransplantation for patients who have inadvertently received thoracic organs from donors harboring occult malignancies.

Background. Graft-resident antigen presenting cells (APCs) are potent stimulators of the alloresponse. To test whether replacement of graft-resident donor-type APCs with those of recipient-type alters allorecogni- tion and the pathogenesis of both acute and chronic rejection, we created chimeric hearts for transplanta- tion into naive recipients. Methods. To replace donor-type APCs with those of recipient-type, chimeric animals were created by bone marrow transplantation (BMT) in fully allogeneic mouse and rat strain combinations. The degree of APC replacement in chimeric organs was assessed pheno- typically and functionally. Chimeric hearts were transplanted heterotopically into untreated recipi- ents. Results. Flow cytometric and immunohistochemical analysis did not detect residual bone marrow recipi- ent-type APCs in mouse BMT chimeras. Although semi-quantitative reverse transcription polymerase chain reaction detected 0.001- 0.01% residual cells, APCs isolated from chimeric organs were functionally unable to stimulate donor-type cells. When trans- planted into naive recipients, chimeric mouse hearts had significantly prolonged survival but were never- theless rejected acutely. Similar results were obtained in the ACI3 LEW rat strain combination. However, in the PVG 3 DA rat model, the majority of chimeric hearts survived >100 days and all long-surviving hearts developed cardiac allograft vasculopathy. Conclusions. BMT leads to near complete replace- ment of organ-resident APCs. The virtual absence of donor-type APCs in chimeric hearts delays or prevents acute rejection in a strain-dependent manner. In con- trast, this type of graft modification does not prevent cardiac allograft vasculopathy. This suggests that, al- though the CD41 direct pathway may play a role in acute rejection, it is not essential for the development of chronic rejection in rodent cardiac allografts.

Cytotoxic T lymphocyte (CTL)-mediated destruction of allogeneic vascular endothelium is important in the pathogenesis of both acute and chronic allograft rejection. Despite the importance of this phenomenon, the effector mechanisms responsible for endothelial cell killing are not well defined, and conflicting conclusions have been reached based on variation in experimental methodology.We used a recently described method for isolating mouse vascular endothelium to evaluate endothelial cell lysis by CTLs. Endothelial cell destruction was assessed in vitro both by 51Cr release and DNA fragmentation using wild-type and lpr (Fas deficient) endothelium of C3H/HeJ (H2(k)) mice by MHC alloantigen-specific T cells of wild-type, gld (Fas ligand deficient), and perforin-deficient mice on a C57BL/6 (H2(b)) background.Although maximal lysis of 56.6+/-0.8% was seen when using wild-type targets and effectors, only a moderate decrease in apoptosis to 37.6+/-4.0% was detected when the Fas/Fas ligand death receptor pathway was eliminated. This decrease in cytotoxicity occurred despite the preserved functional capacity of this pathway. Alternatively, a significant decrease in cytotoxicity to 17.4+/-4.7% was seen when the perforin/granzyme exocytosis pathway was eliminated.These data indicate that CTLs destroy vascular endothelium primarily by the perforin/granzyme exocytosis pathway with only a minor contribution to apoptosis by the Fas/Fas ligand death receptor pathway. These data are critical for the proper interpretation of studies evaluating acute and chronic allograft rejection and for the design of rational strategies to ameliorate vascular injury concomitant to the rejection process.

Although graft-resident passenger leukocytes are known to mediate acute rejection by triggering direct allorecognition, they may also act in an immunomodulatory fashion and play an important role in tolerance induction. Our purpose in the current study was to utilize rat bone marrow chimeras to evaluate the role of the genotype of passenger leukocytes in both acute rejection and tolerance of liver allografts.The fate of livers bearing donor-type, recipient-type, and third-party passenger leukocytes was evaluated in the MHC class I and II mismatched rejector combination ACI--LEW and the acceptor combination PVG--DA.We report that although treatment of ACI liver donors with lethal irradiation does not lead to prolongation of graft survival in the ACI--LEW strain combination, ACI livers bearing recipient-type (LEW) or third-party passenger leukocytes (BN) are rejected at a significantly slower rate. We confirm that lethal irradiation of PVG donor animals leads to abrogation of tolerance induction with acute rejection of their livers by DA recipients. However, the majority of PVG livers carrying donor-type (PVG), recipient-type (DA), or third-party (LEW) passenger leukocytes are accepted for100 days. These DA recipients develop immune tolerance to the donor parenchyma (PVG).Our findings demonstrate that long-term acceptance of liver allografts and tolerance induction is not dependent on the presence of donor-type passenger leukocytes and can be achieved with organs carrying donor-type, recipient-type, or third-party passenger leukocytes. The importance of the MHC framework on the surface of passenger leukocytes as a critical regulator of the immune response after transplantation of chimeric organs is substantiated by the delayed tempo of rejection of ACI livers bearing recipient-type or third-party passenger leukocytes in the ACI--LEW strain combination.

Background Antithymocyte globulin is frequently used as a component of induction therapy in thoracic organ transplantation. This study evaluates the utility of monitoring peripheral CD3 lymphocytes to rationally adjust antithymocyte globulin therapy in this patient population. Methods A total of 17 heart and 19 lung transplant recipients received antithymocyte globulin (ATGAM or thymoglobulin) as induction therapy or to treat steroid-resistant acute or chronic rejection. Absolute CD3 counts were maintained between 50 and 100 cells/microl. Results With CD3 monitoring, the doses of antithymocyte globulin were reduced from 10-15 mg/kg to 1-5 mg/kg during the course of therapy. The total amount of antithymocyte globulin given to each CD3 monitored patient was reduced by 48%. Dose reduction did not alter the number of acute rejection or infectious episodes, and hematological side effects were infrequent. Conclusion CD3 monitoring of antithymocyte globulin therapy in thoracic organ recipients reduced the amount of drug received by each patient, while maintaining CD3 counts less than 100 cells/microl.

We have described that chimeric rat hearts bearing recipient-type antigen-presenting cells (APCs) do not reject acutely, but develop cardiac allograft vasculopathy (CAV) in untreated recipients. This suggests that CAV is triggered either by CD8+ direct allorecognition or by CD4+ indirect allorecognition. To determine the allorecognition pathway responsible for CAV in this model, recipients of chimeric hearts underwent either CD8+ or CD4+ T cell depletion.Chimeric hearts were created via bone marrow transplantation in two fully major histocompatibility-mismatched rat strain combinations. DA recipients were thymectomized and treated with Ox8 and Ox38 murine monoclonal antibodies, which deplete CD8+ and CD4+ T cells, respectively. Chimeric PVG hearts bearing DA APCs, abbreviated PVG(DA), were heterotopically transplanted into recipients undergoing thymectomy alone or recipients undergoing thymectomy plus either CD4+ or CD8+ T cell depletion.PVG(DA) allografts survived 100 days, but developed CAV in thymectomized recipients and in those permanently depleted of CD8+ T cells. In contrast, chimeric hearts transplanted into permanently CD4+ T cell-depleted recipients survived 100 days and demonstrated no evidence of CAV.In this specific strain combination, recipient CD8+ T cells are neither necessary nor sufficient for the development of CAV, whereas recipient CD4+ T cells are required for the development of CAV. These findings suggest that CAV is dependent on CD4+ indirect allorecognition and that CD8+ direct allorecognition stimulated by nonprofessional APCs plays a minor role.

Graft-resident antigen presenting cells (APCs) are potent stimulators of the alloresponse. To test whether replacement of graft-resident donor-type APCs with those of recipient-type alters allorecognition and the pathogenesis of both acute and chronic rejection, we created chimeric hearts for transplantation into naive recipients.To replace donor-type APCs with those of recipient-type, chimeric animals were created by bone marrow transplantation (BMT) in fully allogeneic mouse and rat strain combinations. The degree of APC replacement in chimeric organs was assessed phenotypically and functionally. Chimeric hearts were transplanted heterotopically into untreated recipients.Flow cytometric and immunohistochemical analysis did not detect residual bone marrow recipient-type APCs in mouse BMT chimeras. Although semi-quantitative reverse transcription polymerase chain reaction detected 0.001-0.01% residual cells, APCs isolated from chimeric organs were functionally unable to stimulate donor-type cells. When transplanted into naive recipients, chimeric mouse hearts had significantly prolonged survival but were nevertheless rejected acutely. Similar results were obtained in the ACI --LEW rat strain combination. However, in the PVG --DA rat model, the majority of chimeric hearts survived100 days and all long-surviving hearts developed cardiac allograft vasculopathy.BMT leads to near complete replacement of organ-resident APCs. The virtual absence of donor-type APCs in chimeric hearts delays or prevents acute rejection in a strain-dependent manner. In contrast, this type of graft modification does not prevent cardiac allograft vasculopathy. This suggests that, although the CD4+ direct pathway may play a role in acute rejection, it is not essential for the development of chronic rejection in rodent cardiac allografts.