Immune mechanisms associated with the rejection of encapsulated neonatal porcine islet xenografts

BACKGROUND: The immune mechanisms associated with the rejection of microencapsulated neonatal porcine islets (NPI) are not clearly understood. Therefore, in this study we characterized the immune cells and molecules that are involved in this process by examining the microencapsulated NPI xenografts at various time points post-transplantation in B6 mice. METHODS: Microencapsulated NPI were transplanted into streptozotocin-induced diabetic immune-competent B6 and immune-deficient B6 rag-/- mice and blood glucose levels were monitored twice a week. Encapsulated NPI were then recovered from B6 mice at various time points post-transplantation to characterize the islets and immune response using immunohistochemical and RT-PCR analyses. To determine which T-cell subpopulation is important for the rejection of encapsulated NPI, B6 rag-/- mice with established microencapsulated NPI xenografts were reconstituted with either CD4(+) or CD8(+) T cells and a return to the diabetic state was noted. For controls, adoptive transfer experiments involved reconstitution of B6 rag-/- mice with established microencapsulated NPI with non-fractionated lymph node cells or non-reconstituted mice. RESULTS: All B6 recipients of microencapsulated NPI remained diabetic throughout the study while B6 rag-/- recipients achieved normoglycemia and maintained normoglycemia for up to 100 days post-transplantation. Encapsulated NPI recovered from B6 mice at early time points (day 7 and day 14) post-transplantation were surrounded with very few layers of immune cells that increased with time post-transplantation. The extent of cellular overgrowth on the surface of encapsulated NPI has a significant correlation with islet cell death and the presence of CD4(+) T cells, B cells and macrophages. Mouse IgG antibody and complement as well as cytokines [gamma-interferon (IFN-gamma), interleukin10 (IL10)] and chemokines (monocyte chemotactic protein-1 and macrophage inflammatory protein-1alpha and beta) were detected within the microcapsules at several time points post-transplantation suggesting that these molecules can traverse the microcapsule. Mouse anti-porcine IgG antibodies in recipient sera were found to peak at 30 days post-transplantation indicating leakage of porcine xenoantigens. In contrast, microencapsulated NPI recovered from B6 rag-/- mice had no cellular overgrowth on the surface. Complement and cytokines (IL 10 but not IFN-gamma) including chemokines were detected within the microcapsules at several days post-transplantation. We also found that B6 rag-/- mice reconstituted with non-fractionated lymph node cells or CD4(+) T cells but not CD8(+) T cells became diabetic demonstrating that CD4(+) T cells are the necessary T-cell subtype for microencapsulated NPI rejection. In contrast, non-reconstituted B6 rag-/- mice remained normoglycemic for the entire duration of the study. CONCLUSIONS: Our results demonstrate that CD4(+) T cells, B cells and macrophages are the immune cells recruited to and involved in the rejection of encapsulated NPI. Immune molecules secreted by these cells as well as complement can traverse the microcapsule membrane and are responsible for destroying the NPI cells. Treatment regimens which target these molecules may modify the rejection of encapsulated NPI and lead to prolonged islet xenograft survival.     

Role of anti-donor antibody in the rejection of pig proislet xenografts in mice

Abstract: CBA/H mice produced serum anti-pig IgG₁, IgG_2a, and IgG_2b following xenotransplantation of pig proislets beneath the kidney capsule; anti-pig IgM was present as pre-existing antibody in the serum of normal CBA/H mice and was also produced in response to pig proislet xenografts. Serum anti-pig IgG₃was not detected post-xenotransplantation. Rejection of pig proislet xenografts and the production of anti-pig IgG₁, IgG_2a, and IgG_2b isotypes were CD4 T cell-dependent. The capacity for adoptively transferred hyperimmune CBA/H mouse anti-pig PBL serum to induce the rejection of intact pig proislet xenografts in CD4 T cell-depleted mice was dose dependent and correlated with markedly elevated levels of serum anti-pig IgG₃. Levels of anti-pig IgG₁, IgG_2a, IgG_2b, and IgM comparable to control mice acutely rejecting pig proislet xenografts and achieved following adoptive transfer of hyperimmune serum did not correlate with induced xenograft rejection. These findings suggest that anti-pig Ig isotypes produced during the conventional process of acute proislet xenograft rejection do not play a major role in mediating graft damage. The acute rejection of pig proislet xenografts in the absence of serum anti-pig Ig in μMT-/- hosts confirmed that anti-pig antibody is not essential for proislet xenograft destruction.     

Histopathology of hyperacute rejection of the heart: experimental and clinical observations in allografts and xenografts

The histologic findings in a total of 112 experimental heart transplants comprising allografts (baboon to baboon: n = 37), concordant xenografts (vervet monkey to baboon: n = 52), and discordant xenografts (pig to baboon: n = 23), in which the roles of ABO blood group incompatibility, corcordance, and immunosuppression were evaluated, are described. Hyperacute (vascular, humoral) rejection was characterized by disruption of the microcirculation, with interstitial hemorrhage and edema, rather than by intravascular thrombosis; the features were basically similar whether hyperacute rejection occurred in an ABO-incompatible allograft, concordant xenograft, or discordant xenograft. Hyperacute rejection was noted in all 23 discordant xenografts, in 12 to 52 concordant xenografts, and in four of 17 ABO-incompatible allografts. A unique mixture of acute and hyperacute rejection was observed in three ABO-incompatible allografts and in 10 concordant xenografts. Intensive antirejection therapy was associated with a reduced incidence of hyperacute rejection in corcordant xenografts but also with a significant number of fatal treatment-related complications.     

Immune mechanisms in organ allograft rejection. II. T helper cells, delayed-type hypersensitivity, and rejection of renal allografts

The cellular requirements for renal allograft rejection have been assessed by adoptive transfer studies in a rat model. Sublethally irradiated (780 rads) LEW (RT1l) recipients of WF (RT1u) renal allografts were selectively reconstituted with spleen cells from specifically sensitized donors. In some experiments the reconstituting inocula were depleted of SIg+ cells by passage over anti-Ig columns or subjected to additional depletion of cytotoxic T cells (Tc) and their precursors reactive with monoclonal antibody OX8. WF renal allografts underwent acute rejection in the unmodified LEW recipient with day 7 serum creatinines of 6.8 +/- 0.9 mg/dL (mean +/- SD; n = 7), graft histology characterized by marked mononuclear cell infiltration and evidence of a brisk humoral response (complement-dependent cytotoxicity (CDC) titer greater than 2(6] and generation of Tc demonstrable by in vitro monitoring. Sublethally irradiated recipients mounted no detectable immune response, and day 7 serum creatinine and graft histological findings were not significantly different from those obtained in isograft controls. Renal allografts were, however, rejected in sublethally irradiated recipients reconstituted with unfractionated immune spleen cells, as evidenced by functional and histologic criteria (day 7 serum creatinine of 5.5 +/- 1.2 mg/dL; histology characterized by extensive interstitial hemorrhage, fibrinoid necrosis of blood vessels, and polymorphic infiltration). Neither antibody nor Tc appear, in this model, to be required to effect rejection, because recipients reconstituted with inocula depleted of SIg+ cells (day 7 CDC titer less than 2l) or subjected to additional depletion of Tc and their precursors (day 7 lymphocyte-mediated cytotoxicity assay: % specific chromium release at 100/1 E/T ratios less than 5%) underwent acute rejection with a day 7 serum creatinine of 5.0 +/- 1 and 4.3 +/- 1.5 mg/dL, respectively, and histological findings were characterized by marked mononuclear cell infiltration and a paucity of hemorrhage.     

High transforming growth factor-beta and extracellular matrix mRNA response in renal allografts during early acute rejection is associated with absence of chronic rejection

BACKGROUND: A case-control study was performed to investigate whether mRNA levels of transforming growth factor-beta (TGF-beta) and various extracellular matrix molecules in renal transplant biopsy specimens, taken during acute rejection episodes within 6 months of transplantation, discriminate between patients who show deterioration of graft function and develop chronic rejection (CR+ group), and those who do not develop chronic rejection (CR- group). METHODS: Patients in both the CR+ group (n=10) and the CR- group (n=18) had at least one biopsy-proven acute rejection episode within the first 6 months after transplantation. The two groups were similar with respect to donor-, recipient-, and transplantation-related clinical variables. Histologic changes (Banff classification) and the timing of the acute rejection episodes in the biopsies studied did not differ between groups. Renal cortical mRNA levels of TGF-beta1, collagen alpha1(IV), collagen alpha1(I), decorin, and the household gene glyceraldehyde-3-phosphate dehydrogenase in biopsy specimens taken during acute rejection episodes were quantified by real-time polymerase chain reaction. RESULTS: The mean TGF-beta mRNA level in the CR- group was 3.4 times higher than that in the CR+ group (P<0.04). The mean collagen IV, collagen I, and decorin mRNA levels in the CR- group were 4.2 times (P<0.05), 5.1 times (not significant), and 3.2 times (P<0.05) higher, respectively, than those in the CR+ group. The mean TGF-beta to decorin mRNA ratios between the two patient groups did not differ significantly. CONCLUSIONS: In summary, high mRNA levels for TGF-beta, collagen IV, and decorin, but not histopathologic changes, in biopsies taken during acute rejection episodes early after kidney transplantation are associated with absence of chronic rejection. We hypothesize that TGF-beta might have beneficial effects during acute rejection through its known antiinflammatory actions or as an inducer of tissue repair.     

Pancreatic islet isografts, allografts, and xenografts: comparison of morphology and function.

Neonatal rat pancreatic islets were transplanted intraperitoneally into adult streptozotocin-diabetic rats and mice. Isologous pancreatic islet recipients (12 of 12) showed consistent and permanent reconstitution of normoglycemia and normal weight gain as well as readjustment to normal of intake of water, urine volume, and glucose excretion for greater than 10 months when compared to age-matched normal (ten) and diabetic (20) controls. Revascularized isologous islet grafts were found to be adherent to both visceral and parietal peritoneum. Pancreatic islet allografts (ten) and allografts and xenografts did not appear to differ markedly; both were characterized by dense round-cell infiltration within 5 days after transplantation.     

Reconstruction of the RVOT with valved biological conduits: 25 years experience with allografts and xenografts

Objective: The reconstruction of the RVOT in congenital heart disease often requires the implantation of a valved conduit. Although allografts are considered the conduit of choice their availability is limited and therefore xenografts are implanted as well. We compared the long-term durability of both grafts in the RVOT over a 25-year period. Methods: Between January 1974 and August 1999, 505 patients (median age 4.0 years, range 2 days–31 years; median weight 14.5 kg, range 2.2–76.6 kg; median body length 103 cm, range 48–183 cm) with congenital malformations (PA 25.3%, TOF 14.5%, TOF+PA 2.4%, DORV 4.2%, TGA+PS 8.7%, TAC 24.8%, and other 20.2%) received their first valved conduit (174 xenografts: median diameter 14 mm, range 8–27 mm; 331 allografts: median diameter 19 mm, range 8–30 mm). Results: Follow-up is 3017 patient-years. The 10-year survival-probability for all patients. was 66% with a mean reoperation-free interval for conduit-exchange of 13.3 years (mean reoperation-free interval for allografts, 16.0 years; mean reoperation-free interval for xenograft, 10.3 years). One hundred and thirteen patients underwent a conduit-exchange, mostly due to conduit stenosis. Fourteen patients had a second exchange and three patients a third exchange. For patients with conduit diameters <18 mm (n=235: allograft n=116, xenograft n=119; median age 9 months, range 0–27.3 years), the mean reoperation-free interval was 11.2 years (mean interval allograft, 13.1 years; mean interval xenograft, 8.6 years, P=0.03). For conduit diameters ≥18 mm (n=270: allograft n=215, xenograft n=55, median age 7.4 years, range 0–34.3 years) the mean interval from freedom of conduit exchange was 15.1 years (for allografts 14.1 years, for xenografts 12.5 years, P<0.01). Comparing xenografts to allografts, we found no difference in patient survival probability (P=0.62). There was no significant difference between antibiotic (n=198) preserved vs. cryopreserved (n=133) allografts (P=0.06). Blood group compatibility of allografts to recipients had no significant influence on allograft function (P=0.42). The donors allograft origin, whether aortic or pulmonary valve, had also no significant influence on allograft long-term function (P=0.15). Conclusion: For the reconstruction of the right ventricular outflow tract (RVOT) allografts show significantly better long-term durability than xenografts regardless of the age at implantation and the diameter.