RESUMEN
BACKGROUND: We investigated in vitro whether HLA highly sensitized patients with end-stage renal disease will be disadvantaged immunologically after a genetically engineered pig kidney transplant. METHODS: Blood was drawn from patients with a calculated panel-reactive antibody (cPRA) 99% to 100% (Gp1, n = 10) or cPRA 0% (Gp2, n = 12), and from healthy volunteers (Gp3, n = 10). Serum IgM and IgG binding was measured (i) to galactose-α1-3 galactose and N-glycolylneuraminic acid glycans by enzyme-linked immunosorbent assay, and (ii) to pig red blood cell, pig aortic endothelial cells, and pig peripheral blood mononuclear cell from α1,3-galactosyltransferase gene-knockout (GTKO)/CD46 and GTKO/CD46/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (CMAHKO) pigs by flow cytometry. (iii) T-cell and B-cell phenotypes were determined by flow cytometry, and (iv) proliferation of T-cell and B-cell carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction. RESULTS: (i) By enzyme-linked immunosorbent assay, there was no difference in IgM or IgG binding to galactose-α1-3 galactose or N-glycolylneuraminic acid between Gps1 and 2, but binding was significantly reduced in both groups compared to Gp3. (ii) IgM and IgG binding in Gps1 and 2 was also significantly lower to GTKO/CD46 pig cells than in healthy controls, but there were no differences between the 3 groups in binding to GTKO/CD46/CMAHKO cells. (iii and iv) Gp1 patients had more memory T cells than Gp2, but there was no difference in T or B cell proliferation when stimulated by any pig cells. The proliferative responses in all 3 groups were weakest when stimulated by GTKO/CD46/CMAHKO pig peripheral blood mononuclear cell. CONCLUSIONS: (i) End-stage renal disease was associated with low antipig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T-cell and B-cell response.
Asunto(s)
Galactosiltransferasas/genética , Antígenos HLA/inmunología , Fallo Renal Crónico/cirugía , Trasplante de Riñón/métodos , Proteína Cofactora de Membrana/genética , Oxigenasas de Función Mixta/genética , Animales , Animales Modificados Genéticamente , Linfocitos B/inmunología , Estudios de Casos y Controles , Células Cultivadas , Galactosiltransferasas/deficiencia , Galactosiltransferasas/inmunología , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Antígenos HLA/sangre , Xenoinjertos , Humanos , Memoria Inmunológica , Isoanticuerpos/sangre , Isoanticuerpos/inmunología , Fallo Renal Crónico/sangre , Fallo Renal Crónico/diagnóstico , Fallo Renal Crónico/inmunología , Trasplante de Riñón/efectos adversos , Activación de Linfocitos , Proteína Cofactora de Membrana/deficiencia , Proteína Cofactora de Membrana/inmunología , Oxigenasas de Función Mixta/deficiencia , Oxigenasas de Función Mixta/inmunología , Sus scrofa , Linfocitos T/inmunologíaRESUMEN
The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46, with islet beta cell-specific expression of human tissue factor pathway inhibitor and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4 or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n = 5). Immunosuppression was based on anti-CD154 mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only two of five demonstrating function beyond 5 months.