Pretransplant Screening for Prevention of Hyperacute Graft Loss in Pig-to-primate Kidney Xenotransplantation

Abstract

Background: Xenotransplantation using pig organs is now a clinical reality. However, the process for xenograft recipient screening lacks clarity and scientific rigor: no established thresholds exist to determine which levels of preformed antipig natural antibodies (Nabs) will be safe for clinical xenograft transplantation, and hyperacute rejection (HAR) or acute humoral xenograft rejection (AHXR), which still impacts pig-to-primate kidney xenograft survivals, may impede broader application of pig-to-human clinical xenograft transplantation.

Methods: We retrospectively examined 28 cases of pig-to-baboon kidney xenotransplantation using GalTKO±human complement regulatory protein (hCRP)-transgenic (Tg) pig donors, as well as 6 cases of triple-KO multi-Tg (10GE) pig donors, and developed screening algorithms to predict risk of HAR/AHXR based on recipient antipig Nab levels. Preformed Nabs were evaluated using both complement-dependent cytotoxicity and antibody (IgM and IgG) binding flow-cytometry assays.

Results: High complement-dependent cytotoxicity was associated with HAR/AHXR as expected. However, we also found that high levels of IgG were independently associated with HAR/AHXR, and we developed 2 indices to interpret and predict the risk of IgG-mediated HAR/AHXR.

Conclusions: Based on the data in this study, we have established a new 2-step screening, which will be used for future clinical kidney xenotransplantation trials.

Figure1.

Graft findings in representative cases of rejected porcine kidney grafts. (A) The baboon 17p9 received GalTKO kidney and rejected the graft hyperacutely in two days after transplantation. The kidney graft on POD2 was dark, firm and hemorrhagic (left top). Hematoxylin and eosin staining shows hemostasis and interstitial hemorrhage in the kidney graft. Immunofluorescence of IgM, IgG, C3, C4d, and C5b (right column) showed deposition of antibodies and complements on glomeruli which is diagnostic of antibody-mediated rejection. (B) Kidney graft transplanted into the baboon 16p52 from GalTKO+hCRP-Tg pig donor. The graft turned dark purple in an hour after revascularization, and the graft was explanted. Pathological findings revealed severe interstitial hemorrhage and hemostasis in the kidney graft.

Figure2.

CDC. (A, B) In overall cases and Group1 analyses, CDC was significantly lower in the cases without HAR/AHXR (P=0.0165 and 0.0014, respectively). (C) CDC was not significantly different in Group2.

Figure3.

IgM antibody index. IgM indices were not significantly different between the cases with HAR/AHXR and without rejection. (A, B: overall cases, C, D: Group1, E, F: Group2).

Figure4.

IgG antibody index. (A, B) Both IgG index-1 and IgG index-2 are significantly higher in the cases with HAR/AHXR (P=0.0006 and 0.0046, respectively). (C, D) In Group1, IgG index-1 is significantly higher in the case with HAR/AHXR, while modified index is not. (P=0.0076 and 0.2695, respectively) (E, F) In Group2, both IgG index-1 and index-2 are significantly higher in the case with HAR/AHXR, but IgG index-2 appears to have stronger correlation (P=0.0119 and 0.0056, respectively).

Figure5.

Correlations between CDC and IgM/IgG MFI. There was significant correlation between IgM MFI and CDC (P=0.0173, r=0.4462), while there was no correlation between IgG MFI and CDC (P=0.4533, r=0.1477).

Figure6.

Recipient screening flow chart for GalTKO+/−hCRP-Tg source pig

Figure7.

Recipient screening flow chart for triple-KO (GalTKO, B4-KO, CMAH-KO with additional genetic modifications) source pig