Islet xenotransplantation using gal-deficient neonatal donors improves engraftment and function

Abstract

Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose-α1,3-galactose, the gal epitope, has emerged as the principal target of rejection in pig-to-primate models of solid organ transplant, the importance of gal-specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal-expressing wild-type (WT) or gal-deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin-independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies.

Figure 1. Islets from galactosyl transferase-hemizygous neonatal porcine donors express Gal

(a) Islet transplant preparations isolated from WT neonatal donors had significantly higher Gal expression as determined by flow cytometric analysis than GTKO counterparts (p<0.008, n=5); GTKO islet Gal expression was not significantly different than WT islets stained with an isotype control. One WT islet preparation had an MFI of Gal expression that was significantly lower than other WT preparations (denoted with^†). (b) Histogram demonstrating Gal expression phenotype for islet preparations from representative GTKO and WT donors compared to an isotype control. One WT islet preparation demonstrated an intermediate Gal expression phenotype (represented with dashed line). (c,d) Immunohistochemical analysis of transplanted intrahepatic islets demonstrates presence of Gal epitope (brown stain) on WT (d) but not GTKO (c) islets. P-value determined using Mann-Whitney test. **p <0.01; MFI – mean fluorescence intensity; IC – isotype control; Gal-INT^† – intermediate Gal expression phenotype.

Figure 2. Gal-expressing neonatal porcine islets demonstrate increased immunogenicity in vitro

WT NPIs were more susceptible to binding of (a) IgG (p=0.002, n=10) and IgM xenoantibody (p<0.05, n=10) and (b) complement components C1 (p=0.03, n=7) and C3 (p<0.02, n=7) than GTKO NPIs when incubated with naïve recipient sera in a flow cytometric assay. (c) More WT NPIs showed evidence of injury as determined by uptake of 7-AAD viability dye following incubation with naïve monkey sera (p<0.008, n=8). This effect was not noted following incubation with heat-inactivated serum or complement alone. P-values determined using paired wilcoxon signed rank test. *p <0.05; **p <0.01; HIS – heat-inactivated serum; comp – rabbit complement.

Figure 3. Experimental timeline and immunosuppressive protocol

All nonhuman primate xenoislet recipients in this study received identical immunosuppression. Arrows indicate individual infusions, with arrow size indicating relative dose. This regimen was similar to our previously described xenoislet immunosuppressive protocols (2), with several important differences: in the current protocol CTLA4Ig was used instead of belatacept to achieve CD28/B7 costimulatory pathway blockade, oral mycophenolate mofeltil was given instead of IM rapamycin and begun 3 weeks after transplant, TS-1/22 replaced basiliximab, and the anti-CD154 clone 5C8 was used instead of H106. In addition, recipients in the current study were rendered diabetic with streptozocin instead of pancreatectomy. STZ – streptozocin. MMF – mycophenolate mofetil.

Figure 4. Transplantation of Gal-deficient islets results in superior graft function and more rapid return of euglycemia

(a-b) WT islet recipients (a) most often experienced primary graft dysfunction, while transplantation of GTKO NPIs (b) resulted in prolonged insulin-independent normoglycemia, as shown in this representative pair. (c) Time to insulin independence was significantly shorter following transplantation of GTKO NPIs (p=0.03); however, duration of rejection-free survival (d) was similar for the two treatment groups (p=0.3). In (c), censored events represent recipient sacrifice. P-values calculated using logrank test.

Figure 5. Gal-deficient islets induce less liver inflammation but similar xenoantibody levels as Gal-positive islets

(a-b) WT islet recipients experienced a greater increase from baseline in the liver enzymes AST (c) and ALT (d); this difference was significant for AST on days 21 (p=0.03) and 28 (p=0.03) post-transplant. (e-f) All WT and GTKO islet recipients experienced a transient increase in anti-Gal IgM and a smaller increase in anti-Gal IgG antibodies, which peaked ~21 days post-transplant. There was no significant difference in antibody levels between groups at any timepoint. Alloislet recipients (n=4), shown for comparison, demonstrated a similar pattern of increase in anti-Gal IgM and IgG following transplant. P-values calculated using Mann-Whitney Test. *p <0.05; BG – blood glucose; POD – post-op day; AST – aspartate aminotransferase; ALT – alanine aminotransferase; ODU – optical density units.

Figure 6. Gal-deficient islets are not protected from eventual cellular rejection

Immunohistochemical analysis of intrahepatic islets from recipients experiencing functional xenograft rejection demonstrated dense lymphocytic infiltrates with loss of insulin positivity. This infiltrate stained heavily for CD3+ T cells, and moderately for CD68+ macrophages. CD20+ B cells and neutrophils did not appear to comprise a significant portion of the infiltrating cells.