Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model

Abstract

Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.

Keywords: costimulation blockade; human decay-accelerating factor; non-human primate; renal transplantation; transgenic pigs; xenoantigen; xenotransplantation; α-1,3-galactosyltransferase.

Fig. 1

Pre-transplant antibody titers against galactose-α1,3-galactose (αGal) knockout pig cells. Thirty-four non-transplanted rhesus macaques were screened for IgG and IgM antibody titers against porcine αGal knockout peripheral blood mononuclear cells. (A) Four monkeys with the lowest IgG titers were selected to receive a porcine αGal knockout/CD55 transgenic renal xenograft. One monkey with the highest IgG titer was also selected. (B) IgM levels varied across treatment group.

Fig. 2

Flow cytometric analysis of peripheral blood in animals receiving T-cell depletion. Peripheral blood immunophenotypic analysis was performed using flow cytometry. Baseline total T-cell, CD8⁺ T-cell, and CD4⁺ T-cell counts were measured prior to administration of T-cell-depleting antibodies. After xenotransplantation, weekly flow cytometry was performed to monitor for reconstitution of T-cell populations. Low-titer, anti-CD154 monkeys are represented by open and filled squares. Low-titer, belatacept monkeys are depicted as open and filled circles. High-titer, anti-CD154 monkey is shown as open triangles. (A) Successful CD8⁺ T-cell depletion was achieved in each monkey with full reconstitution occurring after POD 60. (B) Variable CD4⁺ T-cell depletion was observed among animals, with slow reconstitution of this population over time.

Fig. 3

Serum creatinine and electrolyte levels after pig-to-primate renal xenotransplantation. Serum chemistries were obtained on a weekly basis post-transplant to monitor graft function. Shading depicts normal electrolyte ranges for rhesus macaques. Low-titer, anti-CD154 monkeys are represented by open and filled squares. Low-titer, belatacept monkeys are depicted as open and filled circles. High-titer, anti-CD154 monkey is shown as open triangles. (A) In monkeys with functioning grafts, creatinine values remained within the expected range. (B) Potassium levels remained constant and within normal limits post-transplant. (C) Phosphorus decreased over time, most notably after postoperative day 60. (D) Calcium levels increased after transplant with levels slightly higher than the anticipated range for macaques.

Fig. 4

Gross appearance, histology, and immunofluorescence of pig-to-primate renal xenografts. Protocol renal biopsies were obtained at postoperative day (POD) 14, 35, 70, and 100. At rejection, animals underwent transplant nephrectomy with subsequent histologic analysis. (A, B) Animals that rejected early (POD 6–21) demonstrated gross congestion and hemorrhage in the xenograft at time of rejection. (C, D) H&E staining of rejected renal xenograft in the high-titer monkey demonstrated interstitial hemorrhage and edema. C4d staining was positive, consistent with antibody-mediated rejection. (E–H) Immunofluorescence in the high-titer monkey showed significant deposition of IgG, but not IgM, in the rejected xenograft. Fixed porcine tissue was stained with DAPI (blue) and AF647-anti-IgG or AF647-anti-IgM (red). (E) IgG and (F) IgM staining in a control non-transplanted αGal knockout porcine kidney showed no antibody deposition. (G) Substantial IgG binding was seen in the transplanted renal xenograft at the time of rejection. (H) Minimal IgM staining was seen in the same rejected xenograft. (I, J) H&E staining of rejected xenograft in low-titer, belatacept-treated monkeys showed evidence of antibody-mediated rejection and acute cellular rejection. Both thrombotic microangiopathy (I) and arteritis (J) were seen. (K) H&E staining of protocol renal biopsies in low-titer, anti-CD154-treated monkeys demonstrated preserved renal architecture and no evidence of rejection at POD 100.

Fig. 5

Hemoglobin and platelet measurements after pig-to-primate renal xenotransplantation. Hemoglobin and platelet counts were monitored on a weekly basis. (A) All monkeys started exogenous rhEpo when hemoglobin dropped below 9.5 g/dl. Time of rhEpo initiation ranged from postoperative day 1–20. Two of three animals with histology-proven rejection developed severe anemia at time of rejection. Animals with functioning grafts maintained normal hemoglobin values. (B) Platelet counts remained stable over time in animals without clinical or histologic evidence of rejection. Two animals with antibody-mediated rejection developed significant thrombocytopenia at time of rejection.

Fig. 6

Cytomegalovirus (CMV) titers following T-cell depletion and costimulation blockade. CMV titers were measured weekly using polymerase chain reaction. Standard cutoff for initiation of antiviral therapy is greater than 10 000 copies/ml. However, given the concern for CMV viremia in the setting of T-cell depletion, all monkeys were started on prophylactic ganciclovir beginning the day of transplant. Despite this, two monkeys with the greatest T-cell depletion developed significant CMV viremia. As T-cell counts recovered, CMV titers improved.