Hayato Iwase

Pig kidney graft survival in a baboon for 136 days: longest life‐supporting organ graft survival to date

The longest survival of a non‐human primate with a life‐supporting kidney graft to date has been 90 days, although graft survival > 30 days has been unusual. A baboon received a kidney graft from an α‐1,3‐galactosyltransferase gene‐knockout pig transgenic for two human complement‐regulatory proteins and three human coagulation‐regulatory proteins (although only one was expressed in the kidney). Immunosuppressive therapy was with ATG+anti‐CD20mAb (induction) and anti‐CD40mAb+rapamycin+corticosteroids (maintenance). Anti‐TNF‐α and anti‐IL‐6R were administered. The baboon survived 136 days with a generally stable serum creatinine (0.6 to 1.6 mg/dl) until termination. No features of a consumptive coagulopathy (e.g., thrombocytopenia, decreased fibrinogen) or of a protein‐losing nephropathy were observed. There was no evidence of an elicited anti‐pig antibody response. Death was from septic shock (Myroides spp). Histology of a biopsy on day 103 was normal, but by day 136, the kidney showed features of glomerular enlargement, thrombi, and mesangial expansion. The combination of (i) a graft from a specific genetically engineered pig, (ii) an effective immunosuppressive regimen, and (iii) anti‐inflammatory agents prevented immune injury and a protein‐losing nephropathy, and delayed coagulation dysfunction. This outcome encourages us that clinical renal xenotransplantation may become a reality.

Systemic inflammation in xenograft recipients precedes activation of coagulation

Dysregulation of coagulation is considered a major barrier against successful pig organ xenotransplantation in non‐human primates. Inflammation is known to promote activation of coagulation. The role of pro‐inflammatory factors as well as the relationship between inflammation and activation of coagulation in xenograft recipients is poorly understood.

Pig‐to‐baboon heterotopic heart transplantation – exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade‐based regimens

Three costimulation blockade‐based regimens have been explored after transplantation of hearts from pigs of varying genetic backgrounds to determine whether CTLA4‐Ig (abatacept) or anti‐CD40mAb+CTLA4‐Ig (belatacept) can successfully replace anti‐CD154mAb.

Clinical significance of regulatory T-cell-related gene expression in peripheral blood after renal transplantation.

Background. Regulatory T cells (Tregs) have been suggested to be deeply associated with immune tolerance and long-term graft survival in transplantation. Some recipients with stable graft function (ST) could possibly minimize immunosuppression during the maintenance period. However, effective assays for assessing the suitability of patients have yet to be established. The purpose of this study was to elucidate the clinical relevance of Treg-related gene expression such as forkhead box P3 (Foxp3) in peripheral blood after renal transplantation. Methods. Several key molecules related to the function of immune cells such as Treg, including Foxp3, transforming growth factor-&bgr;, cytotoxic T-lymphocyte antigen-4, chemokine receptor 7, toll-like receptor 4, granzyme B, T-bet, GATA3, RORC, &agr;1,2-mannosidase, and proteasome subunit &bgr; 10 were examined in the peripheral blood of 272 renal transplant recipients by quantitative real-time reverse-transcriptase polymerase chain reaction. The expression levels were compared between recipients with chronic rejection and ST. Results. Foxp3 messenger RNA (mRNA) levels were reduced immediately after transplantation and gradually recovered. Pretransplantation levels were closely correlated with 1 year posttransplantation levels. Recipients with chronic rejection had significantly lower levels of Foxp3, chemokine receptor 7, and granzyme B mRNA, and higher levels of toll-like receptor 4 and proteasome subunit &bgr; 10 mRNA compared with those with ST, although Foxp3 was the most relevant marker. Conclusion. There is a possibility that monitoring mRNA expression levels of Treg-related molecules in peripheral blood might offer useful information on patient selection and early detection of rejection when immunosuppression minimization strategy is implemented in renal transplantation.

The pathobiology of pig-to-primate xenotransplantation: a historical review.

The immunologic barriers to successful xenotransplantation are related to the presence of natural anti‐pig antibodies in humans and non‐human primates that bind to antigens expressed on the transplanted pig organ (the most important of which is galactose‐α1,3‐galactose [Gal]), and activate the complement cascade, which results in rapid destruction of the graft, a process known as hyperacute rejection. High levels of elicited anti‐pig IgG may develop if the adaptive immune response is not prevented by adequate immunosuppressive therapy, resulting in activation and injury of the vascular endothelium. The transplantation of organs and cells from pigs that do not express the important Gal antigen (α1,3‐galactosyltransferase gene‐knockout [GTKO] pigs) and express one or more human complement‐regulatory proteins (hCRP, e.g., CD46, CD55), when combined with an effective costimulation blockade‐based immunosuppressive regimen, prevents early antibody‐mediated and cellular rejection. However, low levels of anti‐non‐Gal antibody and innate immune cells and/or platelets may initiate the development of a thrombotic microangiopathy in the graft that may be associated with a consumptive coagulopathy in the recipient. This pathogenic process is accentuated by the dysregulation of the coagulation‐anticoagulation systems between pigs and primates. The expression in GTKO/hCRP pigs of a human coagulation‐regulatory protein, for example, thrombomodulin, is increasingly being associated with prolonged pig graft survival in non‐human primates. Initial clinical trials of islet and corneal xenotransplantation are already underway, and trials of pig kidney or heart transplantation are anticipated within the next few years.

Initial in vivo experience of pig artery patch transplantation in baboons using mutant MHC (CIITA-DN) pigs.

BACKGROUND In the pig-to-nonimmunosuppressed baboon artery patch model, a graft from an α1,3-galactosyltransferase gene-knockout pig transgenic for human CD46 (GTKO/CD46) induces a significant adaptive immune response (elicited anti-pig antibody response, increase in T cell proliferation on MLR, cellular infiltration of the graft), which is effectively prevented by anti-CD154mAb-based therapy. METHODS As anti-CD154mAb is currently not clinically applicable, we evaluated whether it could be replaced by CD28/B7 pathway blockade or by blockade of both pathways (using belatacept + anti-CD40mAb [2C10R4]). We further investigated whether a patch from a GTKO/CD46 pig with a mutant human MHC class II transactivator (CIITA-DN) gene would allow reduction in the immunosuppressive therapy administered. RESULTS When grafts from GTKO/CD46 pigs were transplanted with blockade of both pathways, a minimal or insignificant adaptive response was documented. When a GTKO/CD46/CIITA-DN graft was transplanted, but no immunosuppressive therapy was administered, a marked adaptive response was documented. In the presence of CD28/B7 pathway blockade (abatacept or belatacept), there was a weak adaptive response that was diminished when compared with that to a GTKO/CD46 graft. Blockade of both pathways prevented an adaptive response. CONCLUSION Although expression of the mutant MHC CIITA-DN gene was associated with a reduced adaptive immune response when immunosuppressive therapy was inadequate, when blockade of both the CD40/CD154 and CD28/B7 pathways was present, the response even to a GTKO/CD46 graft was suppressed. This was confirmed after GTKO/CD46 heart transplantation in baboons.

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein

We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway‐regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life‐supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri‐transplant platelet activation (as β‐thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts

Genetically engineered pigs could provide a source of kidneys for clinical transplantation. The two longest kidney graft survivals reported to date have been 136 and 310 days, but graft survival >30 days has been unusual until recently.

The role of platelets in coagulation dysfunction in xenotransplantation, and therapeutic options

Xenotransplantation could resolve the increasing discrepancy between the availability of deceased human donor organs and the demand for transplantation. Most advances in this field have resulted from the introduction of genetically engineered pigs, e.g., α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs transgenic for one or more human complement‐regulatory proteins (e.g., CD55, CD46, CD59). Failure of these grafts has not been associated with the classical features of acute humoral xenograft rejection, but with the development of thrombotic microangiopathy in the graft and/or consumptive coagulopathy in the recipient. Although the precise mechanisms of coagulation dysregulation remain unclear, molecular incompatibilities between primate coagulation factors and pig natural anticoagulants exacerbate the thrombotic state within the xenograft vasculature. Platelets play a crucial role in thrombosis and contribute to the coagulation disorder in xenotransplantation. They are therefore important targets if this barrier is to be overcome. Further genetic manipulation of the organ‐source pigs, such as pigs that express one or more coagulation‐regulatory genes (e.g., thrombomodulin, endothelial protein C receptor, tissue factor pathway inhibitor, CD39), is anticipated to inhibit platelet activation and the generation of thrombus. In addition, adjunctive pharmacologic anti‐platelet therapy may be required. The genetic manipulations that are currently being tested are reviewed, as are the potential pharmacologic agents that may prove beneficial.

Comparison of hematologic, biochemical, and coagulation parameters in α1,3-galactosyltransferase gene-knockout pigs, wild-type pigs, and four primate species

Ekser B, Bianchi J, Ball S, Iwase H, Walters A, Ezzelarab M, Veroux M, Gridelli B, Wagner R, Ayares D, Cooper DKC. Comparison of hematologic, biochemical, and coagulation parameters in α1,3‐galactosyltransferase gene‐knockout pigs, wild‐type pigs, and four primate species. Xenotransplantation 2012; 19: 342–354.