Cassandra Long

Impact of Thrombocytopenia on Survival of Baboons with Genetically Modified Pig Liver Transplants: Clinical Relevance

A lack of deceased human donor livers leads to a significant mortality in patients with acute‐on‐chronic or acute (fulminant) liver failure or with primary nonfunction of an allograft. Genetically engineered pigs could provide livers that might bridge the patient to allotransplantation. Orthotopic liver transplantation in baboons using livers from α1,3‐galactosyltransferase gene‐knockout (GTKO) pigs (n = 2) or from GTKO pigs transgenic for CD46 (n = 8) were carried out with a clinically acceptable immunosuppressive regimen. Six of 10 baboons survived for 4–7 days. In all cases, liver function was adequate, as evidenced by tests of detoxification, protein synthesis, complement activity and coagulation parameters. The major problem that prevented more prolonged survival beyond 7 days was a profound thrombocytopenia that developed within 1 h after reperfusion, ultimately resulting in spontaneous hemorrhage at various sites. We postulate that this is associated with the expression of tissue factor on platelets after contact with pig endothelium, resulting in platelet and platelet‐peripheral blood mononuclear cell(s) aggregation and deposition of aggregates in the liver graft, though we were unable to confirm this conclusively. If this problem can be resolved, we would anticipate that a pig liver could provide a period during which a patient in liver failure could be successfully bridged to allotransplantation.

In vitro investigation of pig cells for resistance to human antibody-mediated rejection.

Although human complement‐dependent cytotoxicity (CDC) of α1,3‐galactosyltransferase gene‐knockout (GTKO) pig cells is significantly weaker than that of wild‐type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig – WT, GTKO, GTKO transgenic for H‐transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.

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.

Recipient tissue factor expression is associated with consumptive coagulopathy in pig-to-primate kidney xenotransplantation.

Consumptive coagulopathy (CC) remains a challenge in pig‐to‐primate organ xenotransplantation (Tx). This study investigated the role of tissue factor (TF) expression on circulating platelets and peripheral blood mononuclear cells (PBMCs). Baboons (n = 9) received a kidney graft from pigs that were either wild‐type (n = 2), α1,3‐galactosyltransferase gene‐knockout (GT‐KO; n = 1) or GT‐KO and transgenic for the complement‐regulatory protein, CD46 (GT‐KO/CD46, n = 6). In the baboon where the graft developed hyperacute rejection (n = 1), the platelets and PBMCs expressed TF within 4 h of Tx. In the remaining baboons, TF was detected on platelets on post‐Tx day 1. Subsequently, platelet‐leukocyte aggregation developed with formation of thrombin. In the six baboons with CC, TF was not detected on baboon PBMCs until CC was beginning to develop. Graft histopathology showed fibrin deposition and platelet aggregation (n = 6), but with only minor or no features indicating a humoral immune response (n = 3), and no macrophage, B or T cell infiltration (n = 6). Activation of platelets to express TF was associated with the initiation of CC, whereas TF expression on PBMCs was concomitant with the onset of CC, often in the relative absence of features of acute humoral xenograft rejection. Prevention of recipient platelet activation may be crucial for successful pig‐to‐primate kidney Tx.

Hepatic Function After Genetically Engineered Pig Liver Transplantation in Baboons

Background. If “bridging” to allo-transplantation (Tx) is to be achieved by a pig liver xenograft, adequate hepatic function needs to be assured. Methods. We have studied hepatic function in baboons after Tx of livers from &agr;1,3-galactosyltransferase gene-knockout (GTKO, n=1) or GTKO pigs transgenic for CD46 (GTKO/CD46, n=5). Monitoring was by liver function tests and coagulation parameters. Pig-specific proteins in the baboon serum/plasma were identified by Western blot. In four baboons, coagulation factors were measured. The results were compared with values from healthy humans, baboons, and pigs. Results. Recipient baboons died or were euthanized after 4 to 7 days after internal bleeding associated with profound thrombocytopenia. However, parameters of liver function, including coagulation, remained in the near-normal range, except for some cholestasis. Western blot demonstrated that pig proteins (albumin, fibrinogen, haptoglobin, and plasminogen) were produced by the liver from day 1. Production of several pig coagulation factors was confirmed. Conclusions. After the Tx of genetically engineered pig livers into baboons (1) many parameters of hepatic function, including coagulation, were normal or near normal; (2) there was evidence for production of pig proteins, including coagulation factors; and (3) these appeared to function adequately in baboons although interspecies compatibility of such proteins remains to be confirmed.

Investigation of potential carbohydrate antigen targets for human and baboon antibodies.

Yeh P, Ezzelarab M, Bovin N, Hara H, Long C, Tomiyama K, Sun F, Ayares D, Awwad M, Cooper DKC. Investigation of potential carbohydrate antigen targets for human and baboon antibodies. Xenotransplantation 2010; 17: 197–206.

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.

Human dominant‐negative class II transactivator transgenic pigs – effect on the human anti‐pig T‐cell immune response and immune status

Swine leucocyte antigen (SLA) class II molecules on porcine (p) cells play a crucial role in xenotransplantation as activators of recipient human CD4+ T cells. A human dominant‐negative mutant class II transactivator (CIITA‐DN) transgene under a CAG promoter with an endothelium‐specific Tie2 enhancer was constructed. CIITA‐DN transgenic pigs were produced by nuclear transfer/embryo transfer. CIITA‐DN pig cells were evaluated for expression of SLA class II with/without activation, and the human CD4+ T‐cell response to cells from CIITA‐DN and wild‐type (WT) pigs was compared. Lymphocyte subset numbers and T‐cell function in CIITA‐DN pigs were compared with those in WT pigs. The expression of SLA class II on antigen‐presenting cells from CIITA‐DN pigs was significantly reduced (40–50% reduction compared with WT; P < 0·01), and was completely suppressed on aortic endothelial cells (AECs) even after activation (100% suppression; P < 0·01). The human CD4+ T‐cell response to CIITA‐DN pAECs was significantly weaker than to WT pAECs (60–80% suppression; P < 0·01). Although there was a significantly lower frequency of CD4+ cells in the PBMCs from CIITA‐DN (20%) than from WT (30%) pigs (P < 0·01), T‐cell proliferation was similar, suggesting no significant immunological compromise. Organs and cells from CIITA‐DN pigs should be partially protected from the human cellular immune response.

Genetically-Engineered Pig-to-Baboon Liver Xenotransplantation: Histopathology of Xenografts and Native Organs

Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (α1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4–7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4–7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.

Suppressive efficacy and proliferative capacity of human regulatory T cells in allogeneic and xenogeneic responses.

Background. An understanding of the mechanisms that suppress the human anti-pig cellular response is key for xenotransplantation. We have compared the ability of human regulatory T cells (Tregs) to suppress xenogeneic and allogeneic responses in vitro. Methods. Human peripheral blood mononuclear cells (PBMC), CD4+T cells, or CD4+CD25−T cells were stimulated with irradiated human or wild type (WT) or &agr;1,3-galactosyltransferase gene-knockout (GT-KO) pig PBMC in the presence or absence of human CD4+CD25highTregs. In separate experiments, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester-labeled human CD4+T cells were stimulated with human or pig PBMC. The expansion and precursor frequencies of allo- and xeno-reacitve Tregs were assessed by labeling with FoxP3 mAb and flow cytometric analysis. Results. The responses of human PBMC, CD4+T cells, and CD4+CD25−T cells to pig PBMC were stronger than to human PBMC (P<0.05). Human anti-GT-KO responses were weaker than anti-WT responses (P<0.05). Human CD4+CD25highTregs suppressed proliferation of CD4+CD25−T cells to both human and pig PBMC stimulator cells with the same efficiency. Alloreactive CD4+CD25+FoxP3high responder T cells proliferated more than their xenoreactive counterparts (P<0.05), although xenoreactive CD4+CD25+T cells proliferated more than alloreactive cells (P<0.05). There was no difference in precursor frequency between allo- and xeno-reactive CD4+CD25+FoxP3high cells. Conclusions. Human T-cell responses to pig cells are stronger than to allogeneic cells. The human response to GT-KO PBMC is weaker than to WT PBMC. Although human Tregs can suppress both responses, expansion of CD4+CD25+FoxP3high cells against pig PBMC is weaker than against human PBMC. More human Tregs may be required to suppress the stronger xenogeneic response.