Carbohydrate antigen expression and anti-pig antibodies in New World capuchin monkeys: Relevance to studies of xenotransplantation.

BACKGROUND: Old World non-human primates (OWNHPs) are used for preclinical pig-to-NHP studies. However, like pigs, OWNHPs express Neu5Gc, and therefore do not develop natural anti-Neu5Gc antibodies. New World NHPs (NWNHPs) have been reported not to express Neu5Gc. We investigated the potential of NWNHPs in xenotransplantation research. METHODS: We investigated expression of Gal, Neu5Gc, and Sda antigens on RBCs and PBMCs from humans, selected OWNHPs, and capuchin monkeys (a NWNHP). Serum anti-Gal and anti-Neu5Gc IgM and IgG levels were measured by ELISA. Binding of primate serum IgM and IgG to pig RBCs was measured by flow cytometry. RESULTS: (a) Neither humans, OWNHPs, or capuchin monkeys expressed Gal on their RBCs, but capuchins expressed Gal on PBMCs. Humans and capuchins did not express Neu5Gc on either RBCs or PBMCs, but OWNHPs expressed Neu5Gc on both cells. Sda was not expressed on any RBCs or PBMCs. (b) By ELISA, human and OWNHP, but not capuchin, sera showed IgM and IgG binding to Gal. Human and capuchin, but not OWNHP, sera demonstrated some binding to Neu5Gc. (c) Anti-Sda IgM/IgG antibodies were detected in OWNHP sera. Knockout of Sda on pig RBCs did not significantly reduce human and capuchin antibody binding. CONCLUSION: Capuchin monkeys could be surrogates for humans in experiments using RBCs, islets, neuronal cells, etc, from triple-knockout pigs (but may be too small to be used as recipients of pig organ grafts).

Development of retrocorneal membrane following pig-to-monkey penetrating keratoplasty.

Recent reports of long-term survival after wild-type (WT) pig-to-monkey corneal xenotransplantation are encouraging. We experienced the rapid development of retrocorneal membranes, a rare complication after corneal allotransplantation (although seen in infants and young children). The original specific aim of the study was to determine the factors associated with successful (young) pig corneal transplantation in monkeys. However, when it was obvious that retrocorneal membranes rapidly developed, our aims became to determine the factors involved in its development after both WT and Genetically engineered (GE ) pig corneal xenotransplantation and to investigate the characteristics of the retrocorneal membrane. Rhesus monkeys were recipients of penetrating keratoplasty using WT and GE pigs (n=2, respectively, 1-3 months old). Local/systemic steroids were administered for 3 months. Grafts were evaluated by slit lamp for corneal transparency, edema, and neovascularization. Hematoxylin and eosin, Masson trichrome staining, and immunohistochemical analysis were performed. Gal staining was also carried out to distinguish the origin of the membrane. All penetrating keratoplasty recipients developed fibrous retrocorneal membranes in the early post-transplantation period, regardless of whether the graft was from a WT or GE pig. There were no features of rejection, with no cell infiltrate in the graft or anterior chamber during the three-month follow-up. There was no difference in the clinical course between the two groups (WT or GE corneas). Immunohistochemistry indicated that the retrocorneal membranes were CK negative, α-SMA positive, and vimentin positive, suggesting that they were of fibrous (keratocytic) origin. Also, the membrane was Gal positive, suggesting that it is derived from pig cornea. Following pig-to-monkey corneal xenotransplantation, we report that retrocorneal membranes are derived from donor pig keratocytes. Prevention of retrocorneal membranes will be necessary to achieve successful corneal xenotransplantation.

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells.

The results of the assay for measuring anti-non-Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-non-Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human, naive, and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3 hour) and concentrations (5, 10, 20, and 40 µL) was determined by flow cytometry. An increase in incubation time from 30 minutes to 2 hour was associated with increases in anti-non-Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti-non-Gal IgM (1.5 times) and a minimal increase in anti-non-Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2-hour incubation was 1.5 times and 2 times greater than after 30-minutes incubation, respectively, whereas naïve baboon sera showed minimal (non-significant) increase in anti-non-Gal IgM/IgG antibody binding. With 2-hour incubation, increasing the serum concentration from 5 µL to 20 µL significantly increased antibody binding to non-Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-non-Gal antibodies, without affecting cell viability in vitro.

Therapeutic regulation of systemic inflammation in xenograft recipients.

Inflammation is known to preclude tolerance after transplantation. We have previously shown that systemic inflammation in xenograft recipients (SIXR) precedes activation of coagulation in the absence of T cell responses. Accordingly, SIXR may amplify innate and adaptive immune responses against xenografts after pig-to-primate xenotransplantation, even with efficient immunosuppressive therapy. We evaluated the impact of anti-inflammatory agents on pro-inflammatory cytokines and chemokines in pig artery patch and heart xenograft recipients. Baboons received an artery patch (Group1, n=8) or heart (Group2, n=4) from genetically engineered pigs. All baboons received lymphodepletion with thymoglobulin (ATG) and costimulation blockade-based immunosuppression (anti-CD40 and/or CTLA4Ig). In Group1, baboons received either (i) no anti-inflammatory agents (n=2), (ii) cobra venom factor (CVF, n=2), (iii) α1-antitrypsin (AAT, n=2), or (iv) interleukin (IL)-6 receptor antagonist (IL-6RA, n=2). In Group2, all baboon received corticosteroids, either without (n=2) or with (n=2) IL-6RA. Serum IFN-γ, TNF-α, IL-1ß, IL-17, IL-6, IL-8, MCP-1, and sCD40L levels were measured by Luminex. Fibrinogen, D-dimers, and C-reactive protein (C-RP) were also measured. Recipient baboon T cell proliferation was evaluated by mixed lymphocyte reaction (MLR) before and after transplantation. Pig and baboon tissue factor (TF) mRNA levels in heart xenografts were measured by RT-PCR. In no recipient was a marked increase in T cell response to pig cells observed after transplantation. In Groups 1 and 2, post-transplantation levels of IFN-γ, TNF-α, IL-1ß, and IL-17 remained comparable to or lower than pre-transplant levels, except in one heart recipient that succumbed to CMV infection. In Group1, when no anti-inflammatory agent was administered, post-transplant levels of IL-6, IL-8, and MCP-1 were elevated. After CVF, IL-6, IL-8, and MCP-1 remained low. After IL-6RA, IL-6 and MCP-1 were elevated. After AAT, IL-8 was elevated. sCD40L became elevated intermittently in most recipients irrespective of the administered anti-inflammatory agent. In Group2, IL-6 was transiently elevated, particularly after IL-6RA administration. MCP-1 gradually increased by 2 months in Group2 recipients. sCD40L generally remained low except in one recipient. In Group1 and Group2 recipients, C-RP levels were elevated except after IL-6RA administration, while D-dimers were elevated regardless of administration of anti-inflammatory agent. In Group2, pig TF mRNA levels were increased in heart xenografts compared to naive pig hearts, irrespective of IL-6 receptor antagonist administration. Additionally, baboon TF mRNA levels were detectable in heart xenografts, but not in naive pig hearts. Some pro-inflammatory cytokines and chemokines are elevated in xenograft recipients, even with efficient T cell-directed immunosuppressive therapy. Persistent elevation of D-dimers, and individual cytokines and chemokines suggest a continuous inflammatory response, despite administration of anti-inflammatory agents. Systemic administration of combined anti-inflammatory agents as well as complement regulation may be essential to prevent SIXR after xenotransplantation.

Transplantation of hepatocytes from genetically engineered pigs into baboons.

BACKGROUND: Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. METHODS: Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5 weeks after hepatocyte transplantation. The baboon immune response was monitored by the measurement of anti-non-Gal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. RESULTS: Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low-500-1000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. DISCUSSION AND CONCLUSIONS: As a result of this disappointing experience, the following points need to be considered. (i) Were the isolated pig hepatocytes functionally viable? (ii) Are pig hepatocytes more immunogenic than pig hearts, kidneys, artery patch grafts, or islets? (iii) Does injection of pig cells (antigens) into the spleen and/or lymph nodes stimulate a greater immune response than when pig tissues are grafted at other sites? (iv) Did the presence of the recipient's intact liver prevent survival and proliferation of pig hepatocytes? (v) Is pig CD47-primate SIRP-α compatibility essential? In conclusion, the transplantation of genetically engineered pig hepatocytes into multiple sites in immunosuppressed baboons was associated with very early graft failure. Considerable further study is required before clinical trials should be undertaken.

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

BACKGROUND: 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. METHODS: Donor pigs (n=4) were on an α1,3-galactosyltransferase gene-knockout (GTKO)/human complement regulatory protein (CD46) background (GTKO/CD46). In addition, the pigs were transgenic for at least one human coagulation regulatory protein. Two baboons received a kidney from a six-gene pig (GroupA) and two from a three-gene pig (GroupB). Immunosuppressive therapy was identical in all four cases and consisted of anti-thymoglobulin (ATG)+anti-CD20mAb (induction) and anti-CD40mAb+rapamycin+corticosteroids (maintenance). Anti-TNF-α and anti-IL-6R mAbs were administered to reduce the inflammatory response. Baboons were followed by clinical/laboratory monitoring of immune/coagulation/inflammatory/physiological parameters. At biopsy or euthanasia, the grafts were examined by microscopy. RESULTS: The two GroupA baboons remained healthy with normal renal function >7 and >8 months, respectively, but then developed infectious complications. However, no features of a consumptive coagulopathy, eg, thrombocytopenia and reduction of fibrinogen, or of a protein-losing nephropathy were observed. There was no evidence of an elicited anti-pig antibody response, and histology of biopsies taken at approximately 4, 6, and 7 months and at necropsy showed no significant abnormalities. In contrast, both GroupB baboons developed features of a consumptive coagulopathy and required euthanasia on day 12. CONCLUSIONS: The combination of (i) a graft from a specific six-gene genetically modified pig, (ii) an effective immunosuppressive regimen, and (iii) anti-inflammatory therapy prevented immune injury, a protein-losing nephropathy, and coagulation dysfunction for >7 months. Although the number of experiments is very limited, our impression is that expression of human endothelial protein C receptor (±CD55) in the graft is important if coagulation dysregulation is to be avoided.

Human antibody recognition of xenogeneic antigens (NeuGc and Gal) on porcine heart valves: could genetically modified pig heart valves reduce structural valve deterioration?

BACKGROUND: Glutaraldehyde-fixed bioprosthetic heart valves (GBHVs) derived from wild-type (WT, genetically unmodified) pigs are widely used clinically for heart valve replacement. There is evidence that their failure is related to an immune response. The use of valves from genetically engineered pigs that do not express specific pig antigens may prolong GBHV survival. Our aims were to determine (i) expression of Gal and NeuGc on heart (aortic and pulmonary) valves and pericardium of WT, α1,3-galactosyltransferase gene knockout (GTKO) and GTKO/N-glycolylneuraminic acid gene-knockout (GTKO/NeuGcKO) pigs in comparison with three different commercially available GBHVs and (ii) to determine human antibody binding to these tissues. METHODS: Wild-type, GTKO/CD46, and GTKO/CD46/NeuGcKO pig valves and pericardium were tested (i) fresh and (ii) after fixation with glutaraldehyde (0.02%, 0.2%, 2%). Sections of GBHVs, fresh and fixed valves, and pericardium were stained for Gal and NeuGc expression, and for human IgM and IgG antibody binding. RESULTS: Gal and NeuGc expression was high on all GBHVs and WT pig valves/pericardium, but was absent after antigen-specific-knockout. There was no difference in antigen expression or antibody binding among WT aortic, pulmonary valves, and pericardium as well as GBHVs. Glutaraldehyde fixation did not alter expression of Gal or NeuGc. After incubation with human serum, human IgM and IgG bound to all GBHVs and WT pig valves/pericardium. Valves from GTKO/CD46 pigs and, particularly, GTKO/CD46/NeuGcKO pigs (with/without glutaraldehyde fixation) showed less IgM and IgG binding. CONCLUSION: Compared to WT pigs, GTKO/CD46/NeuGcKO pigs would be preferable sources of GBHVs, because the absence of Gal/NeuGc expression reduces human antibody binding.

Thyroid hormone: relevance to xenotransplantation.

BACKGROUND: It has been well documented that the level of serum/plasma free triiodothyronine (fT3) falls rapidly following brain death or during certain surgical procedures, for example, heart surgery carried out on cardiopulmonary bypass. The level in patients following cardiopulmonary bypass usually recovers within 2 days. METHODS: We have measured serum fT3 in healthy naïve baboons (n = 31), healthy naïve monkeys (n = 5), and after pig-to-baboon heterotopic heart xenotransplantation (xenoTx) (Group 1, n = 9), orthotopic liver xenoTx (Group 2, n = 10), artery patch xenoTx (Group 3, n = 9), and in monkey-to-monkey heterotopic heart alloTx (Group 4, n = 5). RESULTS: The mean level of fT3 in healthy naïve baboons was 3.1 ± 0.9 pg/ml and in healthy naïve monkeys was 2.6 ± 0.3 pg/ml. Following pig heart, liver, and artery patch xenoTx and monkey heart alloTx, there was an immediate rapid fall in fT3 level. Recovery of fT3 was more rapid in Groups 3 and 4 than in Groups 1 and 2. In Group 1, within 4 days fT3 had recovered, but only to the lower limit of normal range, where it remained throughout follow-up (for up to 42 days). In Group 2, no recovery was seen during the 7 days of follow-up. In immunosuppressed baboons with pig patch grafts that received IL-6R blockade (n = 2), the fT3 tended to rise higher than in those that received no IL-6R blockade (n = 6). CONCLUSIONS: Following operative procedures, there is a dramatic fall in serum fT3 levels. The persistent low level of fT3 after pig heart and liver xenoTx may be associated with a continuing inflammatory state. We suggest that consideration should be given to the replacement of T3 therapy to maintain normal fT3 levels, particularly in nonhuman primates undergoing orthotopic pig heart or liver xenoTx.

Initial in vitro studies on tissues and cells from GTKO/CD46/NeuGcKO pigs.

BACKGROUND: The impact that the absence of expression of NeuGc in pigs might have on pig organ or cell transplantation in humans has been studied in vitro, but only using red blood cells (pRBCs) and peripheral blood mononuclear cells (pPBMCs) as the target cells for immune assays. We have extended this work in various in vitro models and now report our initial results. METHODS: The models we have used involve GTKO/hCD46 and GTKO/hCD46/NeuGcKO pig aortas and corneas, and pRBCs, pPBMCs, aortic endothelial cells (pAECs), corneal endothelial cells (pCECs), and isolated pancreatic islets. We have investigated the effect of the absence of NeuGc expression on (i) human IgM and IgG binding, (ii) the T-cell proliferative response, (iii) human platelet aggregation, and (iv) in an in vitro assay of the instant blood-mediated inflammatory reaction (IBMIR) following exposure of pig islets to human blood/serum. RESULTS: The lack of expression of NeuGc on some pig tissues (aortas, corneas) and cells (RBCs, PBMCs, AECs) significantly reduces the extent of human antibody binding. In contrast, the absence of NeuGc expression on some pig tissues (CECs, isolated islet cells) does not reduce human antibody binding, possibly due to their relatively low NeuGc expression level. The strength of the human T-cell proliferative response may also be marginally reduced, but is already weak to GTKO/hCD46 pAECs and islet cells. We also demonstrate that the absence of NeuGc expression on GTKO/hCD46 pAECs does not reduce human platelet aggregation, and nor does it significantly modify the IBMIR to pig islets. CONCLUSION: The absence of NeuGc on some solid organs from GTKO/hCD46/NeuGcKO pigs should reduce the human antibody response after clinical transplantation when compared to GTKO/hCD46 pig organs. However, the clinical benefit of using certain tissue (e.g., cornea, islets) from GTKO/hCD46/NeuGcKO pigs is questionable.

Systemic inflammation in xenograft recipients precedes activation of coagulation.

BACKGROUND: 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. METHODS: Baboons received kidney (n=3), heart (n=4), or artery patch (n=8) xenografts from α1,3-galactosyltransferase gene-knockout (GTKO) pigs or GTKO pigs additionally transgenic for human complement-regulatory protein CD46 (GTKO/CD46). Immunosuppression (IS) was based on either CTLA4Ig or anti-CD154 costimulation blockade. Three artery patch recipients did not receive IS. Pro-inflammatory cytokines, chemokines, and coagulation parameters were evaluated in the circulation after transplantation. In artery patch recipients, monocytes and dendritic cells (DC) were monitored in peripheral blood. Expression of tissue factor (TF) and CD40 on monocytes and DC were assessed by flow cytometry. C-reactive protein (C-RP) levels in the blood and C-RP deposition in xenografts as well as native organs were evaluated. Baboon and pig C-RP mRNA in heart and kidney xenografts were evaluated. RESULTS: In heart and kidney xenograft recipients, the levels of INFγ, TNF-α, IL-12, and IL-8 were not significantly higher after transplantation. However, MCP-1 and IL-6 levels were significantly higher after transplantation, particularly in kidney recipients. Elevated C-RP levels preceded activation of coagulation in heart and kidney recipients, where high levels of C-RP were maintained until the time of euthanasia in both heart and kidney recipients. In artery patch recipients, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were elevated with no IS, while IL-6 was not. With IS, INFγ, TNF-α, IL-12, IL-8, and MCP-1 were reduced, but IL-6 was elevated. Elevated IL-6 levels were observed as early as 2 weeks in artery patch recipients. While IS was associated with reduced thrombin activation, fibrinogen and C-RP levels were increased when IS was given. There was a significant positive correlation between C-RP, IL-6, and fibrinogen levels. Additionally, absolute numbers of monocytes were significantly increased when IS was given, but not without IS. This was associated with increased CD40 and TF expression on CD14+ monocytes and lineage(neg) CD11c+ DC, with increased differentiation of the pro-inflammatory CD14+ CD11c+ monocyte population. At the time of euthanasia, C-RP deposition in kidney and heart xenografts, C-RP positive cells in artery patch xenograft and native lungs were detected. Finally, high levels of both pig and baboon C-RP mRNA were detected in heart and kidney xenografts. CONCLUSIONS: Inflammatory responses precede activation of coagulation after organ xenotransplantation. Early upregulation of C-RP and IL-6 levels may amplify activation of coagulation through upregulation of TF on innate immune cells. Prevention of systemic inflammation in xenograft recipients (SIXR) may be required to prevent dysregulation of coagulation and avoid excessive IS after xenotransplantation.

Glucose metabolism in pigs expressing human genes under an insulin promoter.

BACKGROUND: Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. METHODS: On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. RESULTS: This preliminary study did not show definite evidence of ß-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. CONCLUSIONS: Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models.

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.

Further evidence for sustained systemic inflammation in xenograft recipients (SIXR).

INTRODUCTION: In pig-to-baboon heart/artery patch transplantation models, adequate costimulation blockade prevents a T-cell response. After heart transplantation, coagulation dysfunction (thrombocytopenia, reduced fibrinogen, increased D-dimer) and inflammation (increased C-reactive protein [CRP]) develop. We evaluated whether coagulation dysfunction and/or inflammation can be detected following pig artery patch transplantation. METHODS: Baboons received heart (n = 8) or artery patch (n = 16) transplants from genetically engineered pigs and a costimulation blockade-based regimen. Heart grafts functioned for 15-130 days. Artery recipients were euthanized after 28-84 days. Platelet counts, fibrinogen, D-dimer, and CRP were measured. RESULTS: Thrombocytopenia and reduced fibrinogen developed only in recipients of hearts not expressing a coagulation-regulatory protein (n = 4), but not in other heart or patch recipients. However, in heart recipients (n = 8), there were sustained increases in D-dimer (<0.5 to 1.9 ug/ml [P < 0.01]) and CRP (0.26-2.2 mg/dl [P < 0.01]). In recipients of artery patches, there were also sustained increases in D-dimer (<0.5 to 1.4 ug/ml [P < 0.01]) and CRP (0.26 to 1.5 mg/dl [P < 0.001]). An IL-6R antagonist suppressed the increase in CRP, but not D-dimer. CONCLUSION: The pig artery patch model has proved valuable for determining immunosuppressive regimens that prevent sensitization to pig antigens. This model also provides information on the sustained systemic inflammation in xenograft recipients (SIXR). An IL-6R antagonist may help suppress this response.

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

BACKGROUND: 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. METHODS: All pigs were on an α1,3-galactosyltransferase gene-knockout/CD46 transgenic (GTKO.CD46) background. Hearts transplanted into Group A baboons (n=4) expressed additional CD55, and those into Group B (n=3) expressed human thrombomodulin (TBM). Immunosuppression included anti-thymocyte globulin with anti-CD154mAb (Regimen 1: n=2) or abatacept (Regimen 2: n=2) or anti-CD40mAb+belatacept (Regimen 3: n=2). Regimens 1 and 2 included induction anti-CD20mAb and continuous heparin. One further baboon in Group B (B16311) received a modified Regimen 1. Baboons were followed by clinical/laboratory monitoring of immune/coagulation parameters. At biopsy, graft failure, or euthanasia, the graft was examined by microscopy. RESULTS: Group A baboons survived 15 to 33 days, whereas Group B survived 52, 99, and 130 days, respectively. Thrombocytopenia and reduction in fibrinogen occurred within 21 days in Group A, suggesting thrombotic microangiopathy (TM), confirmed by histopathology. In Group B, with follow-up for >4 m, areas of myofiber degeneration and scarring were seen in two hearts at necropsy. A T-cell response was documented only in baboons receiving Regimen 2. CONCLUSIONS: The combination of anti-CD40mAb+belatacept proved effective in preventing a T-cell response. The expression of TBM prevented thrombocytopenia and may possibly delay the development of TM and/or consumptive coagulopathy.

Role of P-selectin and P-selectin glycoprotein ligand-1 interaction in the induction of tissue factor expression on human platelets after incubation with porcine aortic endothelial cells.

Development of coagulation disorders remains a major challenge in pig-to-primate organ xenotransplantation. Our previous studies demonstrated that porcine aortic endothelial cells (pAEC) activate human platelets to express tissue factor (TF). In this study, we investigated the molecular interaction between human platelets and pAEC to identify possible targets for further genetic modification and/or systemic therapy. Human platelets were incubated with pAEC from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO pigs expressing human CD46, after which the platelets were analyzed for TF expression, TF mRNA level and TF function. pAEC were analyzed for von Willebrand factor (vWF) expression and mRNA level as well. Neutralizing antibodies for P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) were used to block the molecular interaction between platelets and pAEC. GTKO and GTKO/CD46 pAEC-activated human platelets to induce human TF activity equivalently to WT pAEC. Simultaneously, after incubation with pAEC, platelets co-expressed TF and P-selectin. TF expression was blocked when pAEC and platelets were pre-incubated with anti-human P-selectin or anti-human PSGL-1 antibodies, but not by anti-porcine P-selectin antibody. Activated pAEC up-regulated TF on platelets through the interaction of porcine vWF with the human GPIb receptor. Up-regulation of TF on human platelets by GTKO and GTKO/CD46 pAEC was comparable to that by WT pAEC, which is associated with concomitant expression of P-selectin and PSGL-1, forming an auto-augmented loop of pAEC and platelet activation. Blocking of P-selectin and PSGL-1 interaction may be required to prevent up-regulation of recipient TF in vivo after organ xenotransplantation.

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.

Human T cells upregulate CD69 after coculture with xenogeneic genetically-modified pig mesenchymal stromal cells.

Mesenchymal stromal cells (MSC) obtained from α1,3-galactosyltransferase gene knock-out pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46 pMSC) suppress in vitro human anti-pig cellular responses as efficiently as allogeneic human MSC. We investigated the immunoregulatory effects of GTKO/CD46 pMSC on human CD4(+) and CD8(+) T cell proliferation in response to pig aortic endothelial cells (pAEC). pMSC efficiently suppressed T cell proliferation, which was associated with downregulation of granzyme B expression. No induction of CD4(+)CD25(+)Foxp3(hi) regulatory T cells or T cell apoptosis was documented. In correlation with T cell proliferation, CD25 expression was upregulated on T cells in response to pAEC but not to pMSC. In contrast, CD69 expression was upregulated on T cells in response to both pMSC and pAEC, which was associated with a significant increase in the phosphorylation of STAT5. GTKO/CD46 pMSC possibly regulate human T cell responses through modulation of CD69 expression and STAT5 signaling.

Minimal effect of bortezomib in reducing anti-pig antibodies in human leukocyte antigen-sensitized patients: a pilot study.

BACKGROUND: Bortezomib, a proteasome inhibitor used to treat multiple myeloma, has been administered (± plasma exchange ± intravenous immunoglobulin [IVIg]) in attempts to reduce antibodies against human leukocyte antigens (HLA) in sensitized patients undergoing organ transplantation. To our knowledge, bortezomib has not been investigated for its effect on natural anti-pig antibodies. If bortezomib could reduce the production of anti-pig antibodies, this would likely be beneficial to the outcome of pig organ grafts in primates. METHODS: Nine patients received bortezomib either to reduce anti-HLA antibody levels before organ allotransplantation or to treat antibody-mediated rejection. Patients at the Mayo Clinic (Group 1; n = 4) received bortezomib alone, whereas at the UPMC (Group 2; n = 5), this was combined with plasmaphereses ± IVIg in some cases. Anti-pig IgM and IgG levels against wild-type (WT) and α1,3-galactosyltransferase gene knockout (GTKO) pig aortic endothelial cells (flow cytometry-relative mean fluorescence intensity) and anti-Gal IgM and IgG (ELISA-OD480 nm ) were measured pre- and post-bortezomib therapy. RESULTS: Mean anti-pig IgM levels were 11.2 (WT) and 1.9 (GTKO) pre-bortezomib treatment and 9.4 (WT: P = 0.02) and 1.7 (GTKO: P = 0.33) post-bortezomib treatment, respectively. Mean anti-pig IgG levels were 4.3 (WT) and 1.5 (GTKO) pre-bortezomib treatment and 3.6 (WT: P = 0.21) and 1.4 (GTKO: P = 0.20) post-bortezomib treatment, respectively. Mean anti-Gal IgM and IgG levels were 0.7 and 1.1, respectively, pre-treatment, and 0.6 (P = 0.03) and 1.1 (NS), respectively, post-treatment. When the data were analyzed in Groups 1 and 2 separately, there were no significant differences between the pre- and post-bortezomib levels of anti-pig, anti-non-Gal, or anti-Gal IgM or IgG. CONCLUSIONS: From this limited study, we conclude that bortezomib might reduce anti-Gal IgM levels in primates, but, in this respect alone, is unlikely to have any significant effect on the outcome of GTKO pig organ transplantation.

Comparison of proliferative capacity of genetically-engineered pig and human corneal endothelial cells.

PURPOSE: The possibility of providing cultured corneal endothelial cells (CECs) for clinical transplantation has gained much attention. However, the worldwide need for human (h) donor corneas far exceeds supply. The pig (p) might provide an alternative source. The aim of this study was to compare the proliferative capacity of CECs from wild-type (WT) pigs, genetically-engineered (GE) pigs, and humans. METHODS: The following CECs were cultured: hCECs from donors (i) ≤36 years (young), (ii) ≥49 years (old), and WT pCECs from (iii) neonatal (<5 days), (iv) young (<2 months), and (v) old (>20 months) pigs, and CECs from young (vi) GE pigs (GTKO/CD46 and GTKO/CD46/CD55). Proliferative capacity of CECs was assessed by direct cell counting over 15 days of culture and by BrdU assay. Cell viability during culture was assessed by annexin V staining. The MTT assay assessed cell metabolic activity. RESULTS: There was significantly lower proliferative capacity of old CECs than of young CECs (p < 0.01) in both pigs and humans. There was no significant difference in proliferative capacity/metabolic activity between young pCECs and young hCECs. However, there was a significantly higher percentage of cell death in hCECs compared to pCECs during culture (p < 0.01). Young GE pCECs showed similar proliferative capacity/cell viability/metabolic activity to young WT pCECs. CONCLUSIONS: Because of the greater availability of young pigs and the excellent proliferative capacity of cultured GE pCECs, GE pigs could provide a source of CECs for clinical transplantation.

Adipose-derived mesenchymal stromal cells from genetically modified pigs: immunogenicity and immune modulatory properties.

BACKGROUND AIMS: The immunomodulatory and anti-inflammatory effects of mesenchymal stromal cells (MSC) could prove to be a potential therapeutic approach for prolongation of survival of cell xenotransplantation. Adipose (Ad) MSC from genetically modified pigs could be an abundant source of pig donor-specific MSC. METHODS: Pig (p) MSC were isolated from adipose tissue of α1,3-galactosyltransferase gene knock-out pigs transgenic for human (h) CD46 (GTKO/hCD46), a potential source of islets. After characterization with differentiation and flow cytometry (FCM), AdMSC were compared with bone marrow (BM) MSC of the same pig and human adipose-derived (hAd) MSC. The modulation of human peripheral blood mononuclear cell (hPBMC) responses to GTKO pig aortic endothelial cells (pAEC) by different MSC was compared by measuring 3H-thymidine uptake. The supernatants from the AdMSC cultures were used to determine the role of soluble factors. RESULTS: GTKO/hCD46 pAdMSC (i) did not express galactose-α1,3-galactose (Gal) but expressed hCD46, (ii) differentiated into chondroblasts, osteocytes and adipocytes, (iii) expressed stem cell markers, (iv) expressed lower levels of Swine Leucocyte Antigen I (SLAI), Swine Leucocyte Antigen II DR (SLAIIDR) and CD80 than pAEC before and after pig interferon (IFN)-γ stimulation. The proliferative responses of hPBMC to GTKO/hCD46 pAdMSC and hAdMSC stimulators were similar, and both were significantly lower than to GTKO pAEC (P < 0.05). The proliferation of hPBMC to GTKO pAEC was equally suppressed by GTKO/hCD46 pAdMSC and hAdMSC (P > 0.05). The supernatant from GTKO/hCD46 pAdMSC did not suppress the human xenoresponse to GTKO pAEC, which was cell-cell contact-dependent. CONCLUSIONS: Initial evidence suggests that genetically modified pAdMSC function across the xenogeneic barrier and may have a role in cellular xenotransplantation.