Incidence of serum antibodies to xenoantigens on triple-knockout pig cells in different human groups.

BACKGROUND: Xenoantigens other than Gal, Neu5Gc, and Sda may be playing a role in pig graft rejection. We investigated the incidence of antibodies to unknown pig xenoantigen in different human groups. METHODS: We collected blood from TKO/hCD55 pigs (n = 3), and isolated PBMCs and RBCs. Serum samples were collected from (i) healthy human volunteers (n = 43), (ii) patients with end-stage renal disease (ESRD) (n = 87), (iii) the same patients after kidney allotransplantation (n = 50), and (iv) renal allotransplant recipients experiencing T cell-mediated rejection (allo-TCMR, n = 10). The sera were initially incubated with TKO/hCD55 pRBCs (1 × 108 cells) for 1 h to absorb anti-pig antibodies (except against SLA and possibly other antigens not expressed on pRBCs) and then the serum (absorbed or unabsorbed) was tested for antibody binding and complement-dependent cytotoxicity (CDC) to TKO/hCD55 pig PBMCs. RESULTS: A significant reduction in IgM/IgG binding and CDC was observed in the absorbed sera. Serum obtained before and after renal allotransplantation showed no significant difference in IgM or IgG binding to, or in CDC of, TKO/hCD55 pig cells. IgM antibodies (but rarely IgG) against unknown xenoantigens expressed on TKO/hCD55 PBMCs, possibly against swine leukocyte antigens, were documented in healthy humans, patients with ESRD, and those with renal allografts undergoing acute T cell rejection. IgM (but not CDC) was higher in patients experiencing allo-TCMR. CONCLUSION: Human sera contain IgM antibodies against unknown pig xenoantigens expressed on TKO/hCD55 pPBMCs. Although not confirmed in the present study, the targets for these antibodies may include swine leukocyte antigens.

The case for the therapeutic use of mechanistic/mammalian target of rapamycin (mTOR) inhibitors in xenotransplantation.

The mechanistic/mammalian target of rapamycin (mTOR) is one of the systems that are necessary to maintain cell homeostasis, such as survival, proliferation, and differentiation. mTOR inhibitors (mTOR-Is) are utilized as immunosuppressants and anti-cancer drugs. In organ allotransplantation, current regimens infrequently include an mTOR-I, which are positioned more commonly as alternative immunosuppressants. In clinical allotransplantation, long-term efficacy has been established, but there is a significant incidence of adverse events, for example, inhibition of wound healing, buccal ulceration, anemia, hyperglycemia, dyslipidemia, and thrombocytopenia, some of which are dose-dependent. mTOR-Is have properties that may be especially beneficial in xenotransplantation. These include suppression of T cell proliferation, increases in the number of T regulatory cells, inhibition of pig graft growth, and anti-inflammatory, anti-viral, and anti-cancer effects. We here review the potential benefits and risks of mTOR-Is in xenotransplantation and suggest that the benefits exceed the adverse effects.

Antibody-mediated rejection in xenotransplantation: Can it be prevented or reversed?

Antibody-mediated rejection (AMR) is the commonest cause of failure of a pig graft after transplantation into an immunosuppressed nonhuman primate (NHP). The incidence of AMR compared to acute cellular rejection is much higher in xenotransplantation (46% vs. 7%) than in allotransplantation (3% vs. 63%) in NHPs. Although AMR in an allograft can often be reversed, to our knowledge there is no report of its successful reversal in a pig xenograft. As there is less experience in preventing or reversing AMR in models of xenotransplantation, the results of studies in patients with allografts provide more information. These include (i) depletion or neutralization of serum anti-donor antibodies, (ii) inhibition of complement activation, (iii) therapies targeting B or plasma cells, and (iv) anti-inflammatory therapy. Depletion or neutralization of anti-pig antibody, for example, by plasmapheresis, is effective in depleting antibodies, but they recover within days. IgG-degrading enzymes do not deplete IgM. Despite the expression of human complement-regulatory proteins on the pig graft, inhibition of systemic complement activation may be necessary, particularly if AMR is to be reversed. Potential therapies include (i) inhibition of complement activation (e.g., by IVIg, C1 INH, or an anti-C5 antibody), but some complement inhibitors are not effective in NHPs, for example, eculizumab. Possible B cell-targeted therapies include (i) B cell depletion, (ii) plasma cell depletion, (iii) modulation of B cell activation, and (iv) enhancing the generation of regulatory B and/or T cells. Among anti-inflammatory agents, anti-IL6R mAb and TNF blockers are increasingly being tested in xenotransplantation models, but with no definitive evidence that they reverse AMR. Increasing attention should be directed toward testing combinations of the above therapies. We suggest that treatment with a systemic complement inhibitor is likely to be most effective, possibly combined with anti-inflammatory agents (if these are not already being administered). Ultimately, it may require further genetic engineering of the organ-source pig to resolve the problem entirely, for example, knockout or knockdown of SLA, and/or expression of PD-L1, HLA E, and/or HLA-G.

In vitro and in vivo immune assessments of genetically-engineered pig skin grafts in New World (squirrel) monkeys.

Half a million patients in the USA alone require treatment for burns annually. Following an extensive burn, it may not be possible to provide sufficient autografts in a single setting. Genetic manipulations (GM) of pigs offer the possibility of reducing primate humoral and cellular rejection of pig skin xenografts and thus extending graft survival. We compared the survival of skin grafts from pigs with 9-GM with that of autografts and allografts in squirrel monkeys. Monitoring for rejection was by (1) macroscopic examination, (2) histopathological examination of skin biopsies, and (3) measurement of anti-monkey and anti-pig IgM and IgG antibodies. Autografts (n = 5) survived throughout the 28 days of follow-up without histopathological features of rejection. Median survival of allografts (n = 6) was 14 days and of pig xenografts (n = 12) 21 days. Allotransplantation was associated with an increase in anti-monkey IgM, but the anticipated subsequent rise in IgG had not yet occurred at the time of euthanasia. Pig grafts were associated with increases in anti-pig IgM and IgG. In all cases, histopathologic features of rejection were similar. 9-GM pig skin xenografts survive at least as long as monkey skin allografts (and trended to survive longer), suggesting that they are a realistic clinical option for the temporary treatment of burns. Although monkeys with pig skin grafts developed anti-pig IgM and IgG antibodies, these did not cross-react with monkey antigens, indicating that a primary 9-GM pig skin graft would not be detrimental to a subsequent monkey skin allograft.

The first clinical pig heart transplant: Was IVIg or pig cytomegalovirus detrimental to the outcome?

The clinical course of the first patient to receive a gene-edited pig heart transplant was recently reported by the University of Maryland team. Although the pig heart functioned well for >40 days, serum anti-pig antibodies then increased, and the patient sadly died after 60 days. Because of his debilitated pre-transplant state, the patient never thrived despite excellent graft function for several weeks, and the cause of his demise continues to be uncertain. A few days before an increase in anti-pig antibodies was observed, the patient had received intravenous human immunoglobulin (IVIg), and whether this played a role in his cardiac deterioration has been discussed. Furthermore, mcfDNA testing indicated an increase in pig cytomegalovirus (CMV), and its possible role in the development of cardiac dysfunction has also been considered. On the basis of the limited data provided in the publication and on our previous investigations into whether IVIg contains anti-TKO pig antibodies and therefore might be deleterious to TKO pig organ xenografts, we suggest that the steady rise in anti-pig antibody titer was more consistent with the failure of the immunosuppressive regimen to prevent elicited anti-TKO pig antibody production, rather than from the passive transfusion of IVIg or the presence of pig CMV in the graft. Although the outcome of the Maryland experience was disappointing, valuable lessons were learned. Our attention was drawn to the potential risks of heart transplantation in a "deconditioned" patient, the administration of IVIg, the transmission of pig CMV, and of the difficulties in interpreting myocardial biopsy findings.

Observations on hydronephrosis after pig kidney transplantation in baboons.

We have seen hydronephrosis (obstructive nephropathy) at necropsy in 3 of 11 (21%) genetically-engineered pig kidneys that functioned in baboons for >36 days, even when the clinical and histopathological features of rejection were minimal. We briefly report one such case and illustrate the macroscopic and microscopic appearances of such a kidney and ureter. The causes of the observed changes remain uncertain. In our small experience, there seems to be no correlation between the development of hydronephrosis and (i) the surgical technique, (ii) the genotype of the pig, (iii) the length of the pig ureter, or (iv) the immunosuppressive and anti-inflammatory therapy administered. We suggest that the distal ureteric thickening may be the result of an inflammatory response. In two cases, we resolved the problem by carrying out a secondary side-to-side anastomosis between the proximal pig ureter and the baboon bladder.

The potential of genetically engineered pig heart transplantation in infants with complex congenital heart disease.

Despite advances in surgical and medical techniques, complex congenital heart disease in neonates and infants continues to be associated with significant mortality and morbidity. More than 500 infants in the USA are placed on the cardiac transplantation wait-list annually. However, there remains a critical shortage of deceased human donor organs for transplantation with a median wait-time of 4 months. Hence, infant mortality on the heart transplant wait-list in the USA is higher than for any other solid organ transplant group. Orthotopic transplantation of a pig heart as a bridge to allotransplantation might offer the best prospect of long-term survival of these patients. In recent years, there have been several advances in genetic engineering of pigs to mitigate the vigorous antibody-mediated rejection of a pig heart transplanted into a nonhuman primate. In this review, we briefly highlight (i) the history of clinical heart xenotransplantation, (ii) current advances and techniques of genetically engineering pigs, (iii) the current status of pig orthotopic cardiac graft survival in nonhuman primates, and (iv) progress toward pursuing clinical trials of cardiac xenotransplantation. Ultimately, we argue that pig heart xenotransplantation should initially be used as a bridge to cardiac allotransplantation in neonates and infants.

Stable expression of the human thrombomodulin transgene in pig endothelial cells is associated with a reduction in the inflammatory response.

BACKGROUND: Xenotransplantation is associated with an inflammatory response. The proinflammatory cytokine, TNF-α, downregulates the expression of thrombomodulin (TBM), and induces coagulation dysfunction. Although human (h) TBM-transgenic pigs (p) have been developed to reduce coagulation dysfunction, the effect of TNF-α on the expression of hTBM and its functional activity has not been fully investigated. The aims of this study were to investigate (i) whether the expression of hTBM on pig (p) cells is down-regulated during TNF-α stimulation, and (ii) whether cells from hTBM pigs regulate the inflammatory response. METHODS: TNF-α-producing T, B, and natural killer cells in blood from baboons with pig heart or kidney xenografts were investigated by flow cytometry. TNF-α staining in the grafts was detected by immunohistochemistry. Aortic endothelial cells (AECs) from GTKO/CD46 and GTKO/CD46/hTBM pigs were stimulated by hTNF-α, and the expression of the inflammatory/coagulation regulatory protein, TBM, was investigated. RESULTS: After pig organ xenotransplantation, there was a trend to increases in TNF-α-producing T and natural killer cells in the blood of baboons. In vitro observations demonstrated that after hTNF-α stimulation, there was a significant reduction in the expression of endogenous pTBM on pAECs, and a significant increase in the expression of inflammatory molecules. Blocking of NF-κB signaling significantly up-regulated pTBM expression, and suppressed the inflammatory response induced by hTNF-α in pAECs. Whereas the expression of pTBM mRNA was significantly reduced by hTNF-α stimulation, hTBM expression on the GTKO/CD46/hTBM pAECs was not affected. Furthermore, after hTNF-α stimulation, there was significant suppression of expression of inflammatory molecules on GTKO/CD46/hTBM pAECs compared to GTKO/CD46 pAECs. CONCLUSIONS: The stable expression of hTBM in pig cells may locally regulate the inflammatory response. This will help suppress the inflammatory response and prevent coagulation dysregulation after xenotransplantation.

Initial experimental experience of triple-knockout pig red blood cells as potential sources for transfusion in alloimmunized patients with sickle cell disease.

BACKGROUND: Blood transfusion remains important in the treatment of patients with sickle cell disease (SCD). However, alloimmunization after blood transfusion is associated with patient morbidity and mortality. Triple-knockout (TKO) pigs (i.e., pigs in which the three known xenoantigens to which humans have anti-pig antibodies have been deleted) may be an alternative source of RBCs for these patients because many humans have no preformed antibodies to TKO pig RBCs (pRBCs). METHODS AND MATERIALS: In an in vitro study, plasma from alloimmunized (n = 12) or non-alloimmunized (n = 12) SCD patients was used to determine IgM/IgG binding to, and CDC of, TKO pRBCs. In an in vivo study, after an estimated 25% of blood volume was withdrawn from two capuchin monkeys, CFSE-labeled TKO pRBCs were transfused. Loss of TKO pRBCs was monitored by flow cytometry, and 7 weeks later, 25% of blood was withdrawn, and CFSE-labeled monkey RBCs were transfused. RESULTS: The in vitro study demonstrated that plasma from neither alloimmunized nor non-alloimmunized SCD patients bound IgM/IgG to, or induced CDC of, TKO pRBCs. In the in vivo study, survival of TKO pRBCs in the two capuchin monkeys was of 5 and 7 days, respectively, whereas after allotransfusion, survival was >28 days. CONCLUSIONS: In conclusion, (1) in the present limited study, no antibodies were detected that cross-reacted with TKO pRBCs, and (2) TKO pigs may possibly be an alternate source of RBCs in an emergency if no human RBCs are available.

Immunological selection and monitoring of patients undergoing pig kidney transplantation.

Pig kidney xenotransplantation has the potential to alleviate the current shortage of deceased and living human organs and provide patients with end-stage renal disease with a greater opportunity for long-term survival and a better quality of life. In recent decades, advances in the genetic engineering of pigs and in immunosuppressive therapy have permitted the resolution of many historical obstacles to the success of pig kidney transplantation in nonhuman primates. Pig kidney xenotransplantation may soon be translated to the clinic. Given the potential risks of kidney xenotransplantation, particularly of immunologic rejection of the graft, potential patients must be carefully screened for inclusion in the initial clinical trials and immunologically monitored diligently post-transplantation. We provide an overview of the immunological methods we believe should be used to (i) screen potential patients for the first clinical trials to exclude those with a higher risk of rejection, and (ii) monitor patients with a pig kidney graft to determine their immunological response to the graft.

Evidence that sensitization to triple-knockout pig cells will not be detrimental to subsequent allotransplantation.

The current evidence is that sensitization to a pig xenograft does not result in the development of antibodies that cross-react with alloantigens, and therefore, sensitization to a pig xenograft would not be detrimental to the outcome of a subsequent allograft. This evidence relates almost entirely to the transplantation of cells or organs from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. However, it is not known whether recipients of triple-knockout (TKO) pig grafts who become sensitized to TKO pig antigens develop antibodies that cross-react with alloantigens and thus be detrimental to a subsequent organ allotransplant. We identified a single baboon (B1317) in which no (or minimal) serum anti-TKO pig antibodies could be measured-in our experience unique among baboons. We sensitized it by repeated subcutaneous injections of TKO pig peripheral blood mononuclear cells (PBMCs) in the absence of any immunosuppressive therapy. After TKO pig PBMC injection, there was a transient increase in anti-TKO pig IgM, followed by a sustained increase in IgG binding to TKO cells. In contrast, there was no serum IgM or IgG binding to PBMCs from any of a panel of baboon PBMCs (n = 8). We conclude that sensitization to TKO pig PBMCs in the baboon did not result in the development of antibodies that also bound to baboon cells, suggesting that there would be no detrimental effect of sensitization on a subsequent organ allotransplant.

A perspective on the potential detrimental role of inflammation in pig orthotopic heart xenotransplantation.

There is a critical shortage of deceased human donor organs for transplantation. The need is perhaps most acute in neonates and infants with life-threatening congenital heart disease, in whom mechanical support devices are largely unsuccessful. If orthotopic (life-supporting) heart transplantation (OHTx) were consistently successful in the genetically engineered pig-to-nonhuman primate (NHP) model, a clinical trial of bridging with a pig heart in such patients might be justified. However, the results of pig OHTx in NHPs have been mixed and largely poor. We hypothesise that a factor is the detrimental effects of the inflammatory response that is known to develop (a) during any surgical procedure that requires cardiopulmonary bypass, and (b) immediately after an NHP recipient is exposed to a pig xenograft. We suggest that the combination of these two inflammatory responses has a direct detrimental effect on pig heart graft function, but also, and possibly of more importance, on recipient baboon pulmonary function, which further impacts survival of the pig heart graft. In addition, the inflammatory response almost certainly adversely impacts the immune response to the graft. If our hypothesis is correct, the potential steps that could be taken to reduce the inflammatory response or its effects (with varying degrees of efficacy) include (a) white blood cell filtration, (b) complement depletion or inactivation, (c) immunosuppressive therapy, (d) high-dose corticosteroid therapy, (e) cytokine/chemokine-targeted therapy, (f) ultrafiltration or CytoSorb hemoperfusion, (g) reduction in the levels of endogenous catecholamines, (h) triiodothyronine therapy and (i) genetic engineering of the organ-source pig. Prevention of the inflammatory response, or attenuation of its effects, by judicious anti-inflammatory therapy may contribute not only to early survival of the recipient of a genetically engineered pig OHTx, but also to improved long-term pig heart graft survival. This would open the possibility of initiating a clinical trial of genetically engineered pig OHTx as a bridge to allotransplantation.

Histopathology of pig kidney grafts with/without expression of the carbohydrate Neu5Gc in immunosuppressed baboons.

INTRODUCTION: Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs, that is, not expressing the three known carbohydrate xenoantigens) and expressing 'protective' human transgenes are considered a likely source of organs for transplantation into human recipients. Some human sera have no or minimal natural antibody binding to red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs. However, all Old World monkeys exhibit natural antibody binding to TKO pig cells. The xenoantigen targets of Old World monkey natural antibodies are postulated to be carbohydrate moieties exposed when the expression of the carbohydrate N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival in baboons and histopathology of renal grafts from pigs that either (a) expressed Neu5Gc (GTKO pigs; Group 1) or (b) did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group 2). METHODS: Life-supporting renal transplants were carried out using GTKO (n = 5) or DKO/TKO (n = 5) pig kidneys under an anti-CD40mAb-based immunosuppressive regimen. RESULTS: Group 1 baboons survived longer than Group 2 baboons (median 237 vs. 35 days; mean 196 vs. 57 days; p < 0.07) and exhibited histopathological features of antibody-mediated rejection in only two kidneys. Group 2 exhibited histopathological features of antibody-mediated rejection in all five grafts, with IgM and IgG binding to renal interstitial arteries and peritubular capillaries. Rejection-free survival was significantly longer in Group 1 (p < 0.05). CONCLUSIONS: The absence of expression of Neu5Gc on pig kidney grafts is associated with increased binding of baboon antibodies to pig endothelium and reduced graft survival.

Evidence suggesting that deletion of expression of N-glycolylneuraminic acid (Neu5Gc) in the organ-source pig is associated with increased antibody-mediated rejection of kidney transplants in baboons.

Pigs deficient in three glycosyltransferase enzymes (triple-knockout [TKO] pigs) and expressing "protective" human transgenes are likely sources of organs for transplantation into human recipients. Testing of human sera against red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) from TKO pigs has revealed minimal evidence of natural antibody binding. However, unlike humans, baboons exhibit natural antibody binding to TKO pig cells. The xenoantigen specificities of these natural antibodies are postulated to be one or more carbohydrate moieties exposed when N-glycolylneuraminic acid (Neu5Gc) is deleted. The aim of this study was to compare the survival of renal grafts in baboons from pigs that either expressed Neu5Gc (GTKO pigs; Group1, n = 5) or did not express Neu5Gc (GTKO/CMAHKO [DKO] or TKO pigs; Group2, n = 5). An anti-CD40mAb-based immunosuppressive regimen was administered in both groups. Group1 kidneys functioned for 90-260 days (median 237, mean 196 days), with histopathological features of antibody-mediated rejection in two kidneys. Group2 kidneys functioned for 0-183 days (median 35, mean 57), with all of the grafts exhibiting histologic features of antibody-mediated rejection. These findings suggest that the absence of expression of Neu5Gc on pig kidneys impacts graft survival in baboon recipients.

Pig kidney xenotransplantation: Progress toward clinical trials.

Pig organ xenotransplantation offers a solution to the shortage of deceased human organs for transplantation. The pathobiological response to a pig xenograft is complex, involving antibody, complement, coagulation, inflammatory, and cellular responses. To overcome these barriers, genetic manipulation of the organ-source pigs has largely been directed to two major aims-(a) deletion of expression of the known carbohydrate xenoantigens against which humans have natural (preformed) antibodies, and (b) transgenic expression of human protective proteins, for example, complement- and coagulation-regulatory proteins. Conventional (FDA-approved) immunosuppressive therapy is unsuccessful in preventing an adaptive immune response to pig cells, but blockade of the CD40:CD154 costimulation pathway is successful. Survival of genetically engineered pig kidneys in immunosuppressed nonhuman primates can now be measured in months. Non-immunological aspects, for example, pig renal function, a hypovolemia syndrome, and rapid growth of the pig kidney after transplantation, are briefly discussed. We suggest that patients on the wait-list for a deceased human kidney graft who are unlikely to receive one due to long waiting times are those for whom kidney xenotransplantation might first be considered. The potential risk of infection, public attitudes to xenotransplantation, and ethical, regulatory, and financial aspects are briefly addressed.

Clinical Pig Kidney Xenotransplantation: How Close Are We?

Patients with ESKD who would benefit from a kidney transplant face a critical and continuing shortage of kidneys from deceased human donors. As a result, such patients wait a median of 3.9 years to receive a donor kidney, by which time approximately 35% of transplant candidates have died while waiting or have been removed from the waiting list. Those of blood group B or O may experience a significantly longer waiting period. This problem could be resolved if kidneys from genetically engineered pigs offered an alternative with an acceptable clinical outcome. Attempts to accomplish this have followed two major paths: deletion of pig xenoantigens, as well as insertion of "protective" human transgenes to counter the human immune response. Pigs with up to nine genetic manipulations are now available. In nonhuman primates, administering novel agents that block the CD40/CD154 costimulation pathway, such as an anti-CD40 mAb, suppresses the adaptive immune response, leading to pig kidney graft survival of many months without features of rejection (experiments were terminated for infectious complications). In the absence of innate and adaptive immune responses, the transplanted pig kidneys have generally displayed excellent function. A clinical trial is anticipated within 2 years. We suggest that it would be ethical to offer a pig kidney transplant to selected patients who have a life expectancy shorter than the time it would take for them to obtain a kidney from a deceased human donor. In the future, the pigs will also be genetically engineered to control the adaptive immune response, thus enabling exogenous immunosuppressive therapy to be significantly reduced or eliminated.

Is interleukin-6 receptor blockade (tocilizumab) beneficial or detrimental to pig-to-baboon organ xenotransplantation?

The interleukin (IL)-6/IL-6 receptor-α (IL-6Rα)/signal transduction and activation of the transcription 3 (STAT3) pathway plays an important role in inflammation. Anti-human IL-6Rα blockade by tocilizumab (TCZ) has been used in pig-to-baboon organ xenotransplant models, but whether it is beneficial remains uncertain. After xenotransplant, there were significant increases in both baboon and pig IL-6 in the baboon serum, especially in baboons that received TCZ before xenotransplant. In vitro observations demonstrated that human, baboon, and pig IL-6 can activate the IL-6/IL-6Rα/STAT3 pathway in human, baboon, and pig cells, respectively. Activation of the IL-6/IL-6Rα/STAT3 pathway was blocked by TCZ in human and baboon cells but not in pig cells (ie, pig IL-6R). Siltuximab (human IL-6 inhibitor) bound to both human and baboon, but not pig, IL-6 and suppressed activation of the IL-6/IL-6Rα/STAT3 pathway. These results indicate that TCZ and siltuximab do not cross-react with pig IL-6R and pig IL-6, respectively. Rapamycin partially inhibited human, baboon, and pig IL-6/IL-6Rα/STAT3 pathways and suppressed inflammatory gene expression. TCZ treatment increased serum IL-6 because it could no longer bind to baboon IL-6Rα. We suggest that increased serum IL-6 may be detrimental to the pig xenograft because it is likely to bind to pig IL-6R, resulting in activation of pig cells.

Efficacy of ATG and Rituximab in capuchin monkeys (a New World monkey)-An in vitro study relevant to xenotransplantation.

While Old World monkeys, for example, baboons, have antibodies against triple-knockout (TKO) pig cells, thus complicating pig organ transplantation studies, capuchin monkeys (a New World monkey) do not, thus more closely mimicking humans in respect to the response to TKO pig cells. Whether drugs such as anti-thymocyte globulin (ATG) and Rituximab are effective in capuchin monkeys remains uncertain. We measured the binding and cytotoxicity of ATG and Rituximab to human (n = 7), baboon (n = 7), and capuchin monkey (n = 5) peripheral blood mononuclear cells (PBMCs), T cells or B cells by flow cytometry.The effect in vitro of ATG in depleting PBMCs in capuchin monkeys and baboons was significantly less than in humans, but the depletion in capuchin monkeys was not significantly different from that in baboons. In contrast, the effect in vitro of Rituximab in depleting B cells in capuchin monkeys was very limited, and significantly less than in humans and baboons.Although capuchin monkeys mimic the human antibody response to TKO pig cells more closely than baboons, Rituximab had a minimal effect in capuchin monkeys in vitro. This observation may limit the value of New World Monkeys as recipients of pig organs, tissues, or cells in experimental studies of xenotransplantation or, indeed, in allotransplantation.

Immunosuppressive and metabolic agents that influence allo- and xenograft survival by in vivo expansion of T regulatory cells.

The transplanted organs or cells survive if the recipient receives adequate long-term immunosuppressive therapy. Immunosuppressive therapy combined with cell-based strategies (eg, regulatory T cell [Treg]-based therapy) promotes graft survival. A combination of Treg-based therapy and minimal or no immunosuppressive drug therapy would have the potential to minimize the risks of the complications and side effects of these drugs. Fortunately, some immunosuppressive and other agents not only impede the effector T cell response, but also help generate new CD4+ Tregs from conventional effector T cells. These agents include IL-2, TGF-ß, agents that block the CD40/CD40L costimulation pathway, mTOR inhibitors, and histone deacetylase inhibitors. Consequently, a state of relative unresponsiveness to the transplanted organ may be induced through the expansion of Tregs. We here review the effect of these various agents on expansion of CD4+ Tregs in allo- and xenotransplantation. The expansion of Tregs might allow a dose reduction of the standard immunosuppressive drugs.

The final obstacle to successful pre-clinical xenotransplantation?

Genetically engineered pigs are now available for xenotransplantation in which all three known carbohydrate xenoantigens, against which humans have natural antibodies, have been deleted (triple-knockout [TKO] pigs). Furthermore, multiple human transgenes have been expressed in the TKO pigs, all of which are aimed at protecting the cells from the human immune response. Many human sera demonstrate no or minimal antibody binding to, and little or no cytotoxicity of, cells from these pigs, and this is associated with a relatively low T-cell proliferative response. Unfortunately, baboons and other Old World NHPs have antibodies against TKO pig cells, apparently directed to a fourth xenoantigen that appears to be exposed after TKO. In our experience, most, if not all, humans do not have natural antibodies against this fourth xenoantigen. This discrepancy between NHPs and humans is providing a hurdle to successful translation of pig organ transplantation into the clinic, and making it difficult to provide pre-clinical data that support initiation of a clinical trial. The potential methods by which this obstacle might be overcome are discussed. We conclude that, whatever currently available genetically engineered pig is selected for the final pre-clinical studies, this may not be the optimal pig for clinical trials.