Regulatory T cells from allo‐ to xenotransplantation: Opportunities and challenges

Regulatory T cells (Treg) are currently being evaluated in clinical allotransplantation for tolerance induction, with proven safety in humans with autoimmune diseases and graft‐versus‐host disease. A considerable amount of recent data suggests that additional factors may need to be validated, including the stability and commitment of newly discovered Treg subsets under inflammatory conditions, to further warrant safe and effective Treg‐based therapeutic approaches. This review explores the opportunities and challenges of Treg‐based cell therapy in xenotransplantation. The emerging new technologies for genetic modifications of the donor pig offer a major advantage for Treg therapy to improve xenograft protection. Particularly, the feasibility of (i) ex vivo expansion of donor (pig)‐specific Treg for infusion, and (ii) development of Treg in situ for the life of the xenograft. Our understanding of the Treg biology and their role in xenograft protection, under the newly developed immunosuppressive protocols remains limited. The incidence of various Treg subpopulations in xenograft recipients and their suppressive efficacy across species barriers are largely unknown. Finally, deciphering the dynamics of Treg function, and their interaction with adaptive and innate immune cells are of critical importance to design safe, effective and clinically relevant Treg‐based therapeutic approaches in xenotransplantation.     

Gal/non-Gal antigens in pig tissues and human non-Gal antibodies in the GalT-KO era

Our knowledge regarding Gal and non-Gal antigens in GalT-KO pig tissues can be summarized as α3Galactosyl-tranferase gene knock out eliminates the Galα3Galβ4GlcNAc-R antigen expression in pig tissues as well as anti-Gal antibody binding. Other Galα-terminating saccharides (e.g. iGb3 glycolipids and Galα2 determinants) may be present but have not been documented. α3Galactosyl-tranferase gene knock out slightly changes the carbohydrate antigen expression but no "new" antigens recognized by the human immune system have been found. Non-Gal antigens are both of protein and carbohydrate nature but their exact chemical structures are poorly defined. Regarding human non-Gal antibodies our knowledge is as Non-Gal antibodies exist naturally and increase in humans/non-human primate (NHP) receiving WT or GalT-KO pig grafts. Non-Gal antibodies with new antigen epitope recognition can be induced in humans/NHP after challenge by WT or GalT-KO pig grafts. Non-Gal antibodies react with both carbohydrates and proteins. Part of the protein reactivity is directed to glycoprotein carbohydrates chains. Non-Gal antibodies reacting with neuraminic acid terminated saccharides (both N-Acetyl and N-Glycoloyl variants) are present in humans/NHP. Anti-neuraminic acid antibodies are increased, as well as induced, after grafting pig organs into humans/NHP. Non-Gal antibodies does not cause hyperacute xenorejection but can be cytotoxic and cause xenoorgan damage. If humans sensitized to HLA antigens are at a higher risk of rejecting pig xenograft compared with non-sensitized individuals is not fully clarified. Clinical trials are needed to evaluate the relevance of non-Gal antigens/antibodies and for the xenofield to advance.     

Amino acid and lipid profiles following pig‐to‐primate liver xenotransplantation

As outcomes in clinical liver transplantation steadily improve, demand continues to exceed supply, leading to a substantial disparity in organ availability. The translation of porcine liver xenotransplantation (LXT) into a clinical reality aims to address this dilemma. Our laboratory has previously established an applicable model of α‐1,3‐galactosyltransferase knockout (GalT‐KO) pig‐to‐primate LXT with continuous human coagulation factor infusion and costimulation blockade. This report aims to further investigate the post‐LXT lipid and amino acid metabolism profile in our longest surviving recipients (25 and 29 days). Experimental samples and control samples, consisting of pre‐transplant porcine and baboon serum and plasma, were analyzed for standard lipid profiles and for amino acid levels. Lipid profiles of LXT recipients remained stable following xenotransplantation compared to donor porcine baseline levels. Amino acid concentrations also remained similar to baseline controls, with the exception of a 3‐fold increase in l ‐ornithine and more than a 10‐fold decrease in l ‐arginine post‐transplant when compared to both porcine and baboon baseline levels. The observed changes in l ‐arginine are consistent with prior studies investigating the effects of graft preservation injury following liver transplantation. These results indicate that the porcine liver can maintain most biochemical profiles stably post‐operatively in baboons and suggest that arginine supplementation post‐LXT may potentially be useful for further prolongation of xenograft survival.     

Double knockout pigs deficient in N‐glycolylneuraminic acid and Galactose α‐1,3‐Galactose reduce the humoral barrier to xenotransplantation

Background

Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. Galα‐1,3‐Gal (Gal) and N‐glycolylneuraminic acid (Neu5Gc) are two known xenoantigens.

Methods

We report the homozygous disruption of the α1, 3‐galactosyltransferase (GGTA1) and the cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase (CMAH) genes in liver‐derived female pig cells using zinc‐finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody‐binding and antibody‐mediated complement‐dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal.

Results

This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1.

Conclusions

This technology will accelerate the development of pigs for xenotransplantation research.     

Semi‐xenotransplantation: the regenerative medicine‐based approach to immunosuppression‐free transplantation and to meet the organ demand

Although xenografts have always held immeasurable potential as an inexhaustible source of donor organs, immunological barriers and physiological incompatibility have proved to be formidable obstacles to clinical utility. An exciting, new regenerative medicine‐based approach termed “semi‐xenotransplantation” (SX) seeks to overcome these obstacles by combining the availability and reproducibility of animal organs with the biocompatibility and functionality of human allografts. Compared to conventional xenotransplantation wherein the whole organ is animal‐derived, SX grafts are cleansed of their antigenic cellular compartment to produce whole‐organ extracellular matrix scaffolds that retain their innate structure and vascular channels. These scaffolds are then repopulated with recipient or donor human stem cells to generate biocompatible semi‐xenografts with the structure and function of native human organs. While numerous hurdles must be still overcome in order for SX to become a viable treatment option for end‐stage organ failure, the immense potential of SX for meeting the urgent needs for a new source of organs and immunosuppression‐free transplantation justifies the interest that the transplant community is committing to the field.     

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 4: pre‐clinical efficacy and complication data required to justify a clinical trial

In 2009, the International Xenotransplantation Association (IXA) published a consensus document that provided guidelines and “recommendations” (not regulations) for those contemplating clinical trials of porcine islet transplantation. These guidelines included the IXA's opinion on what constituted “rigorous pre‐clinical studies using the most relevant animal models” and were based on “non‐human primate testing.” We now report our discussion following a careful review of the 2009 guidelines as they relate to pre‐clinical testing. In summary, we do not believe there is a need to greatly modify the conclusions and recommendations of the original consensus document. Pre‐clinical studies should be sufficiently rigorous to provide optimism that a clinical trial is likely to be safe and has a realistic chance of success, but need not be so demanding that success might only be achieved by very prolonged experimentation, as this would not be in the interests of patients whose quality of life might benefit immensely from a successful islet xenotransplant. We believe these guidelines will be of benefit to both investigators planning a clinical trial and to institutions and regulatory authorities considering a proposal for a clinical trial. In addition, we suggest consideration should be given to establishing an IXA Clinical Trial Advisory Committee that would be available to advise (but not regulate) researchers considering initiating a clinical trial of xenotransplantation.