Possible risks posed by single‐stranded DNA viruses of pigs associated with xenotransplantation

Routine large‐scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state‐of‐the‐art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross‐species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches. Single‐stranded DNA (ssDNA) viruses are the largest group prevalent in virome studies in mammals. Specifically, the ssDNA viral genomes are characterized by a high rate of nucleotide substitution, which confers a proclivity to adapt to new hosts and cross‐species barriers. Pig‐associated ssDNA viruses include torque teno sus viruses (TTSuV) in the Anelloviridae family, porcine parvoviruses (PPV), and porcine bocaviruses (PBoV) both in the family of Parvoviridae, and porcine circoviruses (PCV) in the Circoviridae family, some of which have been confirmed to be pathogenic to pigs. The risks of these viruses for the human recipient during xenotransplantation procedures are relatively unknown. Based on the scant knowledge available on the prevalence, predilection, and pathogenicity of pig‐associated ssDNA viruses, careful screening and monitoring are required. In the case of positive identification, risk assessments and strategies to eliminate these viruses in xenotransplantation pig stock may be needed.     

Xenoreactive CD4+ memory T cells resist inhibition by anti-CD44 mAb and reject islet grafts via a Th2-dependent pathway

Peng YZ, Chen JB, Shao W, Wang FY, Dai HL, Cheng PP, Xia JJ, Wang F, Huang R, Zhu Q, Qi Z. Xenoreactive CD4⁺ memory T cells resist inhibition by anti‐CD44 mAb and reject islet grafts via a Th2‐dependent pathway. Xenotransplantation 2011; 18: 252–261. © 2011 John Wiley & Sons A/S. Abstract: Background: Memory T cells are a significant barrier to the induction of transplant tolerance. Our previous study demonstrated that multiple applications of anti‐CD44 monoclonal antibody (mAb) could significantly inhibit CD4⁺ memory T cells from mediating rejection of cardiac allografts. Now, we sought to explore the effect and mechanism of anti‐CD44 mAb on the rejection of islet allografts and xenografts mediated by CD4⁺ memory T cells. Methods: In this study, we first engrafted skin grafts of C57BL/6 (B6) mice or Dark Agouti (DA) rats onto BALB/c mice to induce donor‐reactive memory T cells. We adoptively transferred purified CD4⁺ memory T cells to BALB/c origin nude mice and then transplanted islet allografts and xenografts to produce the Allo‐Tx and Xeno‐Tx models, respectively. We subsequently administered multiple anti‐CD44 mAb and observed changes in the survival times of the islet grafts. Results: In the Allo‐Tx model, the mean survival time (MST) of the grafts was 7.7 days in the isotype group, and 20.3 days in the anti‐CD44 group. In the Xeno‐Tx model, the MST of the grafts was 7.2 days in the isotype group and 8.2 days in the anti‐CD44 group. Compared with the isotype group, CD4⁺ T cells on the grafts in the anti‐CD44 group were significantly decreased in both the Allo‐Tx and Xeno‐Tx models, but the proportion of CD4⁺ memory T cells in the spleens and draining lymph nodes of the recipient nude mice in the anti‐CD44 group was significantly decreased in the Allo‐Tx model, while it was increased in the Xeno‐Tx model. The production of donor‐specific IgG antibody in the anti‐CD44 group did not vary in the Allo‐Tx model, while it was markedly elevated in the Xeno‐Tx model. Furthermore, the expression of interferon gamma in the anti‐CD44 group was markedly decreased in both the Allo‐Tx and Xeno‐Tx models, while the expression of IL‐4 in the anti‐CD44 group was significantly increased only in the Xeno‐Tx model. Conclusion: Multiple applications of the anti‐CD44 mAb could significantly inhibit donor‐reactive CD4⁺ memory T cells from rejecting grafts via a Th1‐dependent pathway, but xenoreactive CD4⁺ memory T cells can avoid the effects of anti‐CD44 mAb to reject islet xenografts via a Th2‐dependent pathway.     

Xenotransplantation in Asia

The organ shortage crisis affects most of the world today. In Asia, rates of deceased organ donation are extremely low due to sociocultural factors. In this context, implementing new organ donation policies is not enough; xenotransplantation remains the most promising way to solve the organ crisis. Most of the early research on xenotransplantation was conducted in the US and Europe. Today, however, Asia has caught up on its Western counterparts partly due to the increasing demand for organ transplants. Given the growing influence of countries such as China, South Korea, and Japan in xenotransplantation, this article provides the reader with an essential global understanding of the scientific and ethical issues currently at stake. Furthermore, it sheds light on the beliefs and values that shape the response of the Asian public to both organ donation and xenotransplantation.     

TMVA, a novel GPIb-binding protein, significantly prevents platelet microthrombi formation and prolongs discordant cardiac xenograft survival

In xenotransplantation, donor endothelium is the first target of immunological attack. Activation of the endothelial cell by preformed natural antibodies leads to platelet binding via the interaction of the glycoprotein (GP) Ib and von Willebrand factor (vWF). TMVA is a novel GPIb-binding protein purified from the venom of Trimeresurus mucrosquamatus. In this study, the inhibitory effect of TMVA on platelet aggregation in rats and the effect on discordant guinea pig-to-rat cardiac xenograft survival were investigated. Three doses (8, 20 or 40 microg/kg) of TMVA were infused intravenously to 30 rats respectively. Platelet aggregation rate was assayed 0.5, 12, and 24 h after TMVA administration. Wister rats underwent guinea pig cardiac cervical heterotopic transplantation using single dosing of TMVA (20 microg/kg, i.v., 0.5 h before reperfusion). Additionally, levels of TXB(2) and 6-keto-PGF(1alpha) within rejected graft tissues were determined by radioimmunoassay. Treatment with TMVA at a dose of 20 or 40 microg/kg resulted in complete inhibition of platelet aggregation 0.5 h after TMVA administration. Rats receiving guinea pig cardiac xenografts after TMVA therapy had significantly prolonged xenograft survival. Histologic and immunopathologic analysis of cardiac xenografts in TMVA treatment group showed no intragraft platelet microthrombi formation and fibrin deposition. Additionally, the ratio of 6-keto-PGF(1alpha) to TXB(2) in TMVA treatment group was significantly higher than those in control group. We conclude that the use of this novel GPIb-binding protein was very effective in preventing platelet microthrombi formation and fibrin deposition in a guinea pig-to-rat model and resulted in prolongation of xenograft survival. The increased ratio of PGI(2)/TXA(2) in TMVA treatment group may protect xenografts from the endothelial cell activation and contribute to the prolongation of xenograft survival.     

Potential deleterious role of anti‐Neu5Gc antibodies in xenotransplantation

Human beings do not synthesize the glycolyl form of the sialic acid (Neu5Gc) and only express the acetylated form of the sugar, whereas a diet‐based intake of Neu5Gc provokes a natural immunization and production of anti‐Neu5Gc antibodies in human serum. However, Neu5Gc is expressed on mammal glycoproteins and glycolipids in most organs and cells. We review here the relevance of Neu5Gc and anti‐Neu5Gc antibodies in the context of xenotransplantation and the use of animal‐derived molecules and products, as well as the possible consequences of a long‐term exposure to anti‐Neu5Gc antibodies in recipients of xenografts. In addition, the importance of an accurate estimation of the anti‐Neu5Gc response following xenotransplantation and the future contribution of knockout animals mimicking the human situation are also assessed.     

Triple (GGTA1, CMAH,B2M) modified pigs expressing an SLA class Ilow phenotype—Effects on immune status and susceptibility to human immune responses

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9‐mediated deletion of β₂‐microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8⁺ T cell subset was observed in B2M^low pigs. Cells from B2M^low pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8⁺ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4⁺ T cells, natural killer cells) lysed targets from both SLA class I⁺ wildtype and SLA class I^low pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8⁺ T cells during the induction phase of an anti‐xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.