Elimination of GGTA1,CMAH,β4GalNT2 and CIITA genes in pigs compromises human versus pig xenogeneic immune reactions

Background:Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic,while immunogenic genes need to be eliminated to im-prove the immune compatibility between humans and pigs.Current knockout strat-egies are mainly aimed at the genes causing hyperacute immune rejection(HAR)that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure,in which process the MHC II molecule plays critical roles.Methods:Thus,we generate a 4-gene(GGTA1,CMAH,β4GalNT2,and CIITA)knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously.Results:We successfully obtained 4KO piglets with deficiency in all alleles of genes,and at cellular and tissue levels.Additionally,the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping.Piglets have survived for more than one year in the barrier,and also survived for more than 3 months in the conventional environment,suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment.Conclusions:4KO piglets have lower immunogenicity,are safe in genomic level,and are easier to breed than the model with both MHC I and II deletion.

Identification of the miniature pig inbred line by skin allograft

Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identification of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig （WZSP） inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had D^en cultivated successfully, allogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients＇ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4~ and CD8~ lymphocytes, and conducted hematoxylin-eosin （HE） and Masson＇s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism （SNP） chips （60 000 SNPs）. The result showed that there were no significant differences in graft skin, the plasma interleukin-2, 4, 10, CD4~ and CD8~, HE and Masson＇s staining results between the allograft and autograft groups, and no immune rejection occurred on the allograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex （MHC） I and MHC II were homozygous which confirmed that immune antibody of the allograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the allograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successfully for the first time in the world. The test methods also provide a scientific basis for the identification of swine and mammal inbred lines.

Old game, new players: Linking classical theories to new trends in transplant immunology

The evolutionary emergence of an efficient immune system has a fundamental role in our survival against pathogenic attacks. Nevertheless, this same protective mechanism may also establish a negative consequence in the setting of disorders such as autoimmunity and transplant rejection. In light of the latter, although research has long uncovered main concepts of allogeneic recognition, immune rejection is still the main obstacle to long-term graft survival. Therefore, in order to define effective therapies that prolong graft viability, it is essential that we understand the underlying mediators and mechanisms that participate in transplant rejection. This multifaceted process is characterized by diverse cellular and humoral participants with innate and adaptive functions that can determine the type of rejection or promote graft acceptance. Although a number of mediators of graft recognition have been described in traditional immunology, recent studies indicate that defining rigid roles for certain immune cells and factors may be more complicated than originally conceived. Current research has also targeted specific cells and drugs that regulate immune activation and induce tolerance. This review will give a broad view of the most recent understanding of the allogeneic inflammatory/tolerogenic response and current insights into cellular and drug therapies that modulate immune activation that may prove to be useful in the induction of tolerance in the clinical setting.

Xenogeneic stem cell transplantation:Research progress and clinical prospects

Organ transplantation is the ultimate treatment for end-stage diseases such as heart and liver failure.However,the severe shortage of donor organs has limited the organ transplantation progress.Xenogeneic stem cell transplantation provides a new strategy to solve this problem.Researchers have shown that xenogeneic stem cell transplantation has significant therapeutic effects and broad application prospects in treating liver failure,myocardial infarction,advanced type 1 diabetes mellitus,myelosuppression,and other end-stage diseases by replacing the dysfunctional cells directly or improving the endogenous regenerative milieu.In this review,the sources,problems and solutions,and potential clinical applications of xenogeneic stem cell transplantation will be discussed.

Effects of Danchai formula on regulation of immunologic function

The traditional formula of Danchai was provided by associate professor Dan Chen of Tianjin Medical University,which consisted of Chaihu(Radix Bupleuri)9 g,Baishao(Radix Paeoniae Alba)9 g,Yujin(Radix Curcumae)30 g,Mudanpi(Cortex moutan Radicis)9 g,and Gancao(Radix glycyrrhizae)6 g.After soaking in water for 20 min,the herbs were decocted with medium heat to boiling and for 15 min with low heat.The liquid was poured out and decocted again by adding water.The decoction is mixed and consumed orally 30 min after meals,twice per day.Danchai formula can be used to treat excessive immune response-related diseases,including immune rejection after organ transplantation,autoimmune diseases,and hypersensitivity.

Multiple gene modifications of pigs for overcoming obstacles of xenotransplantation

Xenotransplantation,involving animal organ transplantation into humans to address the human organ shortage,has been studied since the 17th century.Early attempts to obtain organs from animals such as goats,dogs,and non-human primates proved unsuccessful.In the 1990s,scientists agreed that pigs were the most suitable donor animals for xenotransplantation.However,immune rejection between pig and human has hindered the application.To overcome these challenges,researchers developed genetically modified pigs that deactivate xenoreactive antigen genes and express human protective genes.These advances extended xenograft survival from days to years in non-human primates,resulting in the first human heart xenotransplant trial.Using genetically engineered pigs for the organ shortage is promising.This review provides an overview of potential incompatibilities of immunogenicity and functional proteins related to xenotransplantation between humans and pigs.Furthermore,it elucidates possible approaches for multiplex gene modification to breed better-humanized pigs for clinical xenotransplantation.

Immature CD4＋ dendritic cells conditioned with donor kidney antigen prolong renal allograft survival in rats

Background AIIogeneic transplant rejection is currently a major problem encountered during organ transplantation. The dendritic cell （DC） is the most effective powerful known professional antigen-presenting cell, and recent studies have found that DCs can also induce immune tolerance, and avoid or reduce the degree of transplant rejection. The aim of this study was to evaluate the effect of transfused immature CD4~ DCs on renal allografts in the rat model. Methods In this study, we induced CD4~ immature DCs from rat bone marrow cells by a cytokine cocktail. The immature CD4~ DCs were identified by morphological analysis and then the suppressive activity of these cells conditioned with donor kidney antigen was evaluated in vitro and in vivo. Results Immature CD4~ DCs conditioned with donor kidney antigen possessed immunosuppressive activity in vitro and they were able to prolong renal transplant survival in an allograft rat model in vivo. Conclusions Our study provides new information on efficacious renal transplantation, which might be useful for understanding the function of immature CD4~ DCs in modulating renal transplant rejection and improving clinical outcome in future studies.

HLA-G inhibits xenogenetic cytotoxicity mediated by human NK cells and T lymphocytes against PECs

In order to investigate whether the non-classi- cal HLA-G classⅠmolecule protects the porcine endothelial cells (PECs) from the lysis mediated by human immune cells in pig to human discordant xenotransplantation, we have cloned HLA-G cDNA from a human placenta by RT-PCR. Mammalian expression vector, pEFG-neo, was constructed by insertion of HLA-G cDNA in pEF-neo. We obtained effi-ciently expressed PECs by stable transfection. Cytotoxicity assay showed that overexpression of HLA-G on PECs was sufficient to inhibit human NK-92 cell lysis. The level of lysis was equal to or less than that of the lysis of human umbilical vein endothelial cells mediated by human NK-92 cells. It also indicated that HLA-G inhibited the lysis of PECs mediated by xeno-antigen specific T lymphocytes. The reduction of lysis ranged between 59.1% and 88.9%. These findings sug-gest that the transgenic approach to overexpress HLA-G is believed to be a new immunotherapy in overcoming the im-mune rejections in xenotransplantion, including delayed xenograft rejection and cell-mediated rejection.

Transforming one organ into another to overcome challenges in tissue engineering

Tissue engineering(TE)is promising for the regeneration of failed organs.However,immune rejection,shortage of seed cells,and unintegrated blood vessels restrict the development and clinical application of TE.The last factor is the most challenging and intractable.Harnessing the mature blood vessel network in existing dispensable organs could be a powerful approach to effectively overcome the obstacles.After being remodeled to harbor an immunosuppressive and proregenerative niche,these potential target organs can be transformed into other organs with specific physiological functions,compensating the latter's failed native functions.Organ transformation,such as a hepatized spleen,represents an effective and encouraging TE strategy.In this review,we discuss the current development and obstacles of TE and its feasibility and superiority in organ transformation.

Locally controlled release of immunosuppressive promotes survival of transplanted adult spinal cord tissue

Transplantation of adult spinal cord tissue(aSCT)is a promising treatment for spinal cord injury(SCI)basing on various types of neural cells and matrix components inside aSCT.However,long-term systemic administration of immunosuppressors(e.g.tacrolimus,TAC)is required for the survival of allogeneic tissue,which often associated with severe side effects such as infection,liver damageand renal failure.In this study,a triglycerol monostearate(TGM)-based TAC delivery system(e.g.TAC@TGM)with high drug loading concentration was developed,which possessed injectable properties as well as sustainable and immune-responsive drug release behaviors.In complete transected SCI model,locally injected TAC@TGM could reduce the infiltration of inflammation cells,enhance the survival of transplanted aSCT(e.g.Tuj-1^(+)and NF^(+)neurons)and promote the recovery of locomotor function.Moreover,controlled release of TAC by TAC@TGM attenuated side effects of TAC on liver and kidneys compared with traditional systemic administration.More importantly,the developed TAC@TGM system provided a facile single dose of long-term immunosuppressive effect not just for aSCT transplantation,but also for other tissue/organ and cell transplantations.