Comparative Inhibitory Effects of Tacrolimus, Cyclosporine, and Rapamycin on Human Anti-Pig Xenogeneic Mixed Lymphocyte Reactions.

BACKGROUND: Long-term immunosuppressive maintenance therapy is necessary to prevent the rejection of xenografts. However, it is still unclear which oral immunosuppressant is most suitable for pig-to-human xenotransplantation . METHODS: A xenogeneic mixed lymphocyte reaction (MLR) system was established using peripheral blood mononuclear cells (PBMCs) isolated from wildtype (WT) or GTKO/CMAHKO/ß4GalNT2KO (TKO) pigs as stimulator cells and human PBMCs as responder cells. Various concentrations of tacrolimus (Tac), cyclosporine (CsA), or rapamycin (Rapa) were added to the MLR system as interventions. The inhibitory effects of the three immunosuppressants on the proliferation and cytokine production of human T cells were studied and compared. The inhibitory effect of anti-CD154 mAb alone or in combination with Tac/CsA/Rapa on xenoreactive MLR was also investigated. RESULTS: PBMCs from both WT and TKO pigs stimulated significant proliferation of human T cells. Tac had a strong inhibitory effect on human T-cell proliferation stimulated by pig PBMCs. CsA inhibited human T-cell proliferation in a typical dose-dependent manner. When Tac and CsA concentrations reached 5 and 200 ng/mL, respectively, the proliferation rates of CD3+/CD4+/CD8+ T cells were reduced almost to a negative level. Even at high concentrations, Rapa had only a moderate inhibitory effect on xenogeneic MLR. The inhibitory effects of these three immunosuppressants on xenogeneic T-cell responses were further confirmed by the detection of CD25 expression and supernatant cytokines (IL-2, IL-6, IFN-γ, TNF-α, IL-4, IL-10, and IL-17). Although anti-CD154 mAb monotherapy showed only moderate inhibitory effects on xenoreactive T-cell proliferation, low-dose anti-CD154 mAb combined with low-dose Tac, CSA, or Rapa could produce significant synergistic inhibitory effects. CONCLUSION: Tac is more efficient than CsA or Rapa in inhibiting xenogeneic T-cell responses in vitro. If used in combination with anti-CD154 mAb, all the three immunosuppressants can achieve satisfactory synergistic inhibitory effects.

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.

Advances and applications of genetically modified pig models in biomedical and agricultural field.

Compared with rodents, pigs are closer to humans in terms of anatomy, metabolism and physiology, so they are ideal animal models of human diseases and xenotransplantation donors. In addition, as one of the most important livestock in China, pigs are closely related to our lives in terms of breeding improvement, disease prevention and animal welfare. In this review, we mainly summarize the research progress and future application of genetically modified pig models in the fields of xenotransplantation, molecular breeding and human disease models. We wish to take this opportunity to raise the awareness of researchers in related fields on cutting-edge technologies such as gene editing and understand the significance of genetically modified pig models in life science research.

Identification and verification of potential piRNAs from domesticated yak testis.

PIWI-interacting RNAs (piRNA) are small non-coding RNA molecules expressed in animal germ cells that interact with PIWI family proteins to form RNA-protein complexes involved in epigenetic and post-transcriptional gene silencing of retrotransposons and other genetic elements in germ line cells, including reproductive stem cell self-sustainment, differentiation, meiosis and spermatogenesis. In the present study, we performed high-throughput sequencing of piRNAs in testis samples from yaks in different stages of sexual maturity. Deep sequencing of the small RNAs (18-40 nt in length) yielded 4,900,538 unique reads from a total of 53,035,635 reads. We identified yak small RNAs (18-30 nt) and performed functional characterization. Yak small RNAs showed a bimodal length distribution, with two peaks at 22 nt and >28 nt. More than 80% of the 3,106,033 putative piRNAs were mapped to 4637 piRNA-producing genomic clusters using RPKM. 6388 candidate piRNAs were identified from clean reads and the annotations were compared with the yak reference genome repeat region. Integrated network analysis suggested that some differentially expressed genes were involved in spermatogenesis through ECM-receptor interaction and PI3K-Akt signaling pathways. Our data provide novel insights into the molecular expression and regulation similarities and diversities in spermatogenesis and testicular development in yaks at different stages of sexual maturity.

Pig-to-human kidney xenotransplants using genetically modified minipigs.

This study develops an observational model to assess kidney function recovery and xenogeneic immune responses in kidney xenotransplants, focusing on gene editing and immunosuppression. Two brain-dead patients undergo single kidney xenotransplantation, with kidneys donated by minipigs genetically modified to include triple-gene knockouts (GGTA1, ß4GalNT2, CMAH) and human gene transfers (hCD55 or hCD55/hTBM). Renal xenograft functions are fully restored; however, immunosuppression without CD40-CD154 pathway blockade is ineffective in preventing acute rejection by day 12. This rejection manifests as both T cell-mediated rejection and antibody-mediated rejection (AMR), confirmed by natural killer (NK) cell and macrophage infiltration in sequential xenograft biopsies. Despite donor pigs being pathogen free before transplantation, xenografts and recipient organs test positive for porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) by the end of the observation period, indicating reactivation and contributing to significant immunopathological changes. This study underscores the critical need for extended clinical observation and comprehensive evaluation using deceased human models to advance xenograft success.

Loss of ZBED6 Protects Against Sepsis-Induced Muscle Atrophy by Upregulating DOCK3-Mediated RAC1/PI3K/AKT Signaling Pathway in Pigs.

Sepsis-induced muscle atrophy often increases morbidity and mortality in intensive care unit (ICU) patients, yet neither therapeutic target nor optimal animal model is available for this disease. Here, by modifying the surgical strategy of cecal ligation and puncture (CLP), a novel sepsis pig model is created that for the first time recapitulates the whole course of sepsis in humans. With this model and sepsis patients, increased levels of the transcription factor zinc finger BED-type containing 6 (ZBED6) in skeletal muscle are shown. Protection against sepsis-induced muscle wasting in ZBED6-deficient pigs is further demonstrated. Mechanistically, integrated analysis of RNA-seq and ChIP-seq reveals dedicator of cytokinesis 3 (DOCK3) as the direct target of ZBED6. In septic ZBED6-deficient pigs, DOCK3 expression is increased in skeletal muscle and myocytes, activating the RAC1/PI3K/AKT pathway and protecting against sepsis-induced muscle wasting. Conversely, opposite gene expression patterns and exacerbated muscle wasting are observed in septic ZBED6-overexpressing myotubes. Notably, sepsis patients show increased ZBED6 expression along with reduced DOCK3 and downregulated RAC1/PI3K/AKT pathway. These findings suggest that ZBED6 is a potential therapeutic target for sepsis-induced muscle atrophy, and the established sepsis pig model is a valuable tool for understanding sepsis pathogenesis and developing its therapeutics.

Both Natural and Induced Anti-Sda Antibodies Play Important Roles in GTKO Pig-to-Rhesus Monkey Xenotransplantation.

Sda, produced by the B4GALNT2 enzyme, has been recognized as an important xenoantigen for pig-to-nonhuman primate xenotransplantation. However, little is known about Sda expression in pigs and its immunogenicity in xenotransplantation. In this study, peripheral blood mononuclear cells (PBMCs) were isolated from wildtype, GTKO (with high, moderate, and low Sda expression), GTKO/ß4GalNT2KO, GTKO/CMAHKO, or GTKO/CMAHKO/ß4GalNT2KO pigs. Anti-pig IgM/IgG binding and complement-dependent cytotoxicity (CDC) to pig PBMCs was measured by flow cytometry using pooled rhesus monkey sera (n=20) or human sera (n=20). As compared to wild-type pigs (n=12), GTKO pigs (n=17) had a significantly higher mean level of Sda expression on PBMCs and showed a greater individual difference in expression. Both the overall binding of monkey serum IgM/IgG antibody to GTKO pig PBMCs and CDC against these PBMCs decreased significantly with a progressive reduction in Sda expression, showing a clear dose-effect relationship. Both the monkey serum antibody binding and CDC decreased significantly after the additional deletion of Sda, whereas the binding of human serum antibody and CDC against the GTKO pig PBMCs were markedly reduced after the deletion of Neu5Gc in the pigs. In addition, anti-Sda antibody accounted for > 50% of the induced anti-non-Gal antibody at the time of rejection in two rhesus monkeys that received GTKO/hCD55 pig kidney xenotransplantation, and the anti-Sda antibody showed significant cytotoxic activity against GTKO pig cells. We conclude that both natural and induced anti-Sda antibodies play important roles in GTKO pig-to-rhesus monkey xenotransplantation, thus providing further evidence for GTKO/ß4GalNT2KO pigs as the preferred organ source for rhesus monkeys as a preclinical model of xenotransplantation.

Serum Antibody Binding and Cytotoxicity to Pig Cells in Chinese Subjects: Relevance to Clinical Renal Xenotransplantation.

Kidney xenotransplantation is expected to contribute to resolving the shortage of kidneys from deceased human donors. Although progress in experimental life-supporting pig renal xenotransplantation has been encouraging, there are still issues to be considered before a clinical trial can be initiated. We attempted to clarify some of these by an in vitro study. Blood was drawn from healthy volunteers (Volunteers, n=20), patients with end-stage renal disease (ESRD, n=20) pre-operation (Pre), and on Day 1 (POD 1) and Day 14 (POD 14) after renal allotransplantation, brain-dead organ donors (DBD, n=20), and renal allotransplant recipients who were currently experiencing T cell-mediated rejection (Allo-TCMR, n=20). Serum IgM/IgG binding to, and complement-dependent cytotoxicity (CDC) of, PBMCs and RBCs from (a) wild-type (WT), (b) α1,3-galactosyltransferase gene-knockout (GTKO), (c) GTKO/beta-1,4-N-acety1 galactosaminyltransferase 2-knockout (GTKO/ß4GalNT2KO), (d) GTKO/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (GTKO/CMAHKO), and (e) GTKO/ß4GalNT2KO/CMAHKO/hCD55 (TKO/hCD55) pigs were measured by flow cytometry. We obtained the following results: (i) Serum IgM/IgG binding and CDC in Volunteers were significantly greater to WT, GTKO, and GTKO/ß4GalNT2KO PBMCs or RBCs than to GTKO/CMAHKO and TKO/hCD55 cells; (ii) ESRD, DBD, and Allo-TCMR serum antibody binding and CDC to WT pig PBMCs were significantly greater than to GTKO, GTKO/ß4GalNT2KO, GTKO/CMAHKO, and TKO/hCD55 cells; (iii) antibody binding to GTKO/CMAHKO pig cells was significantly lower in hemodialysis than peritoneal dialysis patients. (iv) Two of twenty allotransplantation recipients' serum IgG binding to GTKO pig PBMCs increased on POD14 compared with Pre, but IgG binding to GTKO pig RBCs did not; (v) In all sera, the lowest antibody binding and CDC were to GTKO/CMAHKO and TKO/CD55 pig cells. We conclude (i) CMAHKO in the pig may be critical to the success of clinical pig kidney xenotransplantation, and may be the most important after GTKO, at least in Chinese patients; (ii) subjects with ESRD, or who are immunosuppressed after kidney allotransplantation, and DBD, have lower levels of antibody binding and CDC to genetically-engineered pig cells than do volunteers; (iii) TKO pigs with selected human 'protective' transgenes, e.g., CD55, are likely to prove to be the optimal sources of kidneys for clinical xenotransplantation.