Potential Antigens Involved in Delayed Xenograft Rejection in a Ggta1/Cmah Dko Pig-to-Monkey Model

Abstract

When hyperacute rejection is avoided by deletion of Gal expression in the pig, delayed xenograft rejection (DXR) becomes a major immunologic barrier to successful xenotransplantation. This study was to investigate the potential antigens involved in DXR. We isolated primary renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) from a GGTA1/CMAH double-knockout (DKO) pig (and a GGTA1-KO pig) and immunized cynomolgus monkeys with both of these cells. After sensitization, monkey serum antibody binding and cytotoxicity to RMEC was significantly higher than to AEC(p < 0.05), suggesting that RMEC are more immunogenic than AEC. Transcriptome sequencing of GGTA1/CMAH DKO pigs indicated that the expression of 1,500 genes was higher in RMEC than in AEC, while expression of 896 genes was lower. Next, we selected 101 candidate genes expressed only in pig RMEC, but not in pig AEC or in monkey or human RMEC. When these genes were knocked out individually in GGTA1/CMAH DKO RMEC, 32 genes were associated with reduced antibody binding, indicating that these genes might be primary immunologic targets involved in DXR. These genes may be important candidates for deletion in producing pigs against which there is a reduced primate immune response in pig kidney xenograft.

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes

“…Other anti-inflammatory and anti-apoptosis-related genes in humans have also been expressed in pigs [ 55 – 57 ]. Xenograft rejection of these organs from genetically modified pigs to monkey and baboon models has been successfully delayed [ 58 – 60 ]. Multiple gene modifications, including knock-ins of human genes, will be essential for the maintenance of the function of xenotransplanted organs.…”

Downregulation of Gabarapl1 significantly attenuates antibody binding to porcine aortic endothelial cells

“…To investigate the relationship between potential non‐Gal antigens in PAECs and corresponding antibodies produced in recipient nonhuman primates, PAECs from 2 piglets (#16002, #16014) were subcutaneously injected together into the axillae of 3 cynomolgus monkeys on 3 occasions (at 0, 3, and 7 weeks) . Serum samples were collected from the monkeys before PAEC injection and 8 weeks later for antibody binding assays and mixed before testing.…”

“…The following reagents were obtained from the indicated sources: anti‐Gabarapl1 (26632, applicable to pig, according to sequence identity), anti‐GAPDH (2118, applicable to pig, according to manufacturer's description, Cell Signaling); FITC‐goat anti‐human IgG (628411, applicable for monkey, according to sequence identity), FITC‐goat anti‐human IgM (A18842, suitable for monkey, according to sequence identity, Invitrogen); FITC‐mouse anti‐SLA‐II DR (MCA2314F), FITC‐mouse anti‐pig CD31 (MCA1746F, Bio‐Rad); FITC‐mouse IgG isotype control (556649, BD); collagenase type IV from Clostridium histolyticum (Sigma). PAEC‐immunized monkey sera were prepared, as previously reported . The sera from the different monkeys were mixed before use.…”

“…PAEC-immunized monkey sera were prepared, as previously reported. 17 The sera from the different monkeys were mixed before use.…”

Xenotransplantation: Current Status in Preclinical Research

“…Various types of gene-modified pigs already exist, most of which are being tested in preclinical pig-to-NHP xenotransplantation models. In addition, new xenoreactive antigens are continually being discovered (198,199), from which new knockout and transgenic pigs may be generated. Although assessment of current genetically modified pigs combined with immunosuppressive therapy in NHP models is complex and expensive, we agree with the opinion that substantial results should be obtain in NHP models before clinic application.…”