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

Junfang Zhang,Mingtao Li,Dengke Pan,Zhiming Cai,David K C Cooper,Ying Lu,Jia Xu,Lisha Mou,Da Yao,Yifan Dai,Chongwei Xie,Ming Zhou,Zepeng Qu,Hidetaka Hara,Chuanghua Qiu,Shanshan Ma

doi:10.1038/s41598-017-10805-0

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.

Highlights

The endothelial lining of the vasculature of the graft is a major target for the host’s immune response, characterized by antibody-mediated rejection and/or thrombotic microangiopathy[15, 16]
Preformed and induced antibody directed toward the vascular endothelium is considered to be the primary immune mechanism in the development of delayed xenograft rejection (DXR), which is believed to result from chronic activation or injury to the vascular endothelium mediated by antibody binding and/or complement activation[17]
renal microvascular endothelial cells (RMEC) and aortic endothelial cells (AEC) were isolated from the renal microvascular endothelium and aortic endothelium, respectively

Summary

Introduction

The endothelial lining of the vasculature of the graft is a major target for the host’s immune response, characterized by antibody-mediated rejection and/or thrombotic microangiopathy[15, 16]. Preformed and induced antibody directed toward the vascular endothelium is considered to be the primary immune mechanism in the development of DXR, which is believed to result from chronic activation or injury to the vascular endothelium mediated by antibody binding and/or complement activation[17]. These processes promote the formation of a thrombogenic vasculature, which, if unchecked, leads to microvascular thrombosis and ischemic injury[18]. The primary aim of the study was to immunize monkeys to pig antigens and determine whether antibodies developed to new pig antigens that had not been previously identified

Objectives

Methods

Results

Conclusion