Columbia Center for Translational Immunology, Department of Medicine, Columbia University, New York, NY, USA.
Department of Pathology, Nippon Medical School, Bunkyo-ku, Japan.
Xenotransplantation. 2021 Jan;28(1):e12642. doi: 10.1111/xen.12642. Epub 2020 Sep 9.
Recent advances in gene editing technology have enabled the production of multi-knockout (KO) and transgenic pigs in order to overcome immunologic barriers in xenotransplantation (XTx). However, the genetic manipulations required to produce these changes may have the unintended consequence of producing or revealing neoantigens reactive with natural antibodies present in baboons. In this study, we examined whether the neoantigens that develop in multi-transgenic (mTg) GalT, Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), β-1,4-N-acetyl-galactosaminyl transferase 2 (B4) KO pigs can cause rejection of xenografts in baboons.
Five baboons that had <35% cytotoxicity against GalT-KO peripheral blood mononuclear cells (PBMCs) in a pre-screening assay received pig kidneys and vascularized thymic grafts (VT + K) from multi-transgenic hCD47, human thrombomodulin (hTBM), human endothelial protein C receptor (EPCR) with/without hCD46 and hCD55 with GalT-KO/NeuGC-KO/B4-KO (mTg Tri-KO) swine. In order to further examine the effects of anti-donor non-Gal natural antibody (nAb), anti-pig preformed IgM and IgG nAb binding against the GalT-KO PBMCs was compared with the donor-type PBMCs using donor pretransplant sera as well as 5 additional naïve baboon sera by flow cytometric analysis.
Five baboons that received VT + K grafts had stable renal function in the first 11 days (serum creatinine < 1.5 mg/dL). Two of the five baboons had higher binding of preformed IgG to mTg Tri-KO PBMCs than to GalT-KO PBMCs (mTg Tri-KO > GalT-KO), and they rejected their grafts at POD 20. In contrast, the other three baboons demonstrated either mTg Tri-KO = GalT-KO or mTg Tri-KO < GalT-KO, and they maintained renal function 43, 52, and 154 days without rejection. Among 10 baboon sera, two had less antibody binding against PBMCs that were syngeneic to the mTg Tri-KO than against GalT-KO PBMCs (mTg Tri-KO < GalT-KO); three had similar binding to mTg Tri-KO and GalT-KO PBMCs (mTg Tri-KO = GalT-KO); and five had higher binding to m Tg Tri-KO than to GalT-KO PBMCs (mTg Tri-KO > GalT-KO).
These data suggest that neoantigens associated with mTg Tri-KO promote acute xenograft rejection in a pig-to-baboon VT + K XTx model. The screening assays may be useful to select "safe" recipients to receive mTg Tri-KO kidneys.
基因编辑技术的最新进展使得能够生产多敲除（KO）和转基因猪，以克服异种移植（XTx）中的免疫障碍。然而，产生这些变化所需的基因操作可能会产生或揭示与食蟹猴体内天然抗体反应的新抗原。在这项研究中，我们研究了在多转基因（mTg）GalT、胞苷单磷酸-N-乙酰神经氨酸羟化酶（CMAH）、β-1,4-N-乙酰半乳糖胺基转移酶 2（B4）KO 猪中产生的新抗原是否会导致食蟹猴异种移植物的排斥。
在预筛选试验中，5 只食蟹猴对 GalT-KO 外周血单核细胞（PBMC）的细胞毒性<35%，接受了来自多转基因 hCD47、人血栓调节蛋白（hTBM）、人内皮蛋白 C 受体（EPCR）的猪肾和血管化胸腺移植物（VT+K）），具有/不具有 hCD46 和 hCD55 的 GalT-KO/NeuGC-KO/B4-KO（mTg 三 KO）猪。为了进一步研究抗供体非 Gal 天然抗体（nAb）的影响，使用供体移植前血清以及另外 5 份未致敏食蟹猴血清，通过流式细胞术分析比较了抗 GalT-KO PBMCs 的供体型 PBMCs 与供体型 PBMCs 之间的抗供体预形成 IgM 和 IgG nAb 结合。
接受 VT+K 移植物的 5 只食蟹猴在最初的 11 天内肾功能稳定（血清肌酐<1.5mg/dL）。其中 2 只食蟹猴的预形成 IgG 与 mTg 三 KO PBMCs 的结合高于与 GalT-KO PBMCs 的结合（mTg 三 KO>GalT-KO），它们在 POD 20 时排斥移植物。相比之下，其他 3 只食蟹猴表现出 mTg 三 KO=GalT-KO 或 mTg 三 KO<GalT-KO，它们在没有排斥的情况下维持肾功能 43、52 和 154 天。在 10 份食蟹猴血清中，有 2 份对与 mTg 三 KO 同基因的 PBMCs 的抗体结合低于对 GalT-KO PBMCs 的抗体结合（mTg 三 KO<GalT-KO）；3 份对 mTg 三 KO 和 GalT-KO PBMCs 的结合相似（mTg 三 KO=GalT-KO）；5 份对 mTg 三 KO 的结合高于对 GalT-KO PBMCs 的结合（mTg 三 KO>GalT-KO）。
这些数据表明，与 mTg 三 KO 相关的新抗原促进了猪到食蟹猴 VT+K XTx 模型中的急性异种移植物排斥。筛选试验可能有助于选择“安全”的接受者接受 mTg 三 KO 肾脏。
