Background

Measurement of serum anti-pig antibodies is an important parameter in immune monitoring after pig-to-nonhuman primate xenotransplantation. Pig aortic endothelial cells (pAECs) are commonly used for this purpose. However, human (h) CTLA4-Ig (abatacept/belatacept) could bind to pCD80/86 on the cells, and a secondary antibody (i.e., anti-human IgG) may recognize hCTLA4-Ig (in the absence of serum anti-pig IgG antibody binding to pAECs), potentially leading to misinterpretation of the results. Our aim was to determine whether hCTLA4-Ig binding to pAECs is associated with false-positive results.

Methods

Sera were obtained from (i) naïve baboons (n = 3) and (ii) baboons (n = 2) that had undergone pig artery patch transplantation with/without hCTLA4Ig therapy. Serum IgM and IgG binding to (i) AECs, (ii) red blood cells (RBCs), and (iii) CD3⁺T cells in peripheral blood mononuclear cells (PBMCs) from an α1,3-galactosyltransferase gene-knockout pig expressing human CD46 (GTKO/hCD46) was measured by flow cytometry in the presence or absence of hCTLA-4Ig. Complement-dependent cytotoxicity (CDC) of wild-type (WT) pAECs by hCTLA4Ig was measured by flow cytometry.

Results

Sera containing hCTLA4-Ig demonstrated significantly increased IgG (but not IgM) binding to pAECs (relative geometric mean [rGM] = 1.8) compared to sera without hCTLA-4Ig (rGM =1.3) (p < .01). In contrast, there was no increased binding to pRBCs or CD3⁺T cells. hCTLA4-Ig did not result in cytotoxicity of WT pAECs.

Conclusions

pAECs might not be an optimal cell to investigate anti-pig IgG binding when hCTLA4-Ig is administered to the recipient, as a false-positive result may result from hCTLA4-Ig binding to the pAECs. CD3⁺T cells would be preferable targets (compared to pRBCs) because they express both carbohydrate and MHC class I/II antigens.

中文翻译：

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人CTLA4-Ig治疗可以在异种移植后的灵长类动物中产生假阳性的抗猪抗体结果。

背景

猪抗非人灵长类动物异种移植后，血清抗猪抗体的测量是免疫监测中的重要参数。猪主动脉内皮细胞（pAEC）通常用于此目的。但是，人（h）CTLA4-Ig（abatacept / belatacept）可以与细胞上的pCD80 / 86结合，并且二抗（即抗人IgG）可以识别hCTLA4-Ig（在没有血清抗猪的情况下） IgG抗体与pAEC结合），可能导致对结果的曲解。我们的目的是确定hCTLA4-Ig与pAEC的结合是否与假阳性结果相关。

方法

血清是从（i）幼稚狒狒（n  = 3）和（ii）狒狒（n  = 2）接受过或未进行hCTLA4Ig治疗的猪动脉斑块移植术获得的。血清IgM和IgG与（i）表达人的α1,3-半乳糖基转移酶基因敲除猪的外周血单核细胞（PBMC）中的（i）AEC，（ii）红细胞（RBC）和（iii）CD3 ⁺ T细胞结合在有或没有hCTLA-4Ig的情况下，通过流式细胞术测量CD46（GTKO / hCD46）。通过流式细胞术测量了hCTLA4Ig对野生型（WT）pAEC的补体依赖性细胞毒性（CDC）。

结果

与不含hCTLA-4Ig的血清（rGM = 1.3）相比，含hCTLA4-Ig的血清显示与IgG结合的IgG（而非IgM）显着增加（相对几何平均值[rGM] = 1.8）（p  <.01）。相反，与pRBC或CD3 ⁺ T细胞的结合没有增加。hCTLA4-Ig不会导致野生型pAEC的细胞毒性。

结论

当将hCTLA4-Ig给予接受者时，pAEC可能不是研究抗猪IgG结合的最佳细胞，因为hCTLA4-Ig与pAEC的结合可能导致假阳性结果。CD3 ⁺ T细胞将是优选的靶标（与pRBC相比），因为它们同时表达碳水化合物和MHC I / II类抗原。