Human CTLA4-Ig therapy can give false-positive anti-pig antibody results in primates after xenotransplantation

Qi Li; Takayuki Yamamoto; Hayato Iwase; Huy Quoc Nguyen; Yi Wang; David Ayares; David K C Cooper; Hidetaka Hara

doi:10.1016/j.trim.2019.101243

Human CTLA4-Ig therapy can give false-positive anti-pig antibody results in primates after xenotransplantation

Transpl Immunol. 2019 Dec:57:101243. doi: 10.1016/j.trim.2019.101243. Epub 2019 Aug 28.

Authors

Qi Li¹, Takayuki Yamamoto², Hayato Iwase², Huy Quoc Nguyen², Yi Wang³, David Ayares⁴, David K C Cooper², Hidetaka Hara⁵

Affiliations

¹ Second Affiliated Hospital, University of South China, Hengyang City, Hunan, China; Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA.
² Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA.
³ Second Affiliated Hospital, University of South China, Hengyang City, Hunan, China; The Second Affiliated Hospital of Hainan Medical University, Hainan, China; The Transplantation Institute of Hainan Medical University, Hainan, China. Electronic address: wayne0108@126.com.
⁴ Revivicor, Blacksburg, VA, USA.
⁵ Xenotransplantation Program, Department of Surgery, University of Alabama at Birmingham, Birmingham, AL, USA. Electronic address: harahjp@icloud.com.

PMID: 31472231
DOI: 10.1016/j.trim.2019.101243

Abstract

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.

Keywords: Anti-pig; Antibodies; Flow cytometry; Xenotransplantation; hCTLA4-Ig; pAECs.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Abatacept / metabolism
Abatacept / therapeutic use*
Animals
Animals, Genetically Modified
Antibodies, Heterophile / blood*
Antibody-Dependent Cell Cytotoxicity
Aorta / pathology*
Aorta / transplantation
Cells, Cultured
Disease Models, Animal
Endothelium, Vascular / metabolism*
Endothelium, Vascular / pathology
False Positive Reactions
Gene Knockout Techniques
Humans
Membrane Cofactor Protein / genetics
Papio
Protein Binding
Swine
Tissue Transplantation*
Transplantation, Heterologous

Substances

Antibodies, Heterophile
Membrane Cofactor Protein
Abatacept