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112.4 SLA antibody screening using Auckland island pig cells: Toward standardized diagnostics for xenotransplantation

Gisella L. Puga Yung, Switzerland

Division of Immunology adn Allergology
University Hospitals Geneva

Abstract

SLA antibody screening using Auckland island pig cells: Toward standardized diagnostics for xenotransplantation

Gisella Puga Yung1, Maria V. Leuschen2, Nicole Strobl3, Barbara Kessler4,8, Martin Bender5, Matthias Längin5, Teresa Kauke3, Eckhard Wolf4,8, Jörg D. Seebach1, Bruno Reichart6, Andrea Dick 7.

1Medicine, University Hospitals and Faculty of Medicine Geneva, Geneva, Switzerland; 2Cardiac Surgery, University Hospital LMU , Munich, Germany; 3Thoracic Surgery, University Hospital LMU , Munich, Germany; 4Veterinary Sciences, LMU, Munich, Germany; 5Anesthesiology, University Hospital LMU , Munich, Germany; 6Walter Brendel Centre of Experimental Medicine, LMU, Munich, Germany; 7Transfusion Medicine, Cellular Therapeutics and Hemostaseology, University Hospital LMU , Munich, Germany; 8Center for Innovative Medical Models (CiMM), LMU, Munich, Germany

Introduction: The Auckland Island pig (AIP) breed is of particular interest in xenotransplantation due to its complete homozygosity across the swine leukocyte antigen (SLA) loci, a result of inbreeding and natural selection. This genetic uniformity offers a major advantage over cloned pigs by providing a stable background and functionally uniform expression of introduced human genes. With the primary carbohydrate xenoantigens (Ags) effectively eliminated by genetic modifications, SLAs have emerged as the next major immunologic barrier, capable of inducing antibody (Ab) responses upon sensitization. Additionaly, the generation of de novo Abs following transplantation remains major barriers to long-term xenograft survival. Post-transplant Ab levels against donor Ags are monitored for early signs of rejection or graft injury. The study aimed to test a large cohort of human sera, from both healthy individuals and highly sensitized patients, against cells from AIP, wild-type (WT) and genic modified animals. The ultimate goals were to support SLA matching at the time of xenotransplantation and to enable point-of-care or at-home monitoring through the development of a rapid screening method.
Methods: Peripheral blood mononuclear cells (PBMC) from AIP homozygous for SLA-1*23:03, SLA-2*13:02, SLA-6*10:01, SLA-11*01:01, SLA-DRA*01:01:02, SLA-DRB1*05:01, SLA-DQA*01:03 and SLA-DQB1*08:01. Both wild-type (WT) and triple knockout (TKO) animals (lacking carbohydrate Ags) were included in the study to assess Ab binding. The preparation of a human negative control serum was necessary. A human negative control serum was prepared by pooling sera from multiple donors and immunoadsorbing them first with red blood cells from WT pigs, then with PBMCs from TKO animals to remove anti-carbohydrate, anti-SLA, and other minor Ag-specific Abs. Human sera from individuals on the organ transplant waiting list and the general German population were tested using standardized porcine PBMC-based crossmatch assays, including antibody binding and complement-mediated cytotoxicity, analyzed via flow cytometry.
Results: We successfully adapted and standardized existing allotransplantation crossmatch protocols for use with porcine PBMC to detect anti-pig antibodies in human sera. Strong IgM and IgG binding was consistently observed with WT-derived PBMC. In contrast, most sera showed no reactivity against TKO-derived PBMC, with only a few highly sensitized samples displaying weak or borderline positivity. The use of the AIP breed facilitated compatibility testing by presenting a single, consistent SLA haplotype, thereby reducing screening complexity.
Conclusions: SLA homozygosity in the AIP breed reduces antigenic diversity, aiding compatibility assessment in xenotransplantation. Future work will standardize assays using porcine endothelial cell monolayers and fluorescence microscopy to better reflect xenograft antigens.

Funding. Deutsche Forschungsgemeinschaft (CRC-TR 127). Swiss National Science Foundation (Sinergia grant CRSII5_198577/1). Leducq Foundation (23CVD01). German Heart Foundation.

References:

[1] crossmatch
[2] Auckland Island pig
[3] anti-pig antibodies
[4] Flow cytometry

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