Regulation of Human NK Cell Activation by Expression of HLA Class I Molecules in Pig Endothelial Cells

Authors

  • Benjamin Obando Indiana University School of Medicine https://orcid.org/0000-0001-6544-6538
  • Arthur Cross-Najafi Indiana University School of Medicine
  • Kevin Lopez Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine
  • Deepthi Thadasina Indiana University School of Medicine https://orcid.org/0000-0002-6896-1479
  • Wenjun Zhang Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine
  • A. Kadir Isidan Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine
  • Yujin Park Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine
  • Gonzalo Campana Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine
  • Ping Li, PhD Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine
  • Burcin Esker, MD, PhD Division of Transplant Surgery, Department of Surgery, Indiana University School of Medicine

DOI:

https://doi.org/10.18060/25715

Abstract

Background:  

Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically engineered pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute xenograft rejection (AXR) remains a major barrier to clinical XTx. AXR is mediated by multiple immune cells, of which natural killer (NK) cells play a crucial role. Previous studies have shown that human HLA-E suppresses NK cell activation through the inhibitory receptor NKG2A. We seek to improve pig-to-human compatibility by expressing HLA-E in a genetically modified pig endothelial cell (pEC) line. This cell line 5GKO/ HLA-G+ has mutations in five genes encoding for xenoantigens and expresses HLA-G, an inhibitory ligand of the NK cell receptor KIR2DL4. In this study, the 5GKO/HLA-G+/HLA-E+ pEC line was established to examine whether co-expression of inhibitory ligands promotes NK cell tolerance.   

 

Methods:  

The HLA-Eα/pCDNA3.1 plasmid containing the HLA-E α-chain (HLA-Eα) cDNA driven by a CMV promoter was linearized and introduced into 106 cells of the 5GKO/HLA-G+ pEC line by electroporation. After 48 hours, HLA-E expression was analyzed by flow cytometry. HLA-E+ pECs were isolated by flow cytometry sort and co-cultured with human peripheral blood mononuclear cells (PBMCs) stimulated by IL-2. NK cell degranulation was compared between the 5GKO/HLA-G+ and 5GKO/HLA-G+/HLA-E+ pEC lines by measuring CD107a expression in the CD3- CD56+ cell population. 

 

 

 

 

Results:  

HLA-E molecules were successfully expressed on the pECs surface, indicating the HLA-E a chain can pair with the existing b2-microglobulin (B2M). The transfection efficiency was 38.2%. Three weeks later, the 5GKO/HLA-G+/HLA-E+ pEC was successfully established, confirming via flow cytometric analysis. The analysis of NK cell degranulation (CD107a) is underway.  

 

Conclusion:  

We established a 5GKO/HLA-G+/HLA-E+ pEC line, which is a valuable tool to study human-to-pig xenoreactive immune response in vitro, with the goal of improving pig-to-human xenograft immunotolerance. 

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Published

2021-12-10

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Abstracts