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. 2016 Mar;23(2):137-50.
doi: 10.1111/xen.12229. Epub 2016 Mar 14.

Initial in vitro studies on tissues and cells from GTKO/CD46/NeuGcKO pigs

Whayoung Lee  1 , Hidetaka Hara  1 , Mohamed B Ezzelarab  1 , Hayato Iwase  1 , Rita Bottino  2 , Cassandra Long  1 , Jagdeece Ramsoondar  3 , David Ayares  3 , David K C Cooper  1
Affiliations

Initial in vitro studies on tissues and cells from GTKO/CD46/NeuGcKO pigs

Whayoung Lee et al. Xenotransplantation. 2016 Mar.

Abstract

Background: The impact that the absence of expression of NeuGc in pigs might have on pig organ or cell transplantation in humans has been studied in vitro, but only using red blood cells (pRBCs) and peripheral blood mononuclear cells (pPBMCs) as the target cells for immune assays. We have extended this work in various in vitro models and now report our initial results.

Methods: The models we have used involve GTKO/hCD46 and GTKO/hCD46/NeuGcKO pig aortas and corneas, and pRBCs, pPBMCs, aortic endothelial cells (pAECs), corneal endothelial cells (pCECs), and isolated pancreatic islets. We have investigated the effect of the absence of NeuGc expression on (i) human IgM and IgG binding, (ii) the T-cell proliferative response, (iii) human platelet aggregation, and (iv) in an in vitro assay of the instant blood-mediated inflammatory reaction (IBMIR) following exposure of pig islets to human blood/serum.

Results: The lack of expression of NeuGc on some pig tissues (aortas, corneas) and cells (RBCs, PBMCs, AECs) significantly reduces the extent of human antibody binding. In contrast, the absence of NeuGc expression on some pig tissues (CECs, isolated islet cells) does not reduce human antibody binding, possibly due to their relatively low NeuGc expression level. The strength of the human T-cell proliferative response may also be marginally reduced, but is already weak to GTKO/hCD46 pAECs and islet cells. We also demonstrate that the absence of NeuGc expression on GTKO/hCD46 pAECs does not reduce human platelet aggregation, and nor does it significantly modify the IBMIR to pig islets.

Conclusion: The absence of NeuGc on some solid organs from GTKO/hCD46/NeuGcKO pigs should reduce the human antibody response after clinical transplantation when compared to GTKO/hCD46 pig organs. However, the clinical benefit of using certain tissue (e.g., cornea, islets) from GTKO/hCD46/NeuGcKO pigs is questionable.

Keywords: Islets; N-glycolylneuraminic acid; Pig; Xenotransplantation; cytidine monophospho-N-acetylneuraminic acid hydroxylase; galactose-α1,3-galactose; pancreatic; α1,3-galactosyltransferase gene-knockout.

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Figures

Figure 1
Figure 1
Expression of Gal and NeuGc on heart, lung, kidney, liver, spleen, and lymph nodes of GTKO/hCD46 and GTKO/CD46/NeuGcKO pigs. All tissues from GTKO/hCD46 pigs were negative for Gal, but strongly positive for NeuGc, whereas tissues from two GTKO/hCD46/NeuGcKO pigs were negative for both antigens. (Magnification x200; nuclei, blue; Gal, green; NeuGc, red)
Figure 2
Figure 2
Expression of Gal, NeuGc, and hCD46 on WT, GTKO/hCD46, and GTKO/hCD46/NeuGcKO pig cells and on human cells - (A) RBCs, (B) PBMCs. Human IgM (C) and IgG (D) binding to WT, GTKO/hCD46, and GTKO/hCD46/NeuGcKO pig and human RBCs. Human IgM (E) and IgG (F) binding to WT, GTKO/hCD46, and GTKO/hCD46/NeuGcKO pig PBMCs. (To prevent a false positive result from alloantibody binding, human PBMC were not tested for IgM/IgG binding.) (A, B) Gal was detected only on WT RBCs and PBMCs. NeuGc was detected on RBCs and PBMCs from WT and GTKO/hCD46 pigs. RBCs and PBMCs from GTKO/hCD46/NeuGcKO pigs and humans were negative for both Gal and NeuGc. The hCD46 molecule is not expressed on the surface of RBCs, but it is detected on the PBMCs from GTKO/hCD46 and GTKO/hCD46/NeuGcKO pigs and from humans. (C, D) There was a significant difference in human IgM and IgG binding between WT vs. GTKO/hCD46, GTKO/hCD46/NeuGcKO pRBCs, and human RBCs (*p<0.05, **p<0.01). There was also a significant difference in binding between GTKO/hCD46 and GTKO/hCD46/NeuGcKO pRBCs (‡p<0.05). There was no IgM/IgG binding to GTKO/CD46/NeuGcKO pig and human RBCs (a relative MFI<1 indicates no significant binding of IgM or IgG). (E,F) There were significant differences in human IgM and IgG binding between WT and GTKO/hCD46 pPBMCs (*p<0.05) and GTKO/hCD46 and GTKO/hCD46/NeuGcKO pPBMCs (‡p<0.05).
Figure 3
Figure 3
(A) Expression of Gal, NeuGc, and hCD46 on GTKO/hCD46, and two GTKO/hCD46/NeuGcKO pigs islet cells. GTKO/hCD46 pig islets were negative for Gal, but positive for NeuGc and hCD46. GTKO/hCD46/NeuGcKO pig islets were negative for both Gal and NeuGc expression, and positive for hCD46. (B) Human IgM and IgG binding to GTKO/hCD46, and GTKO/hCD46/NeuGcKO pig islet cells. There was no statistical significance in IgM and IgG binding to islets between the two groups.
Figure 4
Figure 4
Expression of Gal and NeuGc on pig and human aortas (A) and corneas (B) by immunofluorescence, and on cultured AECs (C) and CECs (D) by flow cytometry. (A, B) Gal was detected on the endothelium of WT pig aortas (red arrows) and epithelium and stroma of WT pig corneas. NeuGc was strongly detected on aortas and corneas from WT and GTKO/hCD46 pigs (Magnification x200; nuclei, blue; Gal, green; NeuGc, red). (C, D) Gal was detected on AECs and CECs of WT pigs, whereas NeuGc was detected on AECs from WT and GTKO/hCD46 pigs. Cells from GTKO/hCD46/NeuGcKO pigs and humans were negative for both Gal and NeuGc antigens.
Figure 5
Figure 5
Human IgM and IgG antibody binding to pig and human AECs (A, B) and CECs (C, D) by flow cytometry. (A, B) Human IgM and IgG binding to GTKO/hCD46 pAECs was significantly decreased compared to WT pAECs (*p<0.05), and was further decreased to GTKO/hCD46/NeuGcKO pAECs (*p<0.05). Also, there was a significant difference in IgM and IgG binding to GTKO/hCD46 and GTKO/hCD46/NeuGcKO pAECs (‡p<0.05). There was significantly greater IgM binding to GTKO/hCD46/NeuGcKO pAECs than human AECs (‡p<0.05), but there was no statistical significance in the extent of IgG binding between them. (C, D) Human IgM and IgG binding to WT pCECs was significantly greater than to CECs from the other pigs (*p<0.05), but there was no significant difference in binding between these other pigs.
Figure 6
Figure 6
Human PBMC proliferative response to GTKO/hCD46 and GTKO/hCD46/NeuGcKO pAECs and islet cells. CFSE-labeled human PBMC were cocultured with either GTKO/hCD46 or GTKO/hCD46/NeuGcKO pAECs at 1:10 ratio for 5 days. Similarly, human PBMC were cocultured with islet cells at 1:1 ratio. The proliferative response to pAECs and islet cells was weak. The response to GTKO/hCD46/NeuGcKO AECs was slightly weaker than to GTKO/hCD46 AECs, as was the response to the respective isolated islet cells. (Representative data from experiments using PBMCs from two different humans)
Figure 7
Figure 7
The effect of genetic modification on pAEC-induced human platelet aggregation. When human platelet aggregation associated with WT pAEC (54%) was compared with that to hAEC (4%), a significant difference was observed (***p<0.001). GTKO/hCD46 (42%) and GTKO/hCD46/NeuGcKO (39%) pAEC significantly reduced the aggregation compared to WT pAEC (*p<0.05), but aggregation remained significantly greater than to hAEC (**p<0.01). There was no significant difference in platelet aggregation between GTKO/hCD46 and GTKO/hCD46/NeuGcKO.
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