K. Pham1, N. Wilson-Schlei1, R. Redfield1 1University Of Wisconsin,Department Of Surgery,Madison, WI, USA
Introduction: One possible solution to the current shortage of donor organs is the use of xenografts. A potential model for viable xenotransplantation has been porcine organs but the associated immune barriers has delayed its transition to clinical practice. A key component to this barrier is the difference in carbohydrate structures expressed on porcine cells, which serve as antigens to humans. Even with the use of carbohydrate pathway knock-out pigs in combination with immunosuppression, there are still issues with long term graft survival and function in non-human primates. The goal of our experiment is to characterize the pre-existing immune response of porcine xenotransplantation in vitro.
Methods: Using six samples of naïve rhesus macaque serum, we performed a flow crossmatch assay with porcine peripheral blood mononuclear cells (PBMC). Porcine PBMC’s were incubated with three different groups: media alone (used as a negative control), porcine plasma (self), and rhesus macaque plasma (the experimental group). We analyzed IgG and IgM binding between lymphocytes and macrophages using flow cytometry. Results were reported as median fluorescence intensity (MFI) and unpaired two-tailed T test was used for statistical analysis.
Results: It was found that naturally occurring anti-pig IgG binding had significantly increased MFI in the six rhesus macaque plasma samples when incubated with porcine PBMC (P value < 0.01). Anti-pig IgG binding was also found to be most prevalent in cells outside the lymphocyte gate. In terms of IgG binding, MFI did not change for lymphocytes when compared to negative control (self and no plasma). IgG binding to non-lymphoid cells resulted in an average Δ MFI of 100 when compared to negative controls. Interestingly, there appeared to be a heterogeneous response when comparing individual amounts of anti-pig IgG and IgM. Ranges for IgG were of 737 to 1046 MFI. As for IgM, MFI ranged from 291 to 430.
Conclusion: With this experiment, we confirm previous reports that naturally occurring antibodies do bind to xenoantigens (Burlak, 2014, Ezzelarab, 2006, Rood, 2006). There appears to be an increased presence of IgG binding, particularly among cells that may be macrophages. This is consistent with previous results that implicated cells which appeared to be macrophages and xenoantibody of the IgG isotype. However, we need to analyze the data to determine whether the cells in this gate, in which most binding occurred, bind CD68 to show they are indeed macrophages. Future considerations will involve a more in-depth investigation of the role of macrophages in this flow crossmatch assay and also if PBMC’s represent a valid model for xenograft failure.