Department of Clinical Research
University of Bern
A new xenoantigen: Human natural IgM antibodies bind to non-muscular myosin heavy chain type II on porcine endothelial cells
Pavan S Garimella1, Anjan K Bongoni1,2, Jonas Schnider1,3, David Kiermeir1,3, Hansjoerg Jenni4, Baehr Andrea5, Nikolai Klymiuk5, Eckhard Wolf5, David Ayares6, Esther Voegelin1,3, Mihai A Constantinescu1,3, Robert Rieben1.
1Department of Clinical Research, University of Bern, Bern, Switzerland; 2Immunology Research Centre, St. Vincent’s Hospital, Melbourne, Australia; 3Clinic of Plastic and Hand Surgery, University Hospital, Bern, Switzerland; 4Clinic of Cardiovascular Surgery, University Hospital, Bern, Switzerland; 5Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilian University, Munich, Germany; 6Revivicor, Inc., Blacksburg, VA, United States
Background Non-muscular myosin heavy chain type II (NMHC II) has been reported to play a role in ischemia/reperfusion injury. In animal models NMHC II was shown to be expressed as a neoantigen in ischemic tissue. Upon reperfusion, naturally occurring IgM antibodies bind to this target, activate the complement system and initiate reperfusion injury. We hypothesized that NMHC II might be expressed also on porcine endothelium during xenoperfusion with human blood and contribute to antibody-mediated xenorejection.
Methods Alpha1,3-galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human blood for 12 hours. Snap frozen tissue samples from baseline and after 12 hours of xenoperfusion were analyzed by immunofluorescence for expression of NMHC II and deposition of human IgM using specific antibodies. Cultured porcine aortic endothelial cells (PAEC) of wildtype and transgenic origin (alpha-GalKO homozygous, hCD46/hTM double transgenic) were incubated with 1:10 diluted normal human serum for 1 hour and examined for co-localization of NMHC II with IgM as well as IgG.
Results Expression of the NMHC II antigen was seen in the pig limb xenoperfusion experiments at baseline and, at significantly higher levels, after 12 hours of xenoperfusion with human blood. Inhibition experiments with the NMHC II peptide proved that the staining was specific. Co-localization of NMHC II was observed with human IgM and, particularly in wild-type pig limbs, also IgG. A co-localization of human IgM binding and NMHC II expression was also seen in the in vitro experiments with alpha-GalKO homozygous, hCD46/hTM double transgenic PAEC.
Conclusions Our data show that expression of NMHC II occurs on the endothelium of xenoperfused porcine tissue as well as in vitro on PAEC incubated with human serum. The reduced expression of NMHC II on hCD46 transgenic endothelium supports the idea that complement-mediated activation of the cells triggers appearance of this neoantigen. The observed co-localization with human antibodies suggests that expression of NMHC II might contribute to antibody-mediated xenorejection.
15:30 - 16:30
|Non-Gal Antibodies||A new xenoantigen: Human natural IgM antibodies bind to non-muscular myosin heavy chain type II on porcine endothelial cells||Room 109|