title: Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.
abstract: BACKGROUND
  Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model.
  
  
  METHODS
  α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples.
  
  
  RESULTS
  No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions.
  
  
  CONCLUSIONS
  Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.
authors.affiliation: Department of Clinical Research, University of Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; Clinic of Plastic and Hand Surgery, University Hospital, Bern, Switzerland.
authors.forename: Anjan K
authors.initials: AK
authors.lastname: Bongoni
authors.forename: David
authors.initials: D
authors.lastname: Kiermeir
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Jenni
authors.forename: Andrea
authors.initials: A
authors.lastname: Bähr
authors.forename: David
authors.initials: D
authors.lastname: Ayares
authors.forename: Nikolai
authors.initials: N
authors.lastname: Klymiuk
authors.forename: Eckhard
authors.initials: E
authors.lastname: Wolf
authors.forename: Esther
authors.initials: E
authors.lastname: Voegelin
authors.forename: Mihai A
authors.initials: MA
authors.lastname: Constantinescu
authors.forename: Jörg D
authors.initials: JD
authors.lastname: Seebach
authors.forename: Robert
authors.initials: R
authors.lastname: Rieben
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis; D015415:Biomarkers; D001803:Blood Transfusion / Q000379:methods*; D003165:Complement System Proteins / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D055786:Gene Knockout Techniques; D005819:Genetic Markers; D006084:Graft Rejection / Q000276:immunology / Q000473:pathology / Q000517:prevention & control*; D015395:Histocompatibility Antigens Class I / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020287:In Situ Nick-End Labeling; D066298:In Vitro Techniques; D051916:Membrane Cofactor Protein / Q000235:genetics* / Q000378:metabolism; D013552:Swine / Q000235:genetics*; D014183:Transplantation, Heterologous; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D000097625:HLA-E Antigens
chemical_list: D015415:Biomarkers; D003165:Complement System Proteins; D016207:Cytokines; D005819:Genetic Markers; D015395:Histocompatibility Antigens Class I; D051916:Membrane Cofactor Protein; D019010:Vascular Cell Adhesion Molecule-1; D000097625:HLA-E Antigens; C497857:CD46 protein, human
keywords: HLA-E; apoptosis; complement; endothelial cells; hCD46; pig-to-human xenotransplantation; transgenic pig