title: Production and characterization of transgenic pigs expressing porcine CTLA4-Ig.
abstract: BACKGROUND
  Inhibition of the T-cell-mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig alpha1,3-galactosyltransferase gene-knockout (GTKO) organs. Cytotoxic T lymphocyte-associated antigen (CTLA4) is a co-stimulatory molecule that inhibits T-cell activity and may be useful in prolonging graft rejection.
  
  
  METHODS
  An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4-Ig). Pigs transgenic for pCTLA4-Ig, on either a GTKO or wild-type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy.
  
  
  RESULTS
  Fifteen pCTLA4-Ig-transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4-Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics.
  
  
  CONCLUSIONS
  Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.
authors.affiliation: Revivicor, Inc, Blacksburg, VA 24060, USA.
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Phelps
authors.forename: Suyapa F
authors.initials: SF
authors.lastname: Ball
authors.forename: Todd D
authors.initials: TD
authors.lastname: Vaught
authors.forename: Amy M
authors.initials: AM
authors.lastname: Vance
authors.forename: Michael
authors.initials: M
authors.lastname: Mendicino
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Monahan
authors.forename: Anneke H
authors.initials: AH
authors.lastname: Walters
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Wells
authors.forename: Amy S
authors.initials: AS
authors.lastname: Dandro
authors.forename: Jagdeece J
authors.initials: JJ
authors.lastname: Ramsoondar
authors.forename: David K C
authors.initials: DK
authors.lastname: Cooper
authors.forename: David L
authors.initials: DL
authors.lastname: Ayares
mesh_terms: D000069594:Abatacept; D000818:Animals; D030801:Animals, Genetically Modified; D005260:Female; D006801:Humans; D056724:Immunity, Humoral / Q000276:immunology; D018796:Immunoconjugates / Q000235:genetics* / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000276:immunology*; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D013552:Swine / Q000235:genetics*; D014018:Tissue Distribution; D019076:Transgenes; D014183:Transplantation, Heterologous / Q000276:immunology
chemical_list: D018796:Immunoconjugates; D007074:Immunoglobulin G; D007166:Immunosuppressive Agents; D011993:Recombinant Fusion Proteins; D000069594:Abatacept