title: Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.
abstract: Preexisting, donor-specific antibodies (DSAs) are culprits of hyperacute rejection. Donor-specific antibodies are also formed de novo, and their role in acute and chronic rejection is increasingly appreciated. However, it is difficult to assess damage inflicted exclusively by DSAs when alloreactive T cell and B cell responses coincide. We reasoned that allosensitization with "costimulation-deficient" cells should induce DSA synthesis but not naive cytotoxic T lymphocyte (CTL) precursors' priming via direct allorecognition. Accordingly, we have developed a novel model to quantify DSA-mediated cytotoxicity in vivo.
  
  
  
  C57BL/6 (H-2b) mice were sensitized with H-2 kidney epithelial cells, and a cytofluorimetric killing assay was tailored to the measurement of allocytotoxicity. We took cell/complement depletion, costimulation blockade, and serum transfer approaches to reveal the mediators of cytotoxicity. "Third-party" controls and a skin allotransplantation model were used to confirm DSAs' specificity for allo-major histocompatibility complex. We validated our experimental approach in other mouse strains primed with different allogeneic cell types, including endothelial cells. To demonstrate the usefulness of our model/method for drug efficacy testing, we examined the effect of CTLA4-Ig and rapamycin on DSA-mediated cytolysis.
  
  
  
  Allosensitization of MHC-disparate mouse strains with costimulation-deficient cells led to robust cytotoxicity mediated by complement-fixing DSAs and phagocytic cells. This response was independent of CTLs, natural killer or natural killer T cells. It required CD4 T cell help, CD40 signaling and CD28-based costimulation during allosensitization and could be reversed by sustained rapamycin treatment.
  
  
  
  The unique model described herein should enable mechanistic studies on sensitization and effector phases of humoral alloreactivity as well as efficacy testing of future immunotherapies to prevent DSA-induced pathology.
authors.affiliation: 1 Department of Microbiology and Immunology, Western University, London, Ontario, Canada. 2 Animal Care and Veterinary Services, Western University, London, Ontario, Canada. 3 Division of Clinical Immunology & Allergy, Department of Medicine, Western University, London, Ontario, Canada. 4 Centre for Human Immunology, Western University, London, Ontario, Canada. 5 Lawson Health Research Institute, London, Ontario, Canada.
authors.forename: Arash
authors.initials: A
authors.lastname: Memarnejadian
authors.forename: Courtney E
authors.initials: CE
authors.lastname: Meilleur
authors.forename: Delfina M
authors.initials: DM
authors.lastname: Mazzuca
authors.forename: Ian D
authors.initials: ID
authors.lastname: Welch
authors.forename: S M Mansour
authors.initials: SM
authors.lastname: Haeryfar
mesh_terms: D000069594:Abatacept / Q000737:chemistry; D064591:Allografts; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology; D018106:CD28 Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D019013:CD40 Antigens / Q000378:metabolism; D003165:Complement System Proteins; D004912:Erythrocytes / Q000166:cytology; D005434:Flow Cytometry; D006084:Graft Rejection / Q000276:immunology*; D007518:Isoantibodies / Q000737:chemistry*; D007694:Killer Cells, Natural / Q000166:cytology; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D010587:Phagocytosis; D020123:Sirolimus / Q000737:chemistry; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology*
chemical_list: D018106:CD28 Antigens; D019013:CD40 Antigens; D007518:Isoantibodies; D000069594:Abatacept; D003165:Complement System Proteins; D020123:Sirolimus