title: Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers.
abstract: BACKGROUND
  Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting may be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a major MHC mismatch without the need for long-term immunosuppression.
  
  
  METHODS
  Mixed allogeneic chimeras were prepared by using rat strains with strong MHC incompatibility [WF (RT1Au), ACI (RT1Aa)] WF + ACI-->WF, n=23. The bone marrow (BM) of recipient animals was pretreated with low-dose irradiation (500-700 cGy), followed by reconstitution with a mixture of T cell-depleted syngeneic (WF) and allogeneic (ACI) cells. Additionally, the recipient animals received a single dose of anti-lymphocyte serum (10 mg) 5 days before bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day before BMT to 10 days post-BMT. Hindlimb transplants were performed 12 months after BMT. Five animals received a limb allograft irradiated (1000 cGy) just before transplantation. Rat chimeras were characterized (percentage of donor cells present within the bloodstream) by flow cytometry at 3 and 12 months after BM reconstitution and after hindlimb transplantation.
  
  
  RESULTS
  Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 18/23 animals. Multi-lineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60% (n=18) no sign of limb rejection was present for the duration of the study. All animals with chimerism <20% (n=5) developed moderate signs of rejection clinically and histologically. Gross motor and sensory reinnervation (weight bearing, toe spread) developed at >60 days in 14/21 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n=10). Five out of five animals with irradiated limb transplants showed no sign of GVHD at >100 days.
  
  
  CONCLUSIONS
  Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. Partial functional return should be expected. The BM transplanted as part of the hindlimb allograft plays a role in the etiology of GVHD. Manipulating that BM before transplantation may influence the incidence of GVHD. This represents the first reliable rat hindlimb model demonstrating rejection-free CTA survival in an adult animal across a major MHC mismatch without the long-term need for immunosuppressive agents.
authors.affiliation: University of California, San Francisco, Division of Plastic and Reconstructive Surgery, 533 Parnassus Avenue, U-147, San Francisco, CA 94143-0718, USA. fosterr@surgery.ucsf.edu
authors.forename: R D
authors.initials: RD
authors.lastname: Foster
authors.forename: N L
authors.initials: NL
authors.lastname: Ascher
authors.forename: T H
authors.initials: TH
authors.lastname: McCalmont
authors.forename: M
authors.initials: M
authors.lastname: Neipp
authors.forename: J P
authors.initials: JP
authors.lastname: Anthony
authors.forename: S J
authors.initials: SJ
authors.lastname: Mathes
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation / Q000276:immunology; D002678:Chimera / Q000276:immunology*; D006086:Graft vs Host Disease / Q000209:etiology / Q000276:immunology; D006614:Hindlimb / Q000637:transplantation; D006801:Humans; D007108:Immune Tolerance; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D008285:Major Histocompatibility Complex; D015778:Minor Histocompatibility Antigens; D008954:Models, Biological; D051381:Rats; D011912:Rats, Inbred ACI; D011920:Rats, Inbred WF; D016038:Skin Transplantation / Q000276:immunology; D014184:Transplantation, Homologous
chemical_list: D015778:Minor Histocompatibility Antigens