title: Donor antigen-primed regulatory T cells permit liver regeneration and phenotype correction in hemophilia A mouse by allogeneic bone marrow stem cells.
abstract: BACKGROUND
  Cell replacement therapy may be considered as an alternate approach to provide therapeutic dose of plasma factor VIII (FVIII) in patients with hemophilia A (HA). However, immune rejection limits the use of allogeneic cells in this mode of therapy. Here, we have examined the role of donor major histocompatibility complex (MHC)-stimulated host CD4(+)CD25(+) regulatory T (Treg) cells in suppressing immune responses against allogeneic uncommitted (Lin(-)) bone marrow cells (BMCs) for correction of bleeding disorder in HA mice.
  
  
  METHODS
  Allogeneic donor Lin(-) BMCs were co-transplanted with allo-antigen sensitized Treg cells in HA mice having acetaminophen-induced acute liver injury. Plasma FVIII activity was determined by in vitro functional assay, and correction of bleeding phenotype was assessed on the basis of capillary blood clotting time and tail-clip challenge. The immunosuppression potential of the sensitized Treg cells on CD4(+) T cells was studied both in vitro and in vivo. Suppression of inflammatory reactions in the liver against the homed donor cells by sensitized Treg cells was analysed by histopathological scoring. Allo-specificity of sensitized Treg cells and long-term retention of immunosuppression were examined against a third-party donor and by secondary challenge of allogeneic donor cells, respectively. The engraftment and phenotype change of donor BMCs in the liver and their role in synthesis of FVIII and liver regeneration were also determined.
  
  
  RESULTS
  Co-transplantation of allogeneic Lin(-) BMCs with sensitized Treg cells led to systemic immune modulation and suppression of inflammatory reactions in the liver, allowing better engraftment of allogeneic cells in the liver. Allo-antigen priming led to allo-specific immune suppression even after 1 year of transplantation. Donor-derived endothelial cells expressed FVIII in HA mice, leading to the correction of bleeding phenotype. Donor-derived hepatocyte-like cells, which constitute the major fraction of engrafted cells, supported regeneration of the liver after acute injury.
  
  
  CONCLUSIONS
  A highly proficient FVIII secreting core system can be created in regenerating liver by transplanting allogeneic Lin(-) BMCs in HA mice where transplantation tolerance against donor antigens can be induced by in vitro allo-antigen primed Treg cells. This strategy can be beneficial in treatment of genetic liver disorders for achieving prophylactic levels of the missing proteins.
authors.affiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India. veenak@nii.ac.in.
authors.forename: Veena
authors.initials: V
authors.lastname: Kochat
authors.affiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India. sumod.k@gmail.com.
authors.forename: Sumod
authors.initials: S
authors.lastname: Kanjirakkuzhiyil
authors.affiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India. pnbaligar@gmail.com.
authors.forename: Prakash
authors.initials: P
authors.lastname: Baligar
authors.affiliation: Experimental Animal Facility, National Institute of Immunology, New Delhi, India. nagarajan@nii.ac.in.
authors.forename: Perumal
authors.initials: P
authors.lastname: Nagarajan
authors.affiliation: Stem Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, 110067, India. ashok@nii.ac.in.
authors.forename: Asok
authors.initials: A
authors.lastname: Mukhopadhyay
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D016026:Bone Marrow Transplantation; D056486:Chemical and Drug Induced Liver Injury / Q000473:pathology / Q000628:therapy; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000166:cytology / Q000637:transplantation; D005169:Factor VIII / Q000032:analysis; D005260:Female; D006467:Hemophilia A / Q000628:therapy*; D007108:Immune Tolerance; D007150:Immunohistochemistry; D016753:Interleukin-10 / Q000032:analysis; D007728:Kupffer Cells / Q000166:cytology / Q000637:transplantation; D008099:Liver / Q000378:metabolism / Q000473:pathology; D008115:Liver Regeneration / Q000502:physiology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010641:Phenotype; D013154:Spleen / Q000166:cytology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D014019:Tissue Donors; D014184:Transplantation, Homologous
chemical_list: D016753:Interleukin-10; D005169:Factor VIII