Donor CD4+ Foxp3+ regulatory T cells are necessary for posttransplantation cyclophosphamide-mediated protection against GVHD in mice

Blood. 2014 Sep 25;124(13):2131-41. doi: 10.1182/blood-2013-10-525873. Epub 2014 Aug 18.


Posttransplantation cyclophosphamide (PTCy) is an effective prophylaxis against graft-versus-host disease (GVHD). However, it is unknown whether PTCy works singularly by eliminating alloreactive T cells via DNA alkylation or also by restoring the conventional (Tcon)/regulatory (Treg) T-cell balance. We studied the role of Tregs in PTCy-mediated GVHD prophylaxis in murine models of allogeneic blood or marrow transplantation (alloBMT). In 2 distinct MHC-matched alloBMT models, infusing Treg-depleted allografts abrogated the GVHD-prophylactic activity of PTCy. Using allografts in which Foxp3(+) Tregs could be selectively depleted in vivo, either pre- or post-PTCy ablation of donor thymus-derived Tregs (tTregs) abolished PTCy protection against GVHD. PTCy treatment was associated with relative preservation of donor Tregs. Experiments using combinations of Foxp3(-) Tcons and Foxp3(+) Tregs sorted from different Foxp3 reporter mice indicated that donor Treg persistence after PTCy treatment was predominantly caused by survival of functional tTregs that retained Treg-specific demethylation and also induction of peripherally derived Tregs. Finally, adoptive transfer of tTregs retrieved from PTCy-treated chimeras rescued PTCy-treated, Treg-depleted recipients from lethal GVHD. Our findings indicate that PTCy-mediated protection against GVHD is not singularly dependent on depletion of donor alloreactive T cells but also requires rapidly recovering donor Tregs to initiate and maintain alloimmune regulation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bone Marrow Transplantation / adverse effects*
  • Chemoprevention*
  • Cyclophosphamide / administration & dosage
  • Cyclophosphamide / pharmacology*
  • DNA Methylation
  • Disease Models, Animal
  • Forkhead Transcription Factors / metabolism
  • Graft vs Host Disease / genetics
  • Graft vs Host Disease / immunology*
  • Graft vs Host Disease / prevention & control*
  • Lymphoid Tissue / drug effects
  • Lymphoid Tissue / immunology
  • Mice
  • T-Lymphocytes, Regulatory / immunology*
  • T-Lymphocytes, Regulatory / metabolism
  • Tissue Donors
  • Transplantation Chimera
  • Transplantation, Homologous


  • Forkhead Transcription Factors
  • Foxp3 protein, mouse
  • Cyclophosphamide