title: CD45RA Distinguishes CD4+CD25+CD127-/low TSDR Demethylated Regulatory T Cell Subpopulations With Differential Stability and Susceptibility to Tacrolimus-Mediated Inhibition of Suppression.
abstract: BACKGROUND
  Adoptive transfer of forkhead box protein (FOX)3 regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipient's immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD127 expression enhances identification of Treg in the human CD4CD25 population. CD45RA expression identifies naive CD4CD25 Treg with an enhanced stability of Treg phenotype.
  
  
  METHODS
  We combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4CD127CD25 cells. Regulatory T cells were sorted according to expression of CD25 and CD45RA and expanded in the presence of a physiological relevant concentration of TAC. Regulatory T cell-specific demethylation region (TSDR) demethylation, FOXP3 expression, and suppression were analyzed.
  
  
  RESULTS
  CD4CD127CD25CD45RA Treg cells had a stable TSDR demethylated FOXP3 phenotype after expansion whereas CD4CD127CD25CD45RA Treg cell lost the TSDR demethylated phenotype. CD45RA Treg had a greater capacity to suppress after expansion with TAC.
  
  
  CONCLUSIONS
  Although CD45RA Treg retained a greater suppressive capacity when expanded with TAC, the marked loss of the TSDR demethylated status highlights the potential for loss of stability of these cells in transplant recipients treated with TAC based immunosuppression. We show that a population of CD4CD127CD45RA Regulatory T cell may offer the best compromise between susceptibility to loss of suppression when exposed to TAC and maintenance of a TSDR demethylated phenotype following in vitro expansion.
authors.affiliation: 1 Nuffield Department of Surgical Sciences, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom. 2 Charité-Universitätsmedizin Berlin, Campus Virchow, Institut f. Medizinische Immunologie, Berlin, Germany. 3 Oxford Transplant Centre, Oxford University Hospitals NHS Trust, Oxford, United Kingdom.
authors.forename: Rebeca
authors.initials: R
authors.lastname: Arroyo Hornero
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Betts
authors.forename: Birgit
authors.initials: B
authors.lastname: Sawitzki
authors.forename: Katrin
authors.initials: K
authors.lastname: Vogt
authors.forename: Paul N
authors.initials: PN
authors.lastname: Harden
authors.forename: Kathryn J
authors.initials: KJ
authors.lastname: Wood
mesh_terms: D065095:Calcineurin Inhibitors / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D019175:DNA Methylation; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000276:immunology / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping / Q000379:methods; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology* / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000276:immunology* / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D010641:Phenotype; D050378:T-Lymphocytes, Regulatory / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology*
chemical_list: D065095:Calcineurin Inhibitors; D016207:Cytokines; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C508017:IL2RA protein, human; D007166:Immunosuppressive Agents; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit; D017493:Leukocyte Common Antigens; D016559:Tacrolimus