title: Stability of human rapamycin-expanded CD4+CD25+ T regulatory cells.
abstract: BACKGROUND
  The clinical use of ex vivo-expanded T-regulatory cells for the treatment of T-cell-mediated diseases has gained increasing momentum. However, the recent demonstration that FOXP3(+) T-regulatory cells may contain interleukin-17-producing cells and that they can convert into effector cells once transferred in vivo raises significant doubts about their safety. We previously showed that rapamycin permits the ex vivo expansion of FOXP3(+) T-regulatory cells while impairing the proliferation of non-T-regulatory cells. Here we investigated the Th17-cell content and the in vivo stability of rapamycin-expanded T-regulatory cells as pertinent aspects of cell-based therapy.
  
  
  METHODS
  T-regulatory-enriched cells were isolated from healthy volunteers and were expanded ex vivo with rapamycin with a pre-clinical applicable protocol. T-regulatory cells cultured with and without rapamycin were compared for their regulatory activity, content of pro-inflammatory cells and stability.
  
  
  RESULTS
  We found that CD4(+)CCR6(+)CD161(+) T cells (i.e., precursor/committed Th17 cells) contaminate the T-regulatory cells cultured ex vivo in the absence of rapamycin. In addition, Th17 cells do not expand when rapamycin-treated T-regulatory cells are exposed to a "Th17-favorable" environment. Rapamycin-expanded T-regulatory cells maintain their in vitro regulatory phenotype even after in vivo transfer into immunodeficient NOD-SCID mice despite being exposed to the irradiation-induced pro-inflammatory environment. Importantly, no additional rapamycin treatment, either in vitro or in vivo, is required to keep their phenotype fixed.
  
  
  CONCLUSIONS
  These data demonstrate that rapamycin secures ex vivo-expanded human T-regulatory cells and provide additional justification for their clinical use in future cell therapy-based trials.
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Milan, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Tresoldi
authors.forename: Ilaria
authors.initials: I
authors.lastname: Dell'Albani
authors.forename: Angela
authors.initials: A
authors.lastname: Stabilini
authors.forename: Tatiana
authors.initials: T
authors.lastname: Jofra
authors.forename: Andrea
authors.initials: A
authors.lastname: Valle
authors.forename: Nicola
authors.initials: N
authors.lastname: Gagliani
authors.forename: Attilio
authors.initials: A
authors.lastname: Bondanza
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Roncarolo
authors.forename: Manuela
authors.initials: M
authors.lastname: Battaglia
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D005260:Female; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D020123:Sirolimus / Q000494:pharmacology*; D050378:T-Lymphocytes, Regulatory / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D058504:Th17 Cells / Q000378:metabolism
chemical_list: D015704:CD4 Antigens; D016207:Cytokines; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit; D020123:Sirolimus