title: Selective depletion of FOXP3(high) cells by Fas-Fas-L-induced apoptosis occurs in CD4(+)CD25(+)-enriched populations during repeated expansion.
abstract: OBJECTIVE
  Expansion of anti-CD25 bead-isolated human Tregs culture has paradoxically resulted in reduced suppressive activity, but the mechanism(s) responsible for these observations are poorly defined.
  
  
  METHODS
  Magnetic-bead isolated human CD25(+) cells were expanded with anti-CD3/CD28 beads and high doses of rhIL-2. Detection of Fas and Fas ligand (Fas-L) expression, activation of Caspase 8, cell proliferation and cytokine production was evaluated by multi-color fluorescence-activated cell sorting analysis. The role of Fas-Fas-L-mediated cell death was dissected through the use of agonist or antagonist monoclonal antibodies directed at Fas and Fas-L.
  
  
  RESULTS
  Repeated expansion of bead-enriched CD4(+)CD25(+) cells generated a cellular product with markedly reduced suppressive activity and with significantly increased CD8(+) T cells and CD4(+) T cells producing interferon-γ and/or interleukin-2. We showed that Fas-Fas-L-mediated apoptosis of CD4(+)FOXP3(high) cells and rapid cell-cycling of CD8(+) T cells were collectively responsible for the reduced proportion of CD4(+)FOXP3(high) cells in expanded cultures. The depletion of CD4(+)FOXP3(high) cells and activation of Caspase 8 in CD4(+)FOXP3(high) cells was attenuated by Fas antagonist antibody, ZB4, in short-term culture. However, the loss of CD4(+)FOXP3(high) cells during expansion was not prevented by either Fas or Fas-L antagonist antibodies.
  
  
  CONCLUSIONS
  Taken together, the data show that Fas-Fas-L-mediated apoptosis may limit the expansion of anti-CD25 bead-isolated cells in vitro.
authors.affiliation: Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, John Radcliffe Hospital, Oxford, United Kingdom. wei.zhang@ndcls.ox.ac.uk
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Sindu
authors.initials: S
authors.lastname: Nair
authors.forename: Robert
authors.initials: R
authors.lastname: Danby
authors.forename: Andy
authors.initials: A
authors.lastname: Peniket
authors.forename: David J
authors.initials: DJ
authors.lastname: Roberts
mesh_terms: D017209:Apoptosis; D015704:CD4 Antigens / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D053222:Fas Ligand Protein / Q000378:metabolism*; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D018189:Immunomagnetic Separation; D007167:Immunotherapy / Q000379:methods*; D007376:Interleukin-2 / Q000276:immunology; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D008213:Lymphocyte Activation; D008212:Lymphocyte Depletion; D050378:T-Lymphocytes, Regulatory / Q000276:immunology*; D019014:fas Receptor / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; D016207:Cytokines; C418974:FOXP3 protein, human; D053222:Fas Ligand Protein; D051858:Forkhead Transcription Factors; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D019014:fas Receptor; D053181:Caspase 8
keywords: FOXP3; Fas; Treg; apoptosis; cell therapy; expansion