title: Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets.
abstract: OBJECTIVE
  The ectonucleotidase CD39 is an enzyme involved in adenosine production. Its surface expression on human regulatory T cells (Treg) allows for their flow-cytometry-based isolation from peripheral blood. To further develop and improve this method on a scale supporting translational studies, we introduced capture of CD39(+) Treg on magnetic immunobeads.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used for negative selection of CD4(+) T cells on AutoMACS using antibodies (Abs) specific for all lineage(+) cells. CD4(+)CD39(+) Treg were captured by biotin-conjugated anti-CD39 Abs and anti-biotin Ab-coated magnetic beads. Isolated CD4(+)CD39(+) T cells were phenotyped by flow cytometry for Treg-associated markers: CD39, CD73, FOXP3, CD25, CTLA-4, CCR4, CD45RO and CD121a or for the absence of CD127 and CD49d. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions.
  
  
  RESULTS
  The purity, recovery and viability of the separated CD4(+)CD39(+) T cells were satisfactory. The isolated CD4(+)CD39(+) T cell population consisted of FOXP3(+)CD25(+) T cells which hydrolyzed exogenous ATP and suppressed autologous CD4(+) T cell proliferation and of FOXP3(neg)CD25(neg) T cells without suppressor function. The same two subsets were detectable by flow cytometry in normal PBMC, gating on CD4(+)CD39(+), CD4(+)CD127(neg), CD4(+)CD49d(neg) or CD4(+)CD25(high) Treg.
  
  
  CONCLUSIONS
  CD4(+)CD39(+) Treg capture on immunobeads led to a discovery of two CD39(+) subsets. Similar to CD39(+) Treg in the peripheral blood, half of these cells are CD25(+)FOXP3(+) active suppressor cells, while the other half are CD25(neg)FOXP3(neg) and do not mediate suppression.
authors.affiliation: University of Pittsburgh Cancer Institute and University of Pittsburgh School of Medicine, Department of Pathology, 5117 Centre Ave, 15213 Pittsburgh, PA, USA.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Schuler
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Harasymczuk
authors.forename: Bastian
authors.initials: B
authors.lastname: Schilling
authors.forename: Stephan
authors.initials: S
authors.lastname: Lang
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Whiteside
mesh_terms: D015703:Antigens, CD / Q000276:immunology; D001081:Apyrase / Q000276:immunology; D015415:Biomarkers / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry / Q000166:cytology* / Q000276:immunology*; D002470:Cell Survival; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D008280:Magnetics; D010641:Phenotype; D013499:Surface Properties
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; D001081:Apyrase; C067869:CD39 antigen