title: Clinical grade isolation of regulatory T cells from G-CSF mobilized peripheral blood improves with initial depletion of monocytes.
abstract: Clinical isolation of circulating CD4(+)CD25(+) regulatory T cells (Tregs) from peripheral blood mononuclear cells is usually performed by CD4(+) cell negative selection followed by CD25(+) cell positive selection. Although G-CSF mobilized peripheral blood (G-PBSC) contains a high number of Tregs, a high number of monocytes in G-PBSC limits Treg isolation. Using a small scale device (MidiMACS, Miltenyi) we initially demonstrated that an initial depletion of monocytes would be necessary to obtaina separation of CD4(+)CD25(+)FoxP3(+)CD127(-) cells from G-PBSC (G-Tregs) with a consistent purity >70% and inhibitory activity of T cell alloreactivity in-vitro. We then validated the same approach in a clinical scale setting by separating G-Tregs with clinically available antibodies to perform a CD8(+)CD19(+)CD14(+) cell depletion followed by CD25(+) cell selection (2-step process) or by adding an initial CD14(+) cell depletion (3-step process) using a CliniMACS column. The 3-step approach resulted in a better purity (81±12% vs. 35±33%) and yield (66% vs. 39%). Clinically isolated G-Tregs were also FoxP3(+)CD127(dim) and functionally suppressive in-vitro. Our findings suggest that a better and more consistent purity of Tregs can be achieved from G-PBSC by an initial single depletion of monocytes prior to selection of CD4(+)CD25(+) cells.
authors.affiliation: Division of Hematology/Oncology, University of Illinois at ChicagoChicago, IL, USA; Cancer Center, University of IllinoisChicago, IL, USA.
authors.forename: Pritesh
authors.initials: P
authors.lastname: Patel
authors.affiliation: Division of Hematology/Oncology, University of Illinois at Chicago Chicago, IL, USA.
authors.forename: Dolores
authors.initials: D
authors.lastname: Mahmud
authors.affiliation: Hospital Stem Cell Laboratory, University of Illinois Hospital and Health Sciences System Chicago, IL, USA.
authors.forename: Youngmin
authors.initials: Y
authors.lastname: Park
authors.affiliation: Hospital Stem Cell Laboratory, University of Illinois Hospital and Health Sciences System Chicago, IL, USA.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Yoshinaga
authors.affiliation: Division of Hematology/Oncology, University of Illinois at ChicagoChicago, IL, USA; Cancer Center, University of IllinoisChicago, IL, USA; Hospital Stem Cell Laboratory, University of Illinois Hospital and Health Sciences SystemChicago, IL, USA.
authors.forename: Nadim
authors.initials: N
authors.lastname: Mahmud
authors.affiliation: Division of Hematology/Oncology, University of Illinois at ChicagoChicago, IL, USA; Cancer Center, University of IllinoisChicago, IL, USA.
authors.forename: Damiano
authors.initials: D
authors.lastname: Rondelli
keywords: CliniMACS; G-CSF; Regulatory T cells; stem cell transplantation