![]() To investigate antileukemic activity in vivo, recipient mice were inoculated with 1x10(5) A20 cells (a B-lymphoblastic leukemia of Balb/c origin) 2 days before PBPC transplantation (PBPCT). In some experimental groups, T cell-depleted PBPCs were incubated with 200 U/mL interleukin (IL)-2 and 100 U/mL IL-12 for 24 hours. Selective T cell depletion (TCD) was performed by immunomagnetic purging with a mononoclonal antibody directed against CD3. After pretreatment of Balb/c (H-2d) recipients with 7.5 Gy of total body irradiation, 2x10(7) rhG-CSF-mobilized PBPCs of splenectomized syngeneic or MHC-identical DBA (H-2d) mice were transferred. We determined the influence of selective T cell depletion of allogeneic PBPC grafts on graft-vs.-leukemia (GVL) activity and investigated the effectiveness of ex vivo treatment with NK cell-activating cytokines to compensate for the putative loss of T cell-derived factors stimulating natural cytotoxicity. Using a murine transplantation model, we simulated a clinical situation in which major histocompatibility complex (MHC)-identical allogeneic peripheral blood progenitor cells (PBPCs) are transplanted for the treatment of a malignant disease that is resistant to resting natural killer (NK) cells but sensitive to cytokine-activated NK cells and T cell-mediated antitumor activity. Uharek, L Glass, B Zeis, M Dreger, P Steinmann, J Löffler, H Schmitz, N PMID:27069755Ībrogation of graft-vs.-leukemia activity after depletion of CD3+ T cells in a murine model of MHC-matched peripheral blood progenitor cell transplantation (PBPCT). 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. Clinically isolated G-Tregs were also FoxP3+CD127dim and functionally suppressive in-vitro. The 3-step approach resulted in a better purity (81Â☑2% vs. 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. 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. 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. Patel, Pritesh Mahmud, Dolores Park, Youngmin Yoshinaga, Kazumi Mahmud, Nadim Rondelli, DamianoĬlinical 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. Clinical grade isolation of regulatory T cells from G-CSF mobilized peripheral blood improves with initial depletion of monocytes ![]()
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