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: Although CD4 /CD25 T regulatory cells (Tregs) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated Tregs from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4 /CD25 ) showed a mean purity of 93[middle dot]6% /- 1[middle dot]1. Recovery efficiency was 81[middle dot]52% /- 7[middle dot]4. The CD4 /CD25 bright cells were 28[middle dot]4% /- 6[middle dot]8. The CD4 /CD25 fraction contained a mean of 51[middle dot]9% /- 15[middle dot]1 FoxP3 cells and a mean of 18[middle dot]9% /- 11[middle dot]5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4 CD25 FoxP3 cells were in line with flow cytometric results. In V[beta] spectratyping the complexity scores of CD4 /CD25 cells and CD4 /CD25- cells were not significantly different, indicating that Tregs had a broad T cell receptor repertoire. The inhibition assay showed that CD4 /CD25 cells inhibited CD4 /CD25- cells in a dose-dependent manner (mean inhibition percentages: 72[middle dot]4 /- 8[middle dot]9 [ratio of T responder (Tresp) to Tregs, 1:2]; 60[middle dot]8% /- 20[middle dot]5 (ratio of Tresp to Tregs, 1:1); 25[middle dot]6 /- 19[middle dot]6 (ratio of Tresp to Tregs, 1:0[middle dot]1)). Our study shows that negative/positive Treg selection, performed using the CliniMACS device and reagents, enriches significantly CD4 CD25 FoxP3 cells endowed with immunosuppressive capacities. The CD4 CD25 FoxP3 population is a source of natural Treg cells that are depleted of CD8 and CD4 /CD25- reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.

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