Rapid Clinical Scale Isolation of CD25^hiCD4⁺ Regulatory T Cells.

Several publications during the last few years have reported CD25^hiCD4⁺ regulatory T cells (Tregs) to prevent or to reverse disease in different mouse models of experimental autoimmune encephalomyelitis (EAE), colitis, graft rejection and graft-versus-host-disease (GvHD). As mouse and human Tregs share many phenotypical and functional characteristics, Tregs could provide a promising therapeutic approach for various human autoimmune diseases and pathological alloresponses. Here we have shown that Tregs can be isolated from leukapheresis harvests by CD25 enrichment using the CliniMACS technology (n=13). By this procedure we obtained 2.32x10⁸ (± 1.12x10⁸, range 0.71–4.42x10⁸) cells out of 10¹⁰ mononuclear cells with a mean purity of 52.12% (± 12.11%, range 25.48–66.61%) for CD25^hiCD4⁺ cells. Around 90% of enriched cells were CD25⁺CD4⁺. Among contaminating CD4⁻ cells most cells were CD25⁺ which were further characterized by counterstaining to be mainly CD19⁺ B cells and a few CD8⁺, CD56⁺ or CD123⁺ cells. It is possible to deplete the CD19⁺ or CD8⁺ cells by using CD19 Microbeads or CD8 Microbeads respectively with the CliniMACS Instrument before CD25 enrichment. Combined depletion of different cells, e.g. CD19⁺ and CD8⁺ cells is conceivable. Isolated cells were phenotypically and functionally characterized. The majority of the CD25^hiCD4⁺ T cells expressed glucocorticoid-induced tumor necrosis factor receptor (GITR), CD62L and CD45RO. In addition, isolated cells were able to suppress the proliferation and activation of cocultured conventional CD4⁺ cells after polyclonal stimulation with anti-CD3 antibody. We conclude that the large-scale isolation of CD25^hiCD4⁺ regulatory T cells for clinical applications (e.g. therapy of autoimmune diseases, graft rejection or GvHD) is possible by using the CliniMACS CD25 Reagent and the CliniMACS Instrument.