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The low frequency of naturally occurring regulatory T cells (nTregs) in peripheral blood and the suboptimal protocols available for their ex vivo expansion limit the development of clinical trials based on the adoptive transfer of these cells. We have, therefore, generated a simplified, robust and cost-effective platform for the large-scale expansion of nTregs using a gas permeable static culture flask (G-Rex) in compliance with Good Manufacturing Practice. More than 10(9) putative Tregs co-expressing CD25 and CD4 molecules (92 ± 5%) and FoxP3 (69 ± 19%) were obtained within 21 days of culture. Expanded Tregs showed potent regulatory activity in vitro (80 ± 13% inhibition of CD8(+) cell division) and in vivo (suppression or delay of graft-versus-host disease in a xenograft mouse model) indicating that the cost-effective and simplified production of nTregs we propose will facilitate the implementation of clinical trials based on their adoptive transfer.

Robust and cost effective expansion of human regulatory T cells highly functional in a xenograft model of graft-versus-host disease