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Background  Treg based immunotherapy is of great interest to facilitate tolerance in autoimmunity and transplantation. For clinical trials, it is essential to have a clinical grade Treg isolation protocol in accordance with Good Manufacturing Practice (GMP) guidelines. To obtain sufficient Treg for immunotherapy, subsequent  ex vivo  expansion might be needed.    Methodology/Principal Findings  Treg were isolated from leukapheresis products by CliniMACS based GMP isolation strategies, using anti-CD25, anti-CD8 and anti-CD19 coated microbeads. CliniMACS isolation procedures led to 40–60% pure CD4 pos CD25 high FoxP3 pos  Treg populations that were anergic and had moderate suppressive activity. Such CliniMACS isolated Treg populations could be expanded with maintenance of suppressive function. Alloantigen stimulated expansion caused an enrichment of alloantigen-specific Treg. Depletion of unwanted CD19 pos  cells during CliniMACS Treg isolation proved necessary to prevent B-cell outgrowth during expansion. CD4 pos CD127 pos  conventional T cells were the major contaminating cell type in CliniMACS isolated Treg populations. Depletion of CD127 pos  cells improved the purity of CD4 pos CD25 high FoxP3 pos  Treg in CliniMACS isolated cell populations to approximately 90%. Expanded CD127 neg  CliniMACS isolated Treg populations showed very potent suppressive capacity and high FoxP3 expression. Furthermore, our data show that cryopreservation of CliniMACS isolated Treg is feasible, but that activation after thawing is necessary to restore suppressive potential.    Conclusions/Significance  The feasibility of Treg based therapy is widely accepted, provided that tailor-made clinical grade procedures for isolation and  ex vivo  cell handling are available. We here provide further support for this approach by showing that a high Treg purity can be reached, and that isolated cells can be cryopreserved and expanded successfully.

Clinical Grade Treg: GMP Isolation, Improvement of Purity by CD127pos Depletion, Treg Expansion, and Treg Cryopreservation