title: Clinical grade Treg: GMP isolation, improvement of purity by CD127 Depletion, Treg expansion, and Treg cryopreservation.
abstract: 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.
  
  
  RESULTS
  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
  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.
authors.affiliation: Department of Bloodtransfusion and Transplantation Immunology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Jorieke H
authors.initials: JH
authors.lastname: Peters
authors.forename: Frank W
authors.initials: FW
authors.lastname: Preijers
authors.forename: Rob
authors.initials: R
authors.lastname: Woestenenk
authors.forename: Luuk B
authors.initials: LB
authors.lastname: Hilbrands
authors.forename: Hans J P M
authors.initials: HJ
authors.lastname: Koenen
authors.forename: Irma
authors.initials: I
authors.lastname: Joosten
mesh_terms: D018941:Antigens, CD19 / Q000096:biosynthesis; D001402:B-Lymphocytes / Q000276:immunology; D016827:CD8 Antigens / Q000096:biosynthesis; D019411:Clinical Laboratory Techniques / Q000592:standards*; D018920:Coculture Techniques; D015925:Cryopreservation / Q000379:methods* / Q000592:standards; D051858:Forkhead Transcription Factors / Q000096:biosynthesis; D006801:Humans; D007108:Immune Tolerance; D007167:Immunotherapy / Q000295:instrumentation* / Q000379:methods; D053645:Interleukin-2 Receptor alpha Subunit / Q000096:biosynthesis; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism*; D007519:Isoantigens / Q000737:chemistry; D007937:Leukapheresis; D050378:T-Lymphocytes, Regulatory / Q000378:metabolism*
chemical_list: D018941:Antigens, CD19; D016827:CD8 Antigens; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit; D007519:Isoantigens
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