title: The time is crucial for ex vivo expansion of T regulatory cells for therapy.
abstract: Ex vivo expanded CD4(+)CD25(high)CD127(-) T regulatory cells (Tregs) are recognized as a promising candidate for immunosuppressive therapy in humans. However, due to the plasticity of Tregs lineage and artificial environment present during ex vivo expansion,  Tregs easily lose suppressive activity. Here, we followed expanding CD4(+)CD25(high)CD127(-) Tregs and their naive (CD45RA(+)) and memory-like (CD45RA(-)) subsets in order to establish the best conditions of the expansion. We found that,  regardless of the phenotype sorted, expanding Tregs were undergoing changes resembling homeostatic proliferation and transformed into effector memory-like cells which produced not only suppressive interleukin-10 (IL-10) but also IL-6, IL-17, and interferon-γ (IFN-γ). With the time  ex vivo, Tregs were losing the expression of FoxP3 and suppressive activity both when stimulated and when at rest. The only variable that helped preserve suppressive abilities of Tregs was the limitation of the time of ex vivo cultures to 2 weeks only. According to our study, the highest number  of highly suppressive Tregs could be yielded with CD4(+)CD25(high)CD127(-) Tregs cultured no longer than 2 weeks. Thorough quality check, preferentially with the assessment of FoxP3 expression and IFN-γ suppression assay, should be applied to assess suppressive  activity of the cells.
authors: Department of Clinical Immunology and Transplantology, Medical University of Gdańsk, Gdańsk Poland.
authors: Natalia
authors: N
authors: Marek
authors: Maria
authors: M
authors: Bieniaszewska
authors: Adam
authors: A
authors: Krzystyniak
authors: Jolanta
authors: J
authors: Juścińska
authors: Jolanta
authors: J
authors: Myśliwska
authors: Piotr
authors: P
authors: Witkowski
authors: Andrzej
authors: A
authors: Hellmann
authors: Piotr
authors: P
authors: Trzonkowski
mesh_terms: D015704:CD4 Antigens / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D016753:Interleukin-10 / Q000378:metabolism; D020381:Interleukin-17 / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D053699:Interleukin-7 Receptor alpha Subunit / Q000378:metabolism; D010641:Phenotype; D050378:T-Lymphocytes, Regulatory / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D013997:Time Factors
chemical_list: D015704:CD4 Antigens; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D020381:Interleukin-17; D053645:Interleukin-2 Receptor alpha Subunit; D015850:Interleukin-6; D053699:Interleukin-7 Receptor alpha Subunit; D016753:Interleukin-10; D007371:Interferon-gamma