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. 2018 Feb 15;9:233. doi: 10.3389/fimmu.2018.00233

Figure 2.

Figure 2

Protocols to generate/expand T regulatory type 1 (Tr1) cells. (A) Tr1-enriched cell lines. Donor-derived PBMC or CD4+ T cells are stimulated with host-derived monocytes in the presence of recombinant human IL-10 for 10 days. Alternatively, PBMC or CD4+ T cells are cultured for 10 days with allogenic dendritic cell (DC)-10, differentiated in vitro from peripheral blood monocytes with GM-CSF/IL-4/IL-10, in the presence of recombinant human IL-10 for 10 days (45). To generate T-allo10 cells, donor-derived T cells are cultured with host-derived DC-10 (Bacchetta and Roncarolo, unpublished data), whereas to induce host-derived T10 cells, T cells are stimulated with donor-derived DC-10 (46). (B) Tr1 cell clones. PBMC are stimulated with antigen (Ag; i.e., ovalbumin or collagen II) in the presence of IL-2 and IL-4 to enrich/expand Ag-specific T cells, followed by T cell cloning and expansion of the T cell clones using Schneider cells (4). (C) Tr1-like cell lines. Human CD4+ T cells are preactivated for 48 h with soluble anti-CD3/CD28 mAbs and IL-2 and then transduced with lentiviral vector (LV)-IL-10 overnight. Transduced T cells are isolated and expanded in feeder mixture (47). To generate allospecific IL-10-transduced cells, naive CD4+ T cells are stimulated with allogeneic DC and transduced with LV-IL-10 upon secondary stimulation. After selection, IL-10-trasduced cells are expanded in vitro with feeder mixture (48).