For iTreg and Tactivated cell differentiation, naive T cells were isolated and 24 well sterile tissue culture plates (Corning, Corning, NY) were coated anti-CD3ε (145–2C11, Biolegend) and anti-CD28 (37.51, Biolegend) as described 36 (link). Naive CD4+ T-cells were resuspended in complete RPMI media supplemented with 100 IU/ml recombinant human IL-2 for both iTreg and Tactivated, with additional 5 ng/ml recombinant human TGF-β for iTreg and 10 μg/ml anti-TGF-β (1D11.16.8, BioXcell, West Lebanon, NH) for Tactivated cultures. 3 × 105 cells were added to the wells at a volume of 1 ml/well. Cells were cultured at 37°C 5% CO2 for 3 days. Prior to experiments, live Tactivated and iTreg were FACS sorted based on their Foxp3-GFP expression status.
IL-2/anti–IL-2 mAb (JES6-1-A12, Bioxcell) complexes were prepared and injected as in 37 (link) to expand antigen-specific tTregs in vivo. Antigen-specific Tregs were isolated from spleens of the TCR transgenic animals by FACS sorting based on Foxp3 reporter expression. Alternatively, antigen-specific tTregs were expanded in vitro. For in vitro proliferation, Tregs were cultured for three days in the presence of plate bound anti-CD3ε (145–2C11; 2 ug/mL), anti-CD28 (37.51; 2 ug/mL) and IL-2 (100 U/mL). On day 3, cells were split 1:2 and cultured with only IL-2. Tregs were harvested on day 5 and FACS sorted for Foxp3 reporter.
IL-2/anti–IL-2 mAb (JES6-1-A12, Bioxcell) complexes were prepared and injected as in 37 (link) to expand antigen-specific tTregs in vivo. Antigen-specific Tregs were isolated from spleens of the TCR transgenic animals by FACS sorting based on Foxp3 reporter expression. Alternatively, antigen-specific tTregs were expanded in vitro. For in vitro proliferation, Tregs were cultured for three days in the presence of plate bound anti-CD3ε (145–2C11; 2 ug/mL), anti-CD28 (37.51; 2 ug/mL) and IL-2 (100 U/mL). On day 3, cells were split 1:2 and cultured with only IL-2. Tregs were harvested on day 5 and FACS sorted for Foxp3 reporter.
