Fig. 4

In vitro suppressive activity of CD4⁺CD25⁺FoxP3⁺ (a) and CD4⁺CD25⁻FoxP3⁺ (b) T cells isolated from naive or SM16 treated FoxP3eGFP mice. CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁻FoxP3⁺ cells from donor mice were obtained by FACS sorting. The activity of isolated cells having the ability to suppress naive T cell proliferation in an in vitro assay was tested by adding increasing numbers of CD4⁺CD25⁺FoxP3⁺ or CD4⁺CD25⁻FoxP3⁺ T cells to co-cultures of sorted CD4⁺CD25⁻ (responder cells) and APCs from naive mice in the presence of anti-CD3. After 48 h the plates were pulsed for 18 h with [³H]-thymidine. Subsequently, the cells were harvested on glass fiber filters and assessed for uptake of the labeled thymidine by liquid scintillation (4 and 3 independent experiments respectively). Repeat assays of CD4⁺CD25⁺Foxp3⁺ cells were performed using a single batch of naive responder mice. Similarly, repeat assays of CD4⁺ CD25⁻Foxp3⁺ cells were performed using a separate batch of responder cells from naive mice