Functional analysis of induced FoxP3-expressing CD4 cells in vitro.

<p>(A) CD4⁺CD25⁻ cells isolated from a patient with B-cell NHL were co-cultured with purified autologous B cells for 5 days. Then, CD4⁺CD25⁺ T cells were isolated and cocultured with CFSE-labeled CD4⁺CD25⁻ T cells for 4 days in the presence of γ-irradiated (30 Gy) PBMCs, with each population 2×10⁵ cells. Histograms showed the profile of CFSE-labeled CD4⁺CD25⁻ T cells and the proliferated CD4⁺CD25⁻ T cells was measured by the percentage of CFSE^dim cells, as indicated. The converted Treg could inhibit the proliferation of allogeneic CD4⁺CD25⁻ T cells (<i>P</i><0.01). Two BM samples (patients 8 and 11) and one LN sample (patient 24) were tested, and a representative experiment of three was presented. (B) CD4⁺CD25⁻ cells isolated from a HV or patient with B-cell NHL were co-cultured with purified respective autologous B cells for 5 days. Then, the cells were stimulated with PMA plus ionomycin for 5 hours in the presence of BFA. Cells were harvested and costained with anti-CD3 and anti-CD8. Next, cells were fixed, permeabilized and stained with anti-FoxP3, anti-IL-2, anti-IFN-γ, anti-IL-4, or anti-IL-17. Dot plots were gated on CD3⁺CD4⁺(CD3⁺CD8⁻) T cells, and the numbers within each quadrant represent the percentages of CD4⁺ T cells. Two BM samples (patients 8 and 11) and one LN sample (patient 24) were tested, and the results were representatives of three independent experiments.