IL-10 producing Tregs are apoptosis prone.

<p>(<b>A</b>) CD4⁺CD25⁺ Tregs from CD28SA treated DEREG mice were isolated and stimulated over night with anti-CD3, anti-CD28, IL-2. Next day cells were stained for IL-10 and separated into IL-10⁺Foxp3⁺ (black line) and IL-10⁻Foxp3⁺ (grey line) via FACS Sorter and cultured for 1 to 3 days with anti-CD3, anti-CD28, IL-2 and examined for Annexin V and 7AAD positive cells. As control unseparated CD4⁺CD25⁺ T-cells (dashed line) were used. Graphs show means ± SD from 3 individual experiments. ** p<0.005 compared IL-10⁺Foxp3⁺ to IL-10⁻Foxp3⁺ cells. (<b>B</b>) Flow cytometric analysis of costaining of Annexin V and clone 10-7A (phospholipids). (<b>C</b>) Binding of clone 10-7A given in ΔMFI ( = MFI(marker)−MFI(isotype)) on gated CD4⁺ T-cells 3 days after CD28SA injection dependent on IL-10 and Foxp3 expression. Graphs show means ± SD from 3 or 5 mice assayed individually and results are representative of two independent experiments. ** p<0.005. (<b>D</b>) Gene expression data given in M (ln(fold change)) of functional cluster “apoptosis” (DAVID) of sorted IL-10⁺ and IL-10⁻ CD4⁺CD25⁺ Tregs 3 days after stimulation. Transcripts selected for genes that showed ≥2-fold change and an adjusted p-value<0.001 (n = 2). (<b>E</b>) Bcl-2 expression of gated CD4⁺ T-cells 3 days after CD28SA injection dependent on IL-10 and Foxp3 expression. * p<0.05. Results are representative of three independent experiments with 3–4 mice.