PARP-1^−/− Tregs showed increased survival in co-culture assay.

<p><b>(A)</b> Enhancement of PARP-1^−/− Foxp3⁺ Tregs in the co-culture assay with CD4⁺CD25⁻ T cells. CFSE labeled WT CD4⁺CD25⁻ T cells (5×10⁴) were stimulated with anti-CD3 antibody and WT T-cell depleted splenic APCs in the absence and presence of indicated WT (top panel) or PARP-1^−/− (bottom panel) CD4⁺CD25⁺ Tregs at indicated cell numbers for 3 days. Cells were collected and then stained with anti-CD4 and Foxp3 antibodies and analyzed by flow cytometry. The dot plots are shown as the profile of Foxp3 vs. CFSE in gated live CD4⁺ T cells. The numbers indicate the percentage of CD4⁺Foxp3⁺ Tregs. The data are shown as a representative experiment of two times. (<b>B</b>) Resistance of PARP-1^−/− CD4⁺CD25⁺ Tregs toward activation-induced death. Purified CD4⁺CD25⁺ Tregs were cultured with plate-coated anti-CD3 and soluble anti-CD28 antibodies for 1 day and stained with Annexin-V and 7-AAD for apoptotic cells. Left panel, flow cytometry of WT (top) and PARP-1^−/− (KO, bottom) CD4⁺CD25⁺ Tregs. The numbers in the right quadrants indicate the early apopototic (Annexin-V⁺ 7-AAD⁻, bottom) and late apoptoic/dead (annexin-V⁺ 7-AAD⁺, top) cells. Right panel is the absolute number of viable Tregs (original input number 1×10⁵) after culture. The data shown are representative of three experiments.