Mice deficient in PARP-1 gene expression have more Treg cells. (A-C) Analysis of PARP-1^−/− (CD45.2⁺) and PARP-1^+/+ (CD45.2⁺) CD4⁺ T cells in mixed bone marrow chimeras; representative staining of CD4 vs Foxp3 in CD4⁺ cells in the spleen and thymus (A). Frequency of CD4⁺Foxp3⁺ Tregs in spleen (B) and thymus (C) (mean ± SEM, PARP-1^+/+ chimeras, n = 5; PARP-1^−/− chimeras, n = 5). (D) Quantitative PCR analysis of the expression of Foxp3 (mean ± SEM) in freshly isolated (fresh) and CD4⁺CD25⁻ T cells stimulated with anti-CD3– (5µg/ml) and CD28– (2 µg/mL) specific antibodies in the absence and presence of indicated concentrations of TGF-β1 for 24 hours. (E) CD4⁺CD25⁻ T cells were cultured in the presence of anti-CD3 antibody (0.5 μg/mL) and wild-type APCs with (TGF-β1) or without TGF-β1 (Med) for 2 days. FACS plots show representative staining of CD4 vs Foxp3. (F) Treg induction in PARP-1^−/− T cells in vivo. CD4⁺CD25^– T cells were sorted from the spleens of CD45.1⁺ OTII transgenic PARP-1^−/− or PARP-1^+/+ littermate control mice and 1 × 10⁶ sorted cells were transferred into CD45.2⁺ recipient mice. Recipient mice were received OVA protein in the drinking water for 5 days. On day 6, the small intestine LP lymphocytes (LPLs) were isolated and stained with Foxp3 to assess Treg induction in vivo by flow cytometry analysis. Bar graph shows the frequency of CD45.2⁺ (Host) and CD45.1⁺ (OT-II) CD4⁺Foxp3⁺ T cells in the LPLs of recipient mice. Data are representative of 2 (A-D,F) or 3 (E) independent experiments. *P < .05; **P < .01 (unpaired 2-tailed Student t test). FACS, fluorescence-activated cell sorter; LPL, LP lymphocyte.