Activated CD8+Foxp3− T cells were generated identically except that TGF-β1 and RA were excluded from the cultures and CD8+GFP− cells were sorted on day 4. CD8+GFP+ T cells and CD8+GFP−-activated T cells were generated from male DEREG×Rag1−/−×OTI mice and sorted as described before.
DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and bisulfite sequencing of the TSDR was performed as described previously 23. Cells were restimulated at a concentration of 1×107/mL if not indicated otherwise with 100 ng/mL buy BMS-907351 PMA and 1 μg/mL ionomycin (both Sigma) for 6 h at 37°C. Brefeldin A (eBioscience) was added during the last 2 h, followed by intracellular cytokine staining and FACS analysis. CD4+ T cells and CD8+ T cells were isolated from spleens and lymph nodes of CD45.1+ mice by negative selection (Invitrogen).
CD25+ cells were subsequently depleted by α-CD25-PE and anti-PE microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Responder T cells were then labeled with 5 μM CFSE and seeded at 5×104 cells per 96-round bottom well together with 3×103 BM-derived DC in complete RPMI medium. CD4+GFP+ nTregs were sorted ex vivo from DEREG mice. CD8+GFP+ or CD8+GFP− T cells were induced VX-770 supplier and sorted as detailed before. For the generation of induced CD4+GFP+ Tregs, CD4+ T cells were negatively selected from spleens and lymph nodes of DEREG×OTII mice followed by depletion of CD25+ cells. Cells were cultured in 96-well round-bottom plates at 5×104 T cells per well in the presence of 3×103 BM-DC (generated with FLT3L hybridoma supernatant), 0.06 μg/mL OVA323–339 (Biosynthan), 200 U/mL IL-2, 2 ng/mL TGF-β and 10 nM RA. After 2 days, 200 U/mL IL-2 was supplemented and CD4+GFP+ cells were FACS-sorted on day 4. All populations were added at to responder T cells at indicated ratios (Treg/responder cells). Responder
T cells were activated by the addition Resveratrol of 1 μg/mL α-CD3 antibody. CFSE dilution of CD4+CD45.1+ responder T cells was assessed by flow cytometry on day 4. In case of CD8+ T cells, wells were restimulated on day 4 as described above and CD8+CD45.1+ cells were analyzed for CFSE dilution and IFN-γ production. Unpaired two-tailed Student’s t-test was performed (Microsoft Excel) to determine the statistical significance (*p<0.05; **p<0.005). We thank Stephanie Dippel, Martina Thiele, Christine Jaencke and Esther Ermeling for technical assistance. This work was supported by the SFB587, SFB738 and SFB900. Christian T. Mayer was supported by a stipend from the German National Academic Foundation. We would further like to thank the Cell Sorting Core Facility of the Hannover Medical School supported in part by the Braukmann-Wittenberg-Herz-Stiftung and Deutsche Forschungsgemeinschaft. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset.