carried out according to institutional guidelines approved by the Niedersa¨chsisches Landesamt f u¨r Verbraucherschutz und Lebens- mittelsicherheit or by the Bavarian state authorities (permit number 33.9-42502-04-07/1253). In vitro cytokine stimulation assays CD4+ T cells were isolated from mixed single cell suspensions of spleen and peripheral lymph nodes by MACS cells separation with the CD4+ T Cell Isolation Kit (Miltenyi). CD4+ cells from BALB/c DO11.10 TCR transgenic mice were co-cultured for 6–7 days with irradiated (30 Gy) spleen-derived and sex-matched BALB/c feeder cells in a ratio of 16106 DO11.10 to 36106 BALB/c spleen cells per ml. The mouse cytokines TGF-b (2 ng/ml), IL-6 (10 ng/ml), IL-1b (10 ng/ml), IL-21 (10 ng/ml) and TNF-a (10 ng/ml) were purchased from Peprotech. Ovalbumin peptide (Ova323–339, 5 mg/ml) was from Anaspec and IL-23 (10 ng/ml) (mouse) from R&D Systems. Human rIL-2 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: from Dr. Maurice Gately, Hoffmann – La Roche. For the Th0 condition IL-2 (300 IU/ml) and Ova322–339 were added to the co-cultured cells. The Th17 mix contained TGF-b, IL- 6, IL-23, IL-1b, TNF-a, anti-IL-4, anti-IFN-c and Ova323–339. The Treg cytokine mix was as follows: IL-2, TGF-b (10 ng/ml), RA (5 nM) and Ova323–339. Th1 polarization mix: IL-2, IL-12 (10 ng/ ml, Peprotech), anti-IL-4 and Ova323–339. Th2 polarization mix: IL- 2, IL-4 (20 ng/ml, Peprotech), anti-IFN-c and Ova323–339. Successful polarization was assessed by intracellular staining for the key cytokines IFN-c, IL-4, IL-17 and the Treg signature transcription factor FoxP3 (data not shown). Anti-IL-4 (clone 11B11, 10 mg/ml) [47] and anti-IFN-c (clone XMG1.2, 10 mg/ml) [48] were produced in rat hybridoma cell lines, anti-IL-12 (p40/ p70, 10 mg/ml) was from BD Pharmingen. Retinoic acid (RA) was obtained from Sigma-Aldrich. For measurement of intracellular cytokines, T cells were re- stimulated with 50 ng/ml Phorbol-12-myristate-13-acetate (PMA, Calbiochem) and 2 mg/ml ionomycin (Invitrogen) in the presence of brefeldin A (Sigma). Cells were fixed using the BD Cytofix/ Cytoperm Kit as described in the supplier’s manual. The IL-17A Secretion Assay (Mouse) was carried out according to the manufacturer’s instructions. Briefly, single cell suspensions of spleens and peripheral lymph nodes of C57BL/6N mice were stimulated with PMA/ionomycin (50 ng/ml/2 mg/ml) for 3 hours at 37uC with 5% CO2. In the next step cells were labelled with the IL-17 Catch Reagent, cultured under rotation at 37uC for 45 min, labelled with the IL-17 Detection Antibody (Biotin) and finally labelled with Anti-Biotin-PE and anti-CD4 (GK1.5) antibody in the presence of FcR block (2.4G2). Ex vivo FACS cells sorting and FACS analysis Antibodies directed against abTCR (clone H57-597), CD4 (clone GK1.5), B220 (clone RA3-GB2), IL-17A (clone ebio17B7), and CCR6 (anti-CD196,) were purchased from ebioscience. Antibodies against CD8a (clone 53-6.7) and CD27 (clone LG.3A10) were obtained from BD Pharmingen. Anti-TCRcd (clone GL3), CD3 (clone 17A2) and anti-FcR antibody (clone 2.4G2) were produced in rat hybridoma cell lines. FACS analysis was performed using an LSRII flow cytometer (BD Biosciences) and data were collected with BD FACSDiva software (BD Biosciences). FACS sorting was carried out in the central cell sorting facility of the Hannover Medical School on a FACSAriaIIu (BD) or on a MoFlow XDP (Beckman Coulter). Post acquisition analyses and compensation were conducted using FlowJo software 8.8.7 (Treestar). IL-17 producing and non-producing CD4+ ab T cells were sorted from mixed spleen and peripheral lymph node Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus cells from C57BL/6N mice after revealing IL-17 secreting cells using the mouse IL-17A Secretion Assay (Miltenyi) and subsequent FACS-sorting. cd T cells [14] were sorted from mixed spleen and peripheral lymph node cells from C57BL/6-Tcrd-H2BeGFP mice based on eGFP and CCR6 expression. Rorct positive and negative cells were sorted from mixed spleen and peripheral lymph node cells from heterozygous B6.129P2(Cg)-Rorctm2Litt/J mice based on their GFP expression. IL-23R expressing or non-expressing cells were sorted according to their GFP expression from either homozygous ( = IL-23R2/2) or heterozygous IL-23Rgfp.KI mice. Human samples were sorted as follows: To capture cytokine expressing cells, PBMC were enriched for CD4+ T cells using negative selection magnetic beads (Stemcell technologies) and stimulated for 2 hours with PMA (10 ng/ml, Sigma) and ionomycin (1 mg/ml, Sigma). IL-17A and IFN-c secreting cells were detected by a human Secretion Assay, Miltenyi Biotec, according to manufacturer’s instructions and co-stained for CD4 (CD4-PC7, Beckman Coulter). Quantitative Real-Time PCR (qRT-PCR) In vitro cultured or FACS sorted cells were resuspended in 700 ml QIAzol lysis buffer, if not described differently and total RNA with conservation of small RNAs was isolated by QIAgen miRNeasy Kit. Quantitative Real-Time PCR was carried out using probes for hsa- miR-133b or hsa-miR-206 (Applied Biosystems, Taqman miRNA Assays ID 00510 and ID 002247) on an Applied Biosystems StepOnePlus Real-Time PCR System. The validity of the qRT-PCR assays for miR-133b and miR-206 was assessed by overexpression and subsequent detection of these in BWa2b2 cells (Fig. S5). Fold- difference was calculated by the DDCt method normalized to snoRNA412 as housekeeping gene for mouse samples or RNU48 for human samples (both Applied Biosystems Assay ID 001243 and ID 001006). The SD values for the representative experiments were calculated from either triplicates or quadruplicates of the combined coefficients of variation from sample and calibrator according to the manufacturer’s manual (Applied Biosystems). ELISA Secreted IL-17A protein was detected by standard sandwich ELISA purchased from BioLegend. Concentrations of IL-17A were calculated using recombinant purified IL-17A protein. Standard curve and sample concentrations were calculated based on the mean of triplicates for each dilution or sample. Retroviral infections The retroviral constructs encoding mmu-miR-133b and mmu- miR-206 were generated by inserting the respective pre-miRNA sequences flanked by approximately 125 bp into the 39LTR of the vector MDH-PGK1-GFP_2.0 (Addgene, [35]). miRNA sequences were obtained by PCR from the BAC AC159614 with the following primers: 206-forward: CCGGTTAACTCGAGGATAGTTTCTGG- GAGTGTAG; 206-reverse: CTAGCTAGGAATTCGGAGAAGTCTGTA- GAGGTAG; 133b-forward: CCGGTTAACTCGAGCTCTGTGAGAG- GTTAGTCAG; 133b-reverse: CTAGCTAGGAATTCTGTGACCTGTGA- ACTTGGGC. The resulting vectors encode GFP under control of the PGK promoter and miRNA under control of the human H1 promoter. PLoS ONE | www.plosone.org 9 May 2011 | Volume 6 | Issue 5 | e20171