animals, they were age-matched to within 2 weeks and housed beside the mutant animals for at least 14 days prior to experimentation. Mice were killed by cervical dislocation or asphyxiation with CO2, and spleen and lymph nodes (subman- dibular, axillary, inguinal, popliteal, mesenteric and para-aortic) dissected, from which splenocytes and lymph node cells (LNC) were prepared. Generation of primary T cell blasts, T cell stimulation, nuclear and cytoplasmic extracts and immunoblotting; assay of intracellular IL-2 Mixed splenocytes and LNC were cultured at 86106 cells/ml in complete medium containing anti-CD3e 100 ng/ml at 37 uC and 5% CO2. After 48 hours, cells were washed and resuspended to the same cell density in fresh medium containing recombinant murine IL-2, 20 units/ml. After a further 48 hours, fresh medium and IL-2 were added, to the same final concentration. Three days later, T cell blasts were washed and restimulated with plate-bound anti-CD3e 5 mg/ml (56106 cells per sample) for up to 4 hours at 37 uC and 5% CO2, before harvesting on ice, by scraping, and preparation of nuclear and cytosolic extracts as above (per 56106 cells: 150 ml hypotonic lysis buffer, 75 ml wash buffer, 40 ml nuclear extraction buffer). 15 mg cytosolic proteins, or total nuclear extracts were assayed for NF-kB and IkB proteins by immunoblot. Relative expression of nuclear and cytosolic proteins was calculated from quantitative densitometric scans (GS710 Calibrat- ed Imaging Densitometer with Quantity One software [Bio-Rad Laboratories, Hemel Hempstead, UK]) using the Phoretix 1D software (Non-Linear Dynamics, Newcastle-upon-Tyne, UK). In parallel experiments, cells (2.56105 cells per point) were stimulated with PMA 10 ng/ml and ionomycin 500 ng/ml in the presence of brefeldin A 10 mg/ml for 6 hours at 37 uC and 5% CO2. Cells were then washed and labelled with anti-CD4-PE-Cy7 and anti-CD8-PE labelled antibodies, both at 1:100 in FACS buffer (2% BSA, 0.02% sodium azide in PBS), for 20 minutes at 4 uC in the dark. Surface-labelled cells were washed and fixed in paraformaldehyde (2%) for 10 minutes at room temperature, washed, permeabilised with saponin 2% in FACS buffer, and labelled with anti-IL-2-FITC 1:50 in permeabilisation buffer for 1 hour at 4uC in the dark. Labelled cells were washed and flow cytometry data acquired in a BDTM LSR II flow cytometer prior to analysis using FACSDivaTM software (BD Biosciences). Thymidine incorporation assay Splenocytes or LNC were plated at 56104 cells per well in 200 ml complete medium and stimulated for 48 hours with soluble anti-CD3e and anti-CD28 at a range of concentrations at 37 uC and 5% CO2. 100 ml conditioned medium was then removed from each well, for cytokine analysis, and 1 mCi 3H-thymidine added. After 18 hours’ further incubation, 3H-thymidine incorporation was measured in a MicroBeta JET plate-reader (Perkin Elmer, Buckinghamshire, UK). Lymphocyte phenotyping and B cell selection and survival Splenocytes, LNC, or B and T cell numbers and viability were assayed by trypan blue exclusion under light microscopy or by flow cytometry (FSc/SSc live gate). Cell phenotypes were analysed by flow cytometry after labelling with anti-B220-FITC, anti-CD11b- APC and anti-CD4/CD8 antibodies as above. B cells were prepared from spleens by positive selection using anti-CD19-coated magnetic beads according to the manufacturer’s instructions (Miltenyi Biotech Ltd, Bisley, UK). B cells - more than 90% pure by B220-FITC labelling - were cultured without antigen receptor Regulation of c-Rel by IkBe in Murine Lymphocytes stimulation at 106 cells per well (per ml) in 24 well plates for 48 hours at 37 uC and 5% CO2 and assayed daily by flow cytometry for viability and receptor expression using either anti-B220-FITC plus anti-CD40-APC, or anti-BAFF-R-FITC plus anti-CD40-APC. In other experiments, purified B cells were cultured as above but in the presence of 10 mg/ml anti-IgM F(ab’)2 fragment (Jackson ImmunoResearch Europe, Ltd, Newmarket, UK ) plus 2 units/ml mIL-4 (R&D Systems, Oxford, UK) for up to 72 hours and assayed daily by flow cytometry for viability and receptor expression using anti-B220-FITC plus anti-CD40-PE and anti-BAFF-R-APC. B cell stimulation, nuclear and cytoplasmic extracts and immunoblotting Splenic B cells were prepared as above from 3 age- and sex- matched WT and IkBe2/2 mice plated at 107 cells per ml in a 12-well plate in complete medium at 37 uC and 5% CO2, and allowed to recover for 2 hours. Cells were then stimulated (107 per condition) for 0, 0.5 and 1 hour with 10 mg/ml anti-IgM F(ab’)2 fragment, and nuclear and cytoplasmic extracts prepared as for T cells. Total nuclear extracts or 10 mg cytosolic proteins were immunoblotted for c-Rel, p65, NFAT2, c-Fos, IkBe and -a and actin. Detection was by chemiluminescence in a BioRad Chemi DocTM XRS+ Molecular ImagerH and c-Rel expression relative to actin expression analysed with Image LabTM software. Results TNF inhibits IL-2 gene transcription and attenuates induction of AP-1 and NFAT When studying effects of TNF on T cell function, we observed that pre-treatment of mouse T cell hybridoma 11A2 with picomolar concentrations of TNF suppressed induction of secreted IL-2 protein by more than 90%, relative to untreated cells. Major suppression occurred whether T cells were activated via TCR or with two non-toxic concentrations of PMA and ionomycin, being partly recovered when the higher of the two was used (P+I)high, and was therefore independent of any attenuation of TCR-proximal signalling that we have reported previously (Figure 1A and [41,42]). Inhibition of IL-2 induction was at the level of mRNA expression, as determined by ribonuclease protection assay, peak expression of IL- 2 mRNA being reduced by at least 90% in TNF-treated as compared to control cells, for all stimuli (Figure 1B). We tested the effect of TNF on stability of induced IL-2 mRNA and found that, for cells stimulated with PMA plus ionomycin, IL-2 mRNA was stable for up to 3 hours after addition of actinomycin D, even though its peak expression in TNF-treated cells was ,10% that of control cells (Figure S1, shown for (P+I)low ). This indicated that inhibition of IL-2 induction in TNF-treated cells was not due to reduced mRNA stability. Therefore, we focussed our studies on the effects of TNF on transcriptional regulation of IL-2. The IL-2 proximal promoter (pIL-2) contains simple and composite binding sites for NFAT, AP-1 (Fos/Jun) and NF-kB transcription factors. We compared induction and nuclear translocation of AP-1, NFAT and NF-kB proteins in control and TNF pre-treated cells by immunoblotting. AP-1 exists as homodimers of jun (c-Jun, Jun B, Jun D) or heterodimers of Fos (c-Fos, Fos B, Fra-1 and Fra-2) and Jun. Induction of nuclear c-Fos by PMA and ionomycin was rapid and transient in control 11A2 cells, being detectable at 30 minutes, peaking at 2 hours and diminishing at 4 hours. In TNF-treated cells, by contrast, its induction was delayed and more transient (Figure 1C, panel 1). Nuclear Fos B was also attenuated in TNF-treated cells as was, to a lesser extent, c-Jun (panels 2 and 3). However, inducible nuclear Jun B was unaffected by TNF (panel 4). Fra-1 and Fra-2 were not PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24504