DNA methylation abrogates the activity of both SNP variant TNFα promoters but does not readily explain their functional differences.

<p>(a) Reporter luciferase genes under the control of TNFα promoters carrying either the −237A or G variant were methylated, or demethylated prior to transfection into Jurkat cells. Following 24 hours the cells were stimulated with PMA (for a further 4 hours) prior to the evaluation of luciferase activity. Fold changes (stimulated/non-stimulated) in TNFα production for unmethylated promoters are noted in parentheses (b) Outline of the proximal TNFα promoter adapted from previous reports <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040100#pone.0040100-Falvo1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040100#pone.0040100-Barthel1" target="_blank">[43]</a>, illustrating the various transcription factors known to bind in this region. CpG dinucleotides including those which encompass the −237 and −1030 SNPs are noted as black strips, and their positioning relative to the Transcription Start Site (TSS) is displayed numerically (in red). (c) DNA isolated from −237 AA homozygous, −237GG homozygous and GA heterozygous BCLs was bisulfite converted, PCR amplified and sequenced to assess the methylation status of TNFα promoter sequences. A representative sequencing screen encompassing the −1030 SNP denotes the frequency of methylated cytidines (noted as black circles) within individual templates sequenced for a single donor, with a tabular version of summary data for a selection of BCL lines denoting the percentage of methylation at each CpG dinucleotide motif. (d) Three BCL lines encoding different combinations of the TNFα promoter −237 SNP variants (GG, GA and AA) were either left untreated or pre-incubated for 48 hours with 5-Azacytidine prior to PMA stimulation for 4 hours. sTNFα was subsequently measured by ELISA, and both absolute (bar chart) and sTNFα fold increase compared to their respective non-drug treated, PMA stimulated backgrounds (embedded values) are reported. Two biological and technical replicates were analysed per experiment, and median TNFα production plus SEM is reported.</p

−237A homozygosity associates with reduced TNFα production in PMA-activated B cell lines.

<p>(a) Immortalised BCL lines generated from healthy donors who were −237 AA homozygous (n = 4), −237GG homozygous (n = 11) or GA heterozygous (n = 4) were stimulated for 4 hours with PMA, or left untreated, following which mRNA was isolated for qPCR analysis. TNFα mRNA fold induction (stimulated/non-stimulated) is reported. (b) One million BCLs from −237GG homozygous (n = 11) and GA heterozygous (n = 4) donors were stimulated with PMA for 4 hours, following which soluble TNFα (sTNFα) levels were measured by ELISA. The data is presented as absolute differences in sTNFα secretion in the −237 homozygous versus heterozygous group. (c) Stimulation of TNFα production on a −237AA background was reduced relative to −237GG in the presence of different stimuli. The data is presented as absolute differences in soluble TNFα secretion (pg/ml) for a single −237GG and −237AA homozygous BCL line. Wilcoxon-Mann-Whitney tests were used for statistical comparisons, and two-tailed P values are indicated in (a) and (b).</p

Reduced sTNFα production on a−237A SNP background following LPS-activation of PBMCs.

<p>1 million PBMCs isolated from healthy −237GG (n = 9) homozygous or GA heterozygous (n = 9) donors were either left untreated, or stimulated for 4 hours with LPS, following which TNFα levels were estimated by ELISA. sTNFα datasets were evaluated relative to the number of CD14 positive monocytes present in each sample, and TNFα levels were adjusted to represent TNFα production per 1million CD14+ cells. (a) Absolute sTNFα production (stimulated minus non-stimulated) and (b) fold induction (stimulated/non-stimulated) were compared. Wilcoxon-Mann-Whitney tests were used for statistical comparisons, and two-tailed P values are indicated.</p

TNFα promoters encoding the −237A SNP display reduced activity following stimulation in a luciferase reporter assay system.

<p>Jurkat cells were transfected with reporter luciferase plasmids under the control of TNFα promoters carrying either the −237A or G variant. Following 24 hours, transfected cells were stimulated for 4 hours with PMA/ionomycin or left untreated following which reporter gene activity was measured. The data is presented as fold change (in relative light units (RLU)), and represents differences in RLU between stimulated and non-stimulated cells for each of the transfected variants. The cat whisker plots illustrate pooled results obtained from 4 independent transfection assays, where each transfection included 5 replicas, and denotes median luciferase induction, standard deviation, the upper and lower quartiles and the data range. Wilcoxon-Mann-Whitney tests were used for statistical comparisons, and two-tailed P values are indicated.</p

Observed TNFα promoter haplotypes and frequencies in HIV-1 infected patient groups.

<p>Five of the common Caucasian ancestral TNFα promoter SNPs were typed by sequencing and confirmed by RFLP analysis. Statistical analysis was based on the total number of haplotypes (2n) within each group. P values were calculated using the Fisher’s exact test. Statistically significant values are shown in bold. The −237A promoter haplotype was exclusively observed in patients who carried HLA-B*5701.</p

TCR response and AICD is unaffected in T cells from Prr7-deficient mice.

<p>(A) Flow cytometry analysis of the activation marker CD69 in Prr7-deficient CD4⁺ or CD8⁺ T cells stimulated with 1 μg of plate-bound anti-CD3 for 24 or 36 h. (B) Proliferation of Prr7^+/+ and Prr7^-/- splenocytes in response to TCR stimulation with plate-bound anti-CD3 measured as [³H]thymidine incorporation (DNA synthesis). cpm, counts per minute. (C) Schema of the <i>in vitro</i> AICD induction protocol. (D) Representative examples of flow cytometry analysis of AICD in T cells upon restimulation. Live = PI^-Annexin V^-, Apoptotic = PI^-Annexin V⁺, Dead = PI⁺Annexin V⁺ (E) Quantification and statistical analysis of AICD performed as shown in (D). (F) Immunoblotting of c-Jun total levels in restimulated T cells isolated from three different wild-type and knockout mice (#1, #2, #3). Tubulin served as a loading control. Data in (A, B, D) represent the mean + SEM of at least three animals per group. n.s., not significant.</p

Absence of Prr7 does not affect the susceptibility of mice to EAE.

<p>(A) Clinical EAE scores in 8–12 week old WT and Prr7 knockout female mice upon immunization with MOG peptide over time. (B) Cumulative EAE score from (A). (C) Maximum EAE scores from (A). Data are represented as mean +/- SEM of 13 (Prr7^-/-) and 14 (Prr7^+/+) mice per group from two independent experiments. n.s. not significant.</p

Prr7 deficiency does not influence T cell response to <i>Listeria monocytogenes</i> infection.

<p>(A) <i>Prr7</i>^-/- mice and <i>Prr7</i>^+/+ control mice were i.v. infected with 1x10⁴ Lm ova. On day 9 post infection, colony forming units were determined in spleen and liver. (B) Representative dot plot of CD4⁺ and CD8⁺ T cells in the spleen of infected mice. (C) Frequency of CD4⁺ and CD8⁺ T cells in the spleen of infected mice. (D) Absolute number of CD4⁺ and CD8⁺ T cells in the spleen of infected mice. (E) Frequency of CD8⁺ naive (CD62L⁺CD44^-), activated (CD62L^-CD44⁺) and memory (CD62L⁺CD44⁺) T cells in the spleen of infected mice. (F) Absolute number of CD8⁺ naive, activated and memory T cells in the spleen of infected mice. (G-K) Splenocytes of infected mice were restimulated with Ova_257-264-peptide (SIINFEKL, 10⁻⁸ M) or left unstimulated for 12 h in the presence of Brefeldin A, to allow for the intracellular accumulation of cytokines. (G) Dot plot of IFN-γ and TNF-producing CD8⁺ T cells without restimulation. (H) Dot plot of TNF-producing CD8⁺ T cells after restimulation. (I) Dot plot of IFN-γ producing CD8⁺ T cells after restimulation. (J) Frequency of IFN-γ and TNF-producing CD8⁺ T cells in the spleen of infected mice. (K) Absolute number of IFN-γ and TNF-producing CD8⁺ T cells in the spleen of infected mice. Data are represented as mean ± SEM of 4–5 mice per group. n.s. not significant.</p

T cell development is largely unaffected in Prr7-deficient mice.

<p>(A) Total numbers of nucleated cells in the thymus (left) and spleen (right) isolated from Prr7^+/+ and Prr7^-/- mice as counted using a haemocytometer. (B) Schematic representation of T cell developmental stages in the thymus. DN, double negative, DP, double positive, SP, single positive. Lower panels with dot plots are representative examples of flow cytometry analysis of thymocyte subpopulations. Percentages of DN subpopulations (C), DP subpopulations (D), and SP subpopulations (E) in thymi of Prr7^+/+ and Prr7^-/- mice as analysed by flow cytometry. (F) Flow cytometry analysis of CD4⁺ and CD8⁺ T cells subpopulations in the secondary lymphatic organs spleen and lymph nodes expressed as percentage of total. (G) Flow cytometry analysis of splenic CD3⁺ T cells expressing markers of naïve T cells (CD62⁺CD25^-), activated T cells (CD62L^-CD25⁺), or memory T cells (CD62L^-CD25⁺). Data in (A-G) represent the mean +SEM, n = 3–8. *p < 0.05, n.s., not significant.</p