Predicted properties of CD1d proteins coded by different mRNA transcripts in human respiratory epithelial cells.

<p>V1–6: CD1D spliced variants; NS: Non-spliced.</p

Expression of six CD1D alternatively spliced CD1D variants in human respiratory epithelial cells.

<p>Panel A: Schematic illustration of primer pairs “C/C”, “D/D”, and “D/E” annealing sites on CD1D mRNA, and corresponding full-length product sizes. Panel B: RT-PCR on primary bronchial epithelial cells detected a 305-bp band. Same primers amplified the expected full-length (572-bp) product plus a shorter amplicon (305-bp) both in oligo dT– and CD1D specific primer–reverse transcribed cDNA from Beas2B. Direct nucleotide sequencing of the smaller band (lower panel) identified a CD1D variant lacking α1 exon (“V1”). Panel C: Primer pairs “D/D” and “D/E” detected five other CD1D variants (“V2–6”) in Beas2B. Panel D: Direct nucleotide sequencing of “V4–6” revealed the splicing junction in these variants.</p

Human respiratory epithelial cells express CD1d protein.

<p>Panel A: Flow cytometry analysis of CD1d expression on Beas2B and A549 epithelial cell lines, using three anti-human CD1d mAbs – clones 42, 51.1.3, and NOR3.2. Panel B: CD1b staining on Beas2B cells and CD1b-expressing monocyte-derived dendritic cells (DC). Panel C: Flow cytometry analysis of CD1d expression on primary human lung epithelial cells (top panel) and normal human bronchial epithelial (NHBE; bottom panel) cells.</p

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

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

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

Increase of HIV-Gag responses over time.

<p>The cellular immune responses against the control peptide pools CEF (A) and HIV-Gag (B) were followed in longitudinally drawn PBMC samples. No statistically significant differences in CTL responses to non-HIV antigens included in the CEF peptide pool were seen over time (Paired T-test, p = 0.269). However, the increase in CTL response to the HIV-Gag (p55) peptide pool was borderline significant (Paired T-test, p = 0.064). The responses in subjects OP177, OP428 and OP599, who developed multiple HLA-A2-restricted CTL responses and showed evidence of viral escape within targeted epitopes, are indicated with dashed lines.</p

Examination of Influenza Specific T Cell Responses after Influenza Virus Challenge in Individuals Vaccinated with MVA-NP+M1 Vaccine

<div><p>Current influenza vaccines stimulate neutralising antibody to the haemagglutinin antigen but as there is antigenic drift in HA it is difficult to prepare a vaccine in advance against an emergent strain. A potential strategy is to induce CD8⁺ and CD4⁺ T cells that recognize epitopes within internal proteins that are less subject to antigenic drift. Augmenting humoral responses to HA with T cell responses to more conserved antigens may result in a more broadly protective vaccine. In this study, we evaluate the quality of influenza specific T cell responses in a clinical trial using MVA-NP+M1 vaccination followed by influenza virus challenge. In vaccinated volunteers, the expression of Granzyme A, Perforin and CD57 on influenza HLA A*02 M1_58–66 antigen specific cells was higher than non-vaccinated volunteers before and after challenge despite a similar frequency of antigen specific cells. BCL₂ expression was lower in vaccinated volunteers. These data indicate that antigen specific T cells are a useful additional measure for use in human vaccination or immunization studies.</p></div