Comparison of lymphocyte sub-sets between CF and healthy control subjects.

<p>*After exclusion of MAIT and γ/δ T-cells. Values expressed as percentage of parent population, Median (interquartile range). Significance of between group differences determined by Mann-Whitney U test.</p><p>Comparison of lymphocyte sub-sets between CF and healthy control subjects.</p

MAIT Cell percentage in CF subjects based on A. Presence of <i>P. aeruginosa</i> in sputum cultures, B. Clinical status.

<p>PE: pulmonary exacerbations, *two and ∧one not infected with <i>P. aeruginosa</i>, between group differences determined by Mann-Whitney U test.</p

Lymphocyte sub-set phenotypes in CF subjects based on the presence of <i>P. aeruginosa</i> infection compared to healthy control subjects.

<p>∧CF No <i>P. aeruginosa versus</i> CF <i>P. aeruginosa</i> infection. ^$CF No <i>P. aeruginosa versus</i> Non-CF. *After exclusion of MAIT and γ/δ T-cells. Values expressed as percentage of parent population, Median (interquartile range). Significance of between group differences determined by Mann-Whitney U test.</p><p>Lymphocyte sub-set phenotypes in CF subjects based on the presence of <i>P. aeruginosa</i> infection compared to healthy control subjects.</p

Relationship between MAIT and γ/δ T-cell counts and percentages with lung function.

<p>A. Pearson’s Correlation co-efficient (r) and significance value (p) of MAIT and γ/δ T-cells and B. Correlation plots for MAIT Cell expressed as percentage of T-cell population, with FEV₁ and FVC % predicted, C-reactive protein and body mass index. MAIT: Mucosal invariant T-Lymphocytes, γ/δ T-Cell: Gamma-Delta T-lymphocytes, FEV₁: Forced expiratory volume in one second, FVC: Forced vital capacity.</p

Subject demographics.

<p>Data presented as median (interquartile range).</p><p>*Summary data, subjects may have had more than one pathogen isolated in sputum, individual microbiological data available in Table S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109891#pone.0109891.s002" target="_blank">File S2</a>.</p><p>Subject demographics.</p

Reduced Mucosal Associated Invariant T-Cells Are Associated with Increased Disease Severity and <i>Pseudomonas aeruginosa</i> Infection in Cystic Fibrosis

<div><p>Background</p><p>Primary defects in host immune responses have been hypothesised to contribute towards an inability of subjects with cystic fibrosis (CF) to effectively clear pulmonary infections. Innate T-lymphocytes provide rapid pathogen-specific responses prior to the development of classical MHC class I and II restricted T-cell responses and are essential to the initial control of pulmonary infection. We aimed to examine the relationship between peripheral blood lymphocyte phenotype and clinical outcomes in adults with CF.</p><p>Methods</p><p>We studied 41 subjects with CF and 22, age matched, non-smoking healthy control subjects. Lymphocytes were extracted from peripheral blood samples and phenotyped by flow-cytometry. Lymphocyte phenotype was correlated with sputum microbiology and clinical parameters.</p><p>Results</p><p>In comparison to healthy control subjects, mucosal associated invariant T (MAIT)-lymphocytes were significantly reduced in the peripheral blood of subjects with CF (1.1% versus 2.0% of T-lymphocytes, P = 0.002). MAIT cell concentration was lowest in CF subjects infected with <i>P. aeruginosa</i> and in subjects receiving treatment for a pulmonary exacerbation. Furthermore a reduced MAIT cell concentration correlated with severity of lung disease.</p><p>Conclusion</p><p>Reduced numbers of MAIT cells in subjects with CF were associated with <i>P. aeruginosa</i> pulmonary infection, pulmonary exacerbations and more severe lung disease. These findings provide the impetus for future studies examining the utility of MAIT cells in immunotherapies and vaccine development. Longitudinal studies of MAIT cells as biomarkers of CF pulmonary infection are awaited.</p></div

Lymphocyte sub-sets in CF subjects based on clinical stability and compared to healthy control subjects.

<p>∧CF pulmonary exacerbation <i>versus</i> CF stable, ^$CF stable <i>versus</i> Non-CF. *After exclusion of MAIT and γ/δ T-cells. Values expressed as percentage of parent population, Median (interquartile range). Significance of between group differences determined by Mann-Whitney U test.</p><p>Lymphocyte sub-sets in CF subjects based on clinical stability and compared to healthy control subjects.</p

Rapid degradation of premature translational termination products.

<p><i>A.</i> Denaturing immunoprecipitation of peptidyl-puromycins. 293-K^b cells were radiolabeled for 30 minutes with [³⁵S]-Met, 20 µM puromycin, and 20 µM MG132. Polypeptides were precipitated with TCA, solubilized, then subjected to a denaturing immunoprecipitation using either non-specific rabbit serum (negative IP control) or anti-puromycin serum. Results are representative of three independent experiments. <i>B.</i> 293-K^b cells were pulse labeled with [³⁵S]-Met and 20 µM puro and chased as described in Fig. 3B. Solubilized TCA precipitates were subjected to denaturing immunoprecipitation using anti-puromycin serum. [³⁵S] in the anti-puromycin immunoprecipitates was measured by liquid scintillation counting (<i>n</i> = 4; mean ± s.e.m.).</p

High Peripheral Blood Th17 Percent Associated with Poor Lung Function in Cystic Fibrosis

<div><p>People with cystic fibrosis (CF) have been reported to make lung T cell responses that are biased towards T helper (Th) 2 or Th17. We hypothesized that CF-related T cell regulatory defects could be detected by analyzing CD4⁺ lymphocyte subsets in peripheral blood. Peripheral blood mononuclear cells from 42 CF patients (6 months–53 years old) and 78 healthy controls (2–61 years old) were analyzed for Th1 (IFN-γ⁺), Th2 (IL-4⁺), Th17 (IL-17⁺), Treg (FOXP3⁺), IL-10⁺ and TGF-β⁺ CD4⁺ cells. We observed higher proportions of Treg, IL-10⁺ and TGF-β⁺ CD4⁺ cells in CF adults (≥ 18 years old), but not children/adolescents, compared with controls. Within the CF group, high TGF-β⁺% was associated with chronic <i>Pseudomonas aeruginosa</i> lung infection (<i>p</i> < 0.006). We observed no significant differences between control and CF groups in the proportions of Th1, Th2 or Th17 cells, and no association within the CF group of any subset with sex, CFTR genotype, or clinical exacerbation. However, high Th17% was strongly associated with poor lung function (FEV1 % predicted) (<i>p</i> = 0.0008), and this association was strongest when both lung function testing and blood sampling were performed within one week. Our results are consistent with reports of CF as a Th17 disease and suggest that peripheral blood Th17 levels may be a surrogate marker of lung function in CF.</p></div

Puromycin stimulates the production of truncated polypeptides in a dose-dependent manner.

<p><i>A.</i> 293-K^b cells were radiolabeled with [³⁵S]-Met for 10 minutes in the presence of a linear range of puromycin concentrations from 0 to 20 µM. Radiolabeled polypeptides were visualized as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone-0051968-g001" target="_blank">Fig. 1B</a>. In the later panels, we analyze the [³⁵S] signals from the four regions indicated to the right of the gel. <i>B</i>. PhosphorImager signal intensities (arbitrary units) from selected lanes in <i>A</i>. The left side of the graph corresponds to the top of the gel while the right side of the graph corresponds to the bottom of the gel at the dye front. The highlighted regions correspond to the parts of the gel indicated in <i>A</i>. <i>C</i>. The effects of puromycin concentration on [³⁵S] signal for each of the highlighted gel regions in <i>A</i> and <i>B</i>. Results are representative of three independent experiments.</p