Cytokine production of human memory CD4⁺ T cells in response to <i>P. aeruginosa</i>.

<p><b>A–C</b> Sorted human memory CD4⁺ T cells from healthy volunteers were co-cultured either with dendritic cells that had been infected with <i>P. aeruginosa</i> (DCs +) or with the supernatant from such infected DCs (DC −). Levels of IL-17A (<b>A</b>), IL-22 (<b>B</b>) and IFN-γ (<b>C</b>) were measured in supernatants after 6 days. DCs were infected at a MOI of 60 before culturing with T cells. Columns show the mean of triplicate determinations; error bars are ±1 standard error of mean. Results are representative of experiments in three separate individuals. <b>D–F</b>, secreted cytokine levels following DC infection at a variety of different MOIs. Each point represents a value from an individual healthy individual.</p

Cell proliferation of memory CD4⁺ T cells following co-culture with infected dendritic cells (DCs).

<p>(<b>A</b>) Memory CD4⁺ T cells loaded with proliferation dye were co-cultured with unstimulated DCs, DCs infected with PA103ΔUΔT MOI 30 (PA103), or DCs infected with Yorkhill 5 MOI 30 (Yorkhill 5). Panels show the distribution of the proliferation dye fluorescence in the cell population after 6-days; inset figures show the percentage of the initial population that has undergone one or more cell divisions. (<b>B</b>) Patterns of cytokine expression by non-proliferating CD4⁺ T cells following 6-days of culture with DCs infected with Yorkhill 5. (<b>C</b>) Patterns of cytokine expression by CD4⁺ T cells proliferating in response to culture with DCs infected with Yorkhill 5. Numbers in plot represent per cent cells in each quadrant. The experiment was repeated in 3 independent individuals with the same result.</p

DC Activation following infection with different strains of <i>P. aeruginosa</i>.

<p>Levels of CD86 (<b>A</b>) and CD40 (<b>B</b>) following DC activation 24 hours following treatment with the indicated microbes. The panels show representative plots of isotype staining (solid shading), staining of unstimulated DCs (solid line) and staining following microbial treatment (dotted line). <b>B</b> and <b>C</b>, mean fold-increase in mean fluorescence intensity (MFI) of CD86 and CD40 respectively in 5 independent individuals. This was calculated as the fold-increase in MFI in infected DCs versus uninfected controls. Columns show mean and SEM.</p

Cytokine production by human memory CD4⁺ T cells in healthy controls and cystic fibrosis.

<p>Human memory CD4⁺ T cells were exposed to unstimulated DCs or DCs infected with the laboratory strains (PA103) or clinical <i>Pseudomonas aeruginosa</i> strains (Yorkhill). After 6 days of co-culture levels of (<b>A</b>) IL-17, (<b>B</b>) IL-22 and (<b>C</b>) IFN-γ were measured. Each point represents the mean of triplicate wells and is the result from one individual. The different bacterial strains used are: ♦, unstimulated; ○, PA103 ΔpcrV; •, PA103ΔUΔT; ▴, YH1; ▪, YH2; ×, YH5. The line indicates the median value. Differences between infection conditions (<b>A</b>–<b>C</b>) were evaluated by a Kruskal-Wallis test all of which had p values <0.0009). Differences between unstimulated and infected conditions were then tested using Dunn's multiple comparison test. ** significant p<0.01 and *** significant p<0.001.</p

Memory CD4⁺ T cells subset response to different Pseudomonas strains in CF and controls.

<p>Following co-culture with dendritic cells infected with laboratory Pseudomonas aeruginosa (PA) strains (PA103) or clinical PA strains (Yorkhill), CD4⁺ T cells were classified into T helper cell subsets based on intracellular cytokine straining patterns, as shown for a representative patient in panel <b>A</b>. The Th1, Th17 and Th22 responses to both PA103 and Yorkhill PA strains is shown for healthy controls (<b>B</b>) and patients with CF (<b>C</b>), as a percentage of the total numbers of CD4⁺ memory cells analysed. The columns show the mean values +/− SEM; n = 8 for CF patients and 10 for healthy controls. Panel <b>D</b> shows the proportions of PA-specific Th17 cells in CF patients compared with controls. Each point represents the result from one individual; different bacterial strains are indicated by the symbols as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090263#pone-0090263-g004" target="_blank">Figure 4</a>. The line indicates the median value. Differences between controls and patients with CF (<b>D</b>) were evaluated by a Mann-Whitney test.</p

Tissue-homing characteristics of Pseudomonas aeruginosa-specific memory Th22 and Th17 cells.

<p><b>A–C</b> Human memory CD4⁺ T cells were sorted into CCR6-depleted (CCR6 negative) and CCR6-enriched (CCR6 positive) populations before being exposed to dendritic cells (DCs) infected with Yorkhill 5 MOI30 (<b>A</b>), DCs infected with PA103 ΔUΔT MOI30 (<b>B</b>), or stimulated with anti-CD3 and anti-CD28 which acted as a positive control by polyclonal T cell stimulation (<b>C</b>). Plots show CD4⁺ IFN-γ negative T cells after 6-days of co-culture. <b>D</b> Memory CD4⁺ T cells were cultured with DCs infected with PA103 ΔpcrV MOI30. Pseudomonas-specific memory CD4⁺ T cells were analysed for expression of intracellular IL-22 or IL-17 as shown together with the skin-homing chemokine receptors CCR4, CCR10 and CCR9 as indicated. Numbers in each plot represent per cent cells in each quadrant. Results are representative of those seen in three independent individuals.</p

Patterns and Impact of Hypoglycemia, Hyperglycemia, and Glucose Variability on Inpatients with Insulin-Treated Cystic Fibrosis-Related Diabetes

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