Transcription factors expressed by T cell subtypes.

<p>Numbers of Th subtypes expressing the transcription factors T-bet (Th1; A), GATA-3 (Th2; B) RORγt (Th17; C) and FOXP3 (Treg; D) in the control (healthy non-asthmatics, n = 9), EOS high (asthmatics with elevated blood eosinophils, n = 11), and EOS low (asthmatics with low blood eosinophils, n = 12) groups. The horizontal line represents the median of the group. Filled symbols represent samples obtained in the winter time, while open symbols represent samples obtained during spring time.</p

Demographic and clinical characteristics of study participants.

¶<p>Non-reactive to methacholine up to a dose of 1.96 mg.</p>¶¶<p>One individual, not subjected to methacholine, was selected based on FEV₁ reversability with a value of 25%.</p>¶¶¶<p>Budesonide 400 µg/day at visit 2for all users, except one individual in EOS low group with a usage of 200 µg/day.</p>¶¶¶¶<p>Short acting β₂-agonist is most frequently used.</p>¶¶¶¶¶<p>Budesonide 320 µg/day at visit 2 for all users.</p>†<p>Statistically significant in comparison with EOS low and healthy controls (***P<0.001).</p>††<p>Statistically significant in comparison with healthy controls (*P<0.05).</p

Alternative models for multiple transcription factor Th cells (Multi-TF).

<p>A) Multiple transcription factor cells are between naïve and terminally differentiated cells. B) Multiple transcription factor cells are differentiated cells that are transitioning from one cell type to another, going through a stage of multi expression before reaching the new type.</p

The distribution of T cell transcription factors differs in the lung microenvironments during allergic inflammation.

<p>A) Photographs of immunohistochemistry of MBP (red) in OVA or PBS exposed sensitised mice, T-bet, GATA-3, Foxp3 (brown) and RORγt (red) in peribronchial, perivascular and alveolar tissue after exposure to OVA in sensitised mice. B) Quantification (cells/mm²) of T-bet, GATA-3, Foxp3 and RORγt expressing cells in different compartments of the lungs, including peribronchial tissue, perivascular tissue and alveolar tissue, after exposure to PBS or OVA. Data shown as mean+SEM, *<0.05. C) Pie chart showing the relative distribution of T-bet, GATA-3, Foxp3 and RORγt expressing cells in the different lung compartments after exposure of mice to OVA.</p

The expression of <i>in vitro</i> transcription factors.

<p>Values are expressed as percent of CD4⁺ T cells.</p>*<p>P<0.05.</p>**<p>P<0.01.</p>***<p>P<0.001 in comparison to medium alone.</p><p>Stim.  =  stimulated. Medium  =  medium alone.</p><p>Bold lettering highlights the transcription factor of interest.</p

Fold change of gene expression in peribronchial, perivascular and alveolar lung tissue of OVA exposed mice compared to PBS control.

<p>Genes showed have fulfilled all quality criteria, including Ct value, melting curve and melting temperature.</p><p>*RefSeq, reference sequence.</p

Proliferating CD4⁺CD25⁺ cells expressing multiple transcription factors in fresh <i>ex vivo</i> whole blood from healthy non-asthmatic controls (n = 5).

<p>Antibody panel D: stained simultaneously with Ki-67/T-bet/GATA-3/RORγt. Antibody panel E: Ki-67/T-bet/RORγt/FOXP3. A) Percentage of Ki-67⁺ (open bar) and Ki-67<b>⁻</b> (filled bar) CD4⁺CD25⁺ cells. B) Percentage of CD4⁺CD25⁺Ki-67⁺ expressing ≥ three transcription factors (open bar) or ≤three transcription factors (filled bar). C) Percentage of CD4⁺CD25⁺Ki-67<b>⁻</b> expressing ≥ three transcription factors (open bar) or ≤three transcription factors (filled bar). Data are expressed as mean±SEM.</p

Th cells increase after <i>in vitro</i> TCR stimulation.

<p>Three subsets of Th cells displayed fold change differences between the study groups: single expressing GATA-3⁺, co-expressingT-bet⁺GATA-3⁺ and GATA-3⁺RORγt⁺FOXP3⁺, after 48 hours <i>in vitro</i> stimulation with anti-CD3/anti-CD28 antibodies. Data are expressed as mean±SEM. *p<0.05.</p

Gene expression in lung microenvironments during allergic inflammation.

<p>A) Fold change of genes related to allergic inflammation in peribronchial, perivascular and alveolar tissue. Fold change is calculated by comparing OVA exposed mice to PBS control. 3 fold change threshold indicated by dotted line was considered biological relevant. B) Network of differentially expressed genes in OVA exposed mice compared to the PBS exposed controls. A network of genes associated with allergic inflammation, generated by Ingenuity Pathways Analysis (3.0 fold change threshold), of OVA/OVA mice compared to OVA/PBS mice. The expression of these genes in three lung microenvironments was examined; peribronchial, perivascular and alveolar tissue. Lines indicate relationships between molecules (solid line = direct interaction; dashed line = indirect interaction). Arrows at the end of these lines indicate the direction of the interaction. Molecules that are up-regulated and down-regulated in the dataset are coloured red and green respectively. Darker colouring of the molecules indicates a higher fold change. Grey molecules did not meet the threshold of 3 fold change. Uncoloured molecules have been added from the Ingenuity Knowledge Base. The exact fold change values of each molecule are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019889#pone-0019889-t001" target="_blank">Table 1</a>.</p

Sub analysis of circulating T-helper cell subtypes.

<p>Numbers of each subtype in the control (healthy non-asthmatics, n = 9), EOS high (asthmatics with elevated blood eosinophils, n = 11), and EOS low (asthmatics with low blood eosinophils, n = 12) groups. CD4⁺CD25⁺ cells positive for only one of the transcription factors T-bet⁺ (A), GATA-3⁺ (B), RORγt⁺ (C) and FOXP3⁺ (D). CD4⁺CD25⁺ cells co-expressing two transcription factors T-bet⁺GATA-3⁺ (E), T-bet⁺RORγt⁺ (F), T-bet⁺FOXP3⁺ (G), GATA-3⁺RORγt⁺ (H), GATA-3⁺FOXP3⁺ (I) and RORγt⁺FOXP3⁺ (J). CD4⁺CD25⁺ cells positive for three transcription factors T-bet⁺GATA-3⁺RORγt⁺ (K), T-bet⁺GATA-3⁺FOXP3⁺ (L), T-bet⁺RORγt⁺FOXP3⁺ (M) and GATA-3⁺RORγt⁺FOXP3⁺ (N). CD4⁺CD25⁺ cells expressing all four transcription factors T-bet⁺GATA-3⁺RORγt⁺FOXP3⁺ (O). The horizontal line represents the median of the group. *p<0.05. Filled symbols represent samples obtained in the winter time, while open symbols represent samples obtained during spring time.</p