Baseline characteristics of the study patient.

<p>^†median (interquartile range)</p><p>^¶The value for βD glucan is missing for one patient</p><p>HIV: human immunodeficiency virus, CMV: cytomegarlovirus, PaO₂: partial pressure of oxygen in arterial blood, A-aDO₂: alveolar-arterial oxygen difference, LDH: lactate dehydrogenase, CRP: C-reactive protein, PCP: pneumocystis pneumonia, cART: combination antiretroviral therapy.</p><p>Baseline characteristics of the study patient.</p

Patient enrollment process.

<p>Patient enrollment process.</p

Baseline characteristics of severe pneumocystis pneumonia patients according to the duration of corticosteroid therapy.

<p>^†median (interquartile range)</p><p>HIV: human immunodeficiency virus, CMV: cytomegarlovirus, PaO₂: partial pressure of oxygen in arterial blood, A-aDO₂: alveolar-arterial oxygen difference, LDH: lactate dehydrogenase, CRP: C-reactive protein, PCP: pneumocystis pneumonia.</p><p>Baseline characteristics of severe pneumocystis pneumonia patients according to the duration of corticosteroid therapy.</p

LPA_1/3 inhibition of human MSCs reduced actin polymerization and increased cell-cycle quiescence.

<p><b>A, B.</b> Phenotypic characteristics of human MSCs treated with Ki16425 or vehicle alone for 48 h after plating. Shown are phase-contrast images (panel <b>A</b>) and fluorescent images in which filamentous actin (F-actin) was visualized with green phalloidin-FITC staining and nuclei were stained with red propidium iodide (panel <b>B</b>). Scale bars, 200 µm. <b>C.</b> Western blotting analysis to evaluate the phosphorylation and activation status of focal adhesion kinase (FAK). <b>D.</b> Cell-cycle analysis. Human MSCs treated with Ki16425 or vehicle alone for 72 h were fixed and then stained for DNA and RNA with 7-AAD and pyronin Y, respectively. Their cell-cycle status was assessed based on their DNA and RNA content by flow cytometry. A representative of three experiments is shown on the left side, and the bar graph summarizes the results of the G₀ proportion on the right side. The data are presented as the means ± standard error (<i>n</i> = 3). <b>E.</b> Western blotting analysis of signaling molecules associated with the Akt pathway. For panels <b>C</b> and <b>E</b>, human MSCs were cultured in the presence or absence of Ki16425 for the indicated times prior to cell lysis.</p

Decreased self-renewal capacity associated with senescence was prevented in human MSCs following treatment with Ki16425, an LPA₁/LPA₃ antagonist.

<p><b>A.</b> Growth kinetics during serial passage. Human MSCs at passage 2 (8.1 population doublings) were serially passaged every 9 days in the presence or absence of Ki16425. Cumulative population doublings are presented as the means of duplicates. <b>B.</b> Western blotting analysis of total and phosphorylated cPLA2 in human MSCs at passage 2. <b>C.</b> Real-time PCR analysis of LPA receptor gene expression in human MSCs at passage 2. Levels of mRNA were quantified relative to the mean of LPA₁ samples. <b>D.</b> CFU-F assay. Human MSCs at passage 2 were cultured in the presence or absence of Ki16425 for two additional passages (27 days). CFU-F colonies initiated from the treated cells (passage 5, 100 cells) were counted after 15 days of normal culture. On the right side are shown representative CFU-F colonies stained with crystal violet. <b>E.</b> SA-β-Gal assay. The total SA-β-Gal activities of Ki16425- and vehicle-treated human MSCs were quantified in the wells of six-well plates as the luminescent intensity (relative luminescence units, RLU). On the right side are shown representative human MSCs stained for SA-β-Gal. Scale bars in inset boxes, 200 µm. <b>F.</b> Telomere measurement. Telomere lengths were determined in Ki16425- and vehicle-treated human MSCs by real-time PCR and quantified relative to the mean of vehicle controls. <b>G.</b> Western blotting analysis of cell-cycle components. Human MSCs at passage 2 were cultured in the presence or absence of Ki16425 for the indicated days prior to cell lysis. Fold-change represents decrease in band intensity of Ki16425 treatment for 18 days compared with a control treatment for the same period of time. For panels <b>C</b>, <b>D</b>, <b>E</b>, and <b>F</b>, the data are presented as the means ± standard error (<i>n</i> = 3).</p

CD8⁺ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma

<div><p>The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8⁺ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8⁺ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8⁺ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female <i>cd8α</i>-disrupted mice. The adaptive transfer of male, but not female, CD8⁺ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8⁺ T cells was observed in female recipient mice. Male CD8⁺ T cells produced higher levels of IFN-γ than female CD8⁺ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4⁺ T cells. Interestingly, IFN-γ receptor expression on CD4⁺ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8⁺ T cells and the lower expression of IFN-γ receptor on CD4⁺ T cells in females compared with males.</p></div

IFN-γ attenuates IL-4 production from BLN male CD4⁺ T cells.

<p>Male or female CD4⁺ T cells (2 x 10⁵ cells/well) and CD11c⁺ cells (2 x 10⁵ cells/well) were cultured with 50 μg/ml of OVA in presence of rIFN-γ (10 ng/ml) or vehicle for 72 h. Data are shown as the mean ± SD of triplicate cultures. Experiments were repeated twice with similar results. Open bars, male CD4⁺ T cells; closed bars, female CD4⁺ T cells. **, P < 0.01; NS, not significant.</p

Sex differences in IFN-γ receptor expression on CD4⁺ T cells from WT mice.

<p>(A) Representative profiles of IFN-γ receptor expression on CD4⁺ T cells in BLN of male and female mice are shown. Cut-off lines were determined on the basis of an IgG isotype-matched control profile. The percentages of IFN-γ receptor α⁺ population (B) and IFN-γ receptor β⁺ population (C) in CD4⁺ T cells were analyzed in each group. Data are shown as the mean ± SD of three to five mice. Experiment were repeated twice with similar results. (D and E) CD4⁺ T cells obtained from naïve WT mice were cultured with rIFN-γ (10 ng/ml) for 72 h. The percentage of IFN-γ receptor α⁺ population (D) and IFN-γ receptor β⁺ population (E) in CD4⁺ T cells were analyzed before and after the culture. Data are shown as the mean ± SD of triplicate cultures. Experiments were repeated twice with similar results. **, P < 0.01 compared with male mice; #, P < 0.05; ##, P < 0.01 compared to vehicle pretreatments.</p

Sex differences in the inhibitory effect of BLN CD8⁺ T cells.

<p>Male (A) or female (B) CD4⁺ T cells (2 x 10⁵ cells/well) were cultured with CD8⁺ T cells (2 x 10⁵ cells/well) and splenic CD11c⁺ cells (2 x 10⁵ cells/well) in the presence of OVA (50 μg/ml) for 72 h. Concentration of IL-4 in the culture supernatants was measured by ELISA. Data are shown as the mean ± SD of triplicate cultures. Experiment were repeated twice with similar results. (C and D) Sensitized male or female CD8KO mice were adoptively transferred with CD8⁺ T cells from BLN of sensitized and challenged male (C) or female (D) WT mice. Saline was used as control of the transfer. Three days later, recipient CD8KO mice were challenged with OVA aerosol, and then sacrificed on day 5 post-OVA challenge. The numbers of inflammatory cells in BAL fluids were counted. Data are shown as the mean ± SEM from two independent experiments (n = 4–9). Open bars, male mice transferred with saline; closed bars, female mice transferred with saline; hatched bars, male mice transferred with male CD8⁺ T cells; horizontal line bars, female mice transferred with male CD8⁺ T cells; dotted bars, male mice transferred with female CD8⁺ T cells; vertical line bars, female mice transferred with female CD8⁺ T cells. −, mice transferred with saline; +, mice transferred with CD8⁺ T cells; *, P < 0.05; **, P < 0.01; NS, not significant.</p

Sex differences in cytokine production in lung.

<p>Male and female WT mice (A) and CD8KO mice (B) were sensitized and challenged. IL-2, IL-4, IL-5, IL-13 and IFN-γ levels in the lung were measured by ELISA one day after OVA challenge. Data are shown as the mean ± SEM from at least three independent experiments (n = 8–11). Open bars, male mice; closed bars, female mice. *, P < 0.05; **, P < 0.01; NS, not significant.</p