Figure 1.
Th9 cells increased in tuberculous pleural effusion (TPE).
(A) Th9 cells within CD4+ T cells were identified based on their expression of CD3 and not of CD8. The representative flow cytometric dot-plots of Th9, Th1, Th2 cells, Th17, and Tregs in TPE and blood are shown. (B) Comparisons of percentages of Th9, Th1, Th2 cells, Th17, and Tregs in TPE and blood (n = 14). Horizontal bars indicate means. The percentages of Th cells represented Th cell numbers in total CD4+ T cell numbers as determined by flow cytometry, comparison was made using a Wilcoxon signed-rank test. The percentages of Th cells were determined by flow cytometry, comparison was made using a Wilcoxon signed-rank test. (C) Th9 cells correlated negatively with Th1, Th2 cells, Th17, and Tregs in TPE (n = 14). Correlations were determined by Spearman rank correlation coefficients.
Table 1.
Comparisons of Th cells in tuberculous pleural effusion (TPE) and blood stimulated with PMA+ionomycin or tuberculosis antigens*.
Figure 2.
Phenotypic characteristics of Th9 in tuberculous pleural effusion (TPE).
(A) The representative dot plots showing expressions of IL-9 and CD45RO, CD45RA, CD62L, or CCR7 on CD4+ T cells. (B) Comparisons of percentages of CD45RO+, CD45RA+, CD62L+, CCR7+ cells in total Th9 cells in TPE and blood from patients with TPE (n = 14). The data are calculated by dividing the numbers in upper right quadrants by the numbers in both upper and lower right quadrants. Horizontal bars indicate means; comparison was made using a Wilcoxon signed-rank test.
Figure 3.
Chemokine receptors expressed on Th9 cells.
(A) Flow cytometric dot-plots of expressions of CCR2, CCR3, CCR4, CCR5, and CCR6 on Th9 cells from tuberculous pleural effusion (TPE) and blood. (B) Comparisons of percentages of CCR2+, CCR3+, CCR4+, CCR5+, and CCR6+ cells in total Th9 cells in TPE and blood from patients with TPE (n = 14). The data are calculated by dividing the numbers in upper right quadrants by the numbers in both upper and lower right quadrants. Horizontal bars indicate means; comparisons of CCR expressions were made using a Wilcoxon signed-rank test.
Figure 4.
Differentiation of human Th9 cells from naïve CD4+ T cells stimulated by different cytokines.
Purified naïve CD4+ T cells isolated from tuberculous pleural effusion (TPE) and blood (both n = 5) were stimulated with plate-bound anti-CD3 and soluble anti-CD28 mAbs in the presence of the indicated cytokines, either alone (A) or in various combinations (B). Seven days after activation, the cells were stimulated with PMA and ionomycin for 5 h and analyzed for IL-9 expression after intracellular staining. The comparisons were determined by Kruskal-Wallis one-way analysis of variance on ranks. * p<0.05 compared with medium control. Naïve CD4+ T cells from TPE and blood (both n = 5) were cultured and stimulated with indicated concentrations of TGF-β for 7 d (C), or with 5 ng/ml of TGF-β for indicated time points (D), the percentages of Th9 cells determined by flow cytometry. Comparisons were determined by Kruskal-Wallis one-way analysis of variance on ranks. * p<0.05 compared with baseline values.
Figure 5.
Chemokine CCL20 in tuberculous pleural effusion (TPE) was chemotactic for Th9 cells in vitro.
TPE and supernatants of cultured pleural mesothelial cells (both n = 5) were used to stimulate chemotaxis of Th9 cells in the absence of presence of anti–CCL20 mAb or an irrelevant isotype control. The comparisons were determined by Kruskal-Wallis one-way analysis of variance on ranks. *p<0.05 compared with the irrelevant isotype control.
Figure 6.
Effects of IL-9, IL-4 and IFN-γ on wound healing of in vitro injury model of pleural mesothelial cells.
(A) Pleural mesothelial cells was identified by expression of calretinin and sideward scatter (SSC) using flow cytometry, the representative flow cytometric dot-plots are showing expression of IL-9R, IL-4R, and IFN-γR1 on PMCs from tuberculous pleural effusion. (B) Summary dada of percentages of IL-9R+, IL-4R+, and IFN-γR1+ PMCs (n = 5). (C) Microscopic photography after wound induction on a confluent monolayer of PMCs in a time course from 16 to 48 h revealed that wound healing was enhanced by IL-9 or IL-4 and retarded by IFN-γ (Original magnification: ×200). (D) Graphs show relative wound closure over time, based on the wound gap compared with initial wound size. Mean ± SEM of 5 independent experiments. The comparisons were determined by Kruskal-Wallis one-way analysis of variance on ranks, *p<0.05 compared with medium control at the same time points.
Figure 7.
Effects of IL-9, IL-4 and IFN-γ on long-term restoring of pleural mesothelial cells (PMCs).
(A) Pleural mesothelial cells were seeded in Petri dishes in complete medium in the presence of IL-9, IL-4 or IFN-γ for 14 d to allow for their growth. Representative of 5 independent experiments (Original magnification: ×400). (B) Comparisons of PMC numbers in each group (n = 5). The comparisons were determined by Kruskal-Wallis one-way analysis of variance on ranks, *p<0.05 compared with medium control at the same time points.
Figure 8.
Effects of IL-9, IL-4 and IFN-γ on apoptosis of pleural mesothelial cells (PMCs).
PMCs were seeded in Petri dishes in complete medium in the presence of one or more of IL-9, IL-4, and IFN-γ for 48 h. (A) The representative flow cytometric dot-plots are showing Annexin V/propidium iodide co-staining for identification of apoptotic PMCs. (B) Comparisons of apoptotic PMCs in each group (n = 5). The comparisons were determined by Kruskal-Wallis one-way analysis of variance on ranks, *p<0.05 compared with medium control, †p<0.05 compared with IFN-γ alone.
Figure 9.
In vitro stimulation of Th9 cell differentiation by antigen presentation of pleural mesothelial cells (PMCs).
Purified naïve CD4+ T cells from blood were cultured with autologous PMCs at a ratio of 5 ∶ 1 for 5 d in the absence or presence of exogenous antigen ESAT-6/CFP-10, IL-9, IL-4 or IFN-γ (A), or anti–CD80, –CD86 mAb, a combination of anti–CD80 and –CD86 mAbs, CTLA-4Ig or control Ig (B) was added into the coculture, the frequencies of Th9 cells were determined by flow cytometry. The results are reported as mean ± SEM from 5 independent experiments. The comparisons were determined by Kruskal-Wallis one-way analysis of variance on ranks. * p<0.05 compared with medium control, †p<0.05 compared with PMCs plus ESAT-6/CFP-10 (or plus control IgG).