Fig. 6

TRAPs derived from malignant pleural effusions or ascites of cancer patients regulate monocytes polarization. a, b The MFIs of (a) CD163 and (b) PD-L1 on CD14⁺ monocytes from peripheral blood of cancer patients (CPs) (n = 14) and healthy donors (HDs) (n = 8) were assessed by flow cytometry. c PBMCs from CPs and HDs were stimulated with LPS (100 ng/ml) for 18 h, breferdin A plus monensin was added for the last 6 h of culture, and the frequency of CD14⁺ IL-10⁺ was determined by flow cytometry. d, e The graphs showed the correlation between concentration of TRAP and MFI of PD-L1 on CD14⁺ monocytes and total IL-10 in malignant pleural effusions or ascites of cancer patients (n = 20). f-h Purified CD14⁺ monocytes from healthy donors were treated with TRAPs (5 μg/ml) derived from pleural effusions or ascites of cancer patients (n = 10) for 3 d. f, g The expression of CD163, PD-L1, CD86 and HLA-DR was detected by flow cytometry. h The amount of IL-10 in the supernatant was assessed by ELISA. i Analysis of T cell proliferation by flow cytometry. Monocytes were left untreated or pretreated with TRAPs for 3 d, then incubated with CFSE-labeled autologous T cells (1:3) for 5 d in the presence of anti-CD3 and anti-CD28, or monocytes and T cells were separated by a transwell chamber. j, k Monocytes were left untreated or pretreated with TRAPs from MDA-MB-231 cells (c-TRAP) and cancer patient (p-TRAP) for 3 d, then incubated with autologous T cells (1:3) for 20 h in the presence of immobilized anti-CD3. j CD25 expression on T cells was examined by flow cytometry. k Breferdin A plus monensin was added during the last 8 h of culture. The percentage of CD4⁺ IFN-γ⁺ and CD8⁺ IFN-γ⁺ T cells and total IFN-γ secretion in the supernatant was detected by flow cytometry and ELISA, respectively. Data (mean ± SEM) are representative of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 by unpaired t test. (a, b, c, g, h and k) and Spearman’s rank correlation (d and e)