Figure 1.

Ascites increases frequency of functional regulatory T cells (Tregs). (A–E) Peripheral blood mononuclear cells (PBMCs) from healthy donors (n = 30) were isolated by Ficoll density centrifugation and incubated in vitro in either AIM V media or cell-free ascites from advanced epithelial ovarian cancer patients for 48 h. Cells were washed, stained with anti-CD3, CD4, CD8, CD25, tumor necrosis factor receptor 2 (TNFR2), and FoxP3, and analyzed with flow cytometry. (F,G) PBMCs from two healthy donors were isolated by Ficoll density centrifugation. Cells were labelled anti-CD3, anti-CD4, anti-CD25, and anti-CD127, and flow cytometric cell sorting was performed. (A) Flow cytometry plots of CD4⁺ and CD8⁺ T cells within CD3⁺ T cells incubated in media and ascites. (B) The frequency (%) of CD4⁺ and CD8⁺ T cells within media (black bar) and ascites (gray bar) (n = 30). (C) Tregs were identified as CD25^hiFoxP3⁺ and effector T cells (Teff) were identified as CD25⁺FoxP3⁺ within CD4⁺ T cells. Flow cytometry plots of Tregs and Teff cells within CD4⁺ T cells incubated in media and ascites. (D,E) The frequency (%) of Tregs (D) and Teff (E) within media (black bar) and ascites (gray bar) (n = 30). (F) Tregs and Teff were identified by gating on CD3⁺CD4⁺ population, followed by CD127 and CD25 to identify CD25^hiCDl27^lo and CD25⁻CD127⁺ T cells respectively (n = 2). (G) Prior to suppression assay, Teff were labelled with carboxyfluorescein diacetate succinimidyl ester (CSFE) and incubated in media, while Tregs were cultured in either complete AIM V media or in cell-free ascites for 48 h (n = 2). Cells were then harvested and washed. The labelled effector cells were cultured either alone or at a 1:2, 1:4, 1:8, and 1:16 ratio with autologous pre-conditioned Tregs in a 96-well plate with anti-CD3/CD28 for 3 days. Cells were washed, stained, and analyzed by flow cytometry. Proliferation of Teff by Tregs incubated in ascites (black line) compared to Tregs incubated in media (gray line and shaded), cultured alone (no Tregs) or in co-culture with several ratios of Teff to Tregs. *p < 0.05 and **p < 0.0001, Wilcoxon matched-pair t-test. Data from PBMCs were pooled from three independent experiments (error bars-SEM), while data of sorted cells are replicates of two donors.