Figure 1.
Triple-positive Tregs infiltrate into the tumor microenvironment of ovarian cancer.
(A) Photo of CD4+CD25+FOXP3+ Tregs in ovarian tumor samples. Top left panel shows CD4 expression (red signal), top right panel shows CD25 expression (green signal), bottom left panel shows FOXP3 expression (blue signal) and bottom right panel shows the combined signals with arrows pointing to triple stained CD4+CD25+FOXP3+ Tregs. In this picture Tregs are seen in close contact to CD4+CD25+FOXP3- cells. (B) Photo of intraepithelial infiltrating CD8+ cytotoxic T cells (left panel) and CD3+ lymphocytes (right panel) in ovarian cancer. This representative scan shows CD8 and CD3 expression (red signal = CD8 or CD3, blue signal = DAPI) in ovarian tumor mass. (C) Correlation plot comparing CD3 counts to either CD8 (filled symbols) and CD4 counts (open symbols). The symbols represent the sum of the 20 fields counted for a unique patient. All patients are represented. The inset lines are the product of linear regression analysis. (D) Shows the overall mean (± SEM, n=52) CD3+, CD4+, and CD8+ T cell counts (sum of 20 fields) for all patients. (E) Correlation plot comparing the total Treg (i.e. CD4+CD25+Foxp3+) counts with the sum of the maximum 3 (MAX3) fields or the maximum field count (MAX1). The symbols represent the sum of the 20 fields counted for a unique patient. All patients are represented. The inset lines are least squares regression lines.
Figure 2.
CD4+ or CD8+ myeloid cells contribute are a minor fraction of the total CD4+ or CD8+ cells, respectively.
(A-B) Bar graphs showing the mean (± SEM, n=3) levels of CD3-CD4+ or CD8+, respectively as a percentage of the total CD4+ or CD8+ T cells, respectively. (C) shown are 4 representative dual color dot plots staining either CD4- or CD8-gated cells. The antibody specificities are noted with the axis. (D) Shown are the mean (± SEM, n=3) levels of CD11C+, BDCA2+ and CD68+ cells as a total CD4+ or CD8+ cells (X-axis).
Figure 3.
The CD8+/Treg ratio correlates with clinical outcome in serous ovarian cancer.
(A) Pie chart showing the distribution of CD4+ T cells with respect to CD25 and FOXP3 staining in all patients. (B-D) Scatter dot plots of the ratios of CD8+ T cell counts to Tregs (CD4+CD25+FOXP3+) (B), CD4+CD25+FOXP3- T cells (C), or combined CD4+CD25+FOXP3+ and CD4+CD25+FOXP3- T cells (D) for both the good outcome and poor outcome groups.
Figure 4.
Tumor-infiltrating CD4+CD25+ cells up-regulate FoxP3 and TGF-β expression upon activation.
(A) Shown are two dots plots of purified tumor-infiltrating CD4+ T cells stimulated with ConA or media alone. Cells are gated on CD4 and CD25. (B) Bar charts showing the mean (± SEM, n=3 unique patient samples) percentage of FoxP3+ and TGF-β T cells among the total CD4+CD25+ T cells stimulated with either ConA or media alone. P value obtained by a paired Student’s T test.