Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins

Figure 3

Secretion of IFN-γ by T-cells in the presence of α-CD3 and PD-1-targeting reagents. T-cells isolated from the ascites () and tumor () were activated and cultured with α-CD3 (OKT-3) and 100 nM of PD-1-directed reagents (nivolumab, Nivo; pembrolizumab, Pembro; DARPin-1; and DARPin-2) or controls (IgG4 and negative control DARPin® protein, NCD) for 48 h. (a) The relative fold increase of IFN-γ when adding PD-1 blockers or control was compared to the release caused by α-CD3 alone (represented by a dashed line set to 1). The results are presented for T-cells isolated from the ascites (circles) or (b) tumor (squares) separately. The number of samples is indicated in each bar (). Corresponding controls for anti-PD-1 reagents are presented next to each anti-PD-1. (c) Significant differences among PD-1 blockers in ascites samples are presented separately and also (d) grouped together with tumor samples. Lines represent paired comparisons in which presented reagents have been assessed in parallel (in the same sample). (e) Comparing fold increase in paired samples of the ascites and tumor from the same patient (). Absolute concentrations of IFN-γ released by T-cells isolated from (f) the ascites or (g) the tumor. (h) Comparing absolute IFN-γ concentrations in paired samples of the ascites and tumor (). All data have been normalized to reflect the same number of T-cells (per 500,000 T-cells). Wilcoxon signed rank test was used, and median values and interquartile ranges are plotted. Significance levels were set to , , and .

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