FIGURE 2 from Dual Blockade of EP2 and EP4 Signaling is Required for Optimal Immune Activation and Antitumor Activity Against Prostaglandin-Expressing Tumors

Abstract

<p>TPST-1495 is more effective than a single EP4 or EP2 antagonists in overcoming PGE2-mediated immune suppression of recovery of cytokine production from murine monocytes and T cells. <b>A,</b> Schematic of PGE2 blockade assay. Murine whole blood (<b>B</b>–<b>F</b>) or splenic and lymph node T cells (<b>G</b>, <b>H</b>) were collected and incubated with inhibitors for 30 minutes at 37°C, followed by incubation with PGE2 at labeled concentrations for 30 minutes at 37°C, followed by stimulation with LPS (0.5 μg/mL, overnight) or SIINFEKL peptide (2 μmol/L, 5 hours). Detection of TNFα production by ELISA (<b>B</b>–<b>D</b>) and maximum percent recovery (<b>E</b>, <b>F</b>) after blockade assay was performed on murine whole blood. Representative data shown from one of three experiments performed with <i>N</i> = 3 technical replicates per experiment. Some technical replicates failed to meet threshold TNFα values in low Stim groups. Percent recovery as calculated Percent recovery = 100*(“Experimental group” − “LPS+PGE2”)/(“LPS” − “LPS+PGE2”). <b>G</b> and <b>H,</b> IFNγ production detected by Luminex in culture supernatants after treatment with TPST-1495 across a PGE2 concentration gradient (<b>F</b>), or with 333 nmol/L PGE2 comparing multiple PGE2 receptor inhibitors (<b>G</b>) in the PGE2 blockade assay performed with a single-cell suspension of OT-1 splenic and lymph node isolates stimulated with SIINFEKL peptide. Data are representative of three experiments with <i>N</i> = 3 technical replicates per experiment. <b>H,</b> IFNγ production levels in OT-1 T cell cultures containing 333 nmol/L PGE2 and incubated with TPST-1495 EP2/EP4 dual antagonist, or EP2 (PF04418948) or EP4 (E7046) single antagonists. Mean and SD are depicted in all graphs and the ordinary one-way ANOVA with Tukey multiple comparisons test was used to assess significance between individual groups. ****, <i>P</i> < 0.0001; ***, <i>P</i> < 0.001; **, <i>P</i> < 0.01; *, <i>P</i> < 0.05.</p>