PMC

OVA MLR T cell proliferation in wild-type and EP2-null cells. DCs were pulsed with the indicated quantity of OVA 323–339 and used to stimulate T cells bearing the OVA-specific TCR (DO11.10). Proliferation of T cells was measured by ³[H] thymidine uptake. Wild-type T cells are represented by filled symbols, EP2 heterozygotes by shaded symbols, and EP2-null T cells by open symbols. The data are the mean ± SEM of triplicate measurements. *EP2-null value was significantly different from wild-type value (P < 0.01). **EP2-null value is significantly different from wild-type value (P < 0.001).

CTL function is elevated in EP2^–/– mice. Spleen cells from MC26-immunized EP2^–/– (white bars) and wild-type mice (black bars) were cocultured with irradiated MC26 cells for 4 days. The cells were then washed and incubated with different ratios of ⁵¹Cr-labeled MC26 cells. After a 6-h incubation, supernatants were harvested and radioactivity was measured using a gamma counter. The maximum and spontaneous release and the percentage of specific ⁵¹Cr release were determined. Data shown are representative of two independent experiments with similar results.

Analysis of tumor angiogenesis in EP2^–/– and wild-type animals. (a) Staining of MC26 tumor sections with anti-CD31 Ab. Tumor samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut and stained using an anti-CD31 Ab. Blood vessels were visualized by the brown color of the CD31-labeled endothelial cells (×400). Slides were lightly counterstained with Mayer’s hematoxylin. (b) VEGF concentration of tumors from EP2^–/– and wild-type mice. Tumor tissue lysates were prepared by homogenization. Mouse VEGF was quantitated using ELISA. Data show VEGF production in pg per milligram of tumor proteins. Three mice each of EP2^–/– and wild type were used, with triplicates for each sample in the immunoassay.

EP2^–/– DCs are refractory to mediated PGE₂ functional inhibition in vitro. PGE₂ induced inhibition of DC function signals through the EP2 receptor, shown by allogenic MLR. Day 9 DCs from EP2^–/– (white bars) and wild-type (black bars) mice on the BALB/c background, treated with PGE₂, were irradiated with 20 Gy, plated in triplicate, and incubated with T cells from wild-type mice with C57BL/6 background for 72 h. All cultures were pulsed with 1 μM [³H] thymidine 18 h before harvesting. Incorporation of [³H] thymidine was counted using a liquid scintillation counter and expressed as counts per minute. *EP2-null value was significantly different from wild-type value (P < 0.02). **EP2-null value is significantly different from EP2-null untreated control (P < 0.01).

Increased DC numbers draining lymph nodes of EP2-null mice. The number of (a) DCs, (b) CD4⁺, and (c) CD8⁺ T cells obtained from draining lymph nodes of normal or tumor-bearing EP2^–/– mice (white bars) is increased compared with wild-type control mice (black bars). For tumor-bearing mice, single-cell suspensions from inguinal lymph nodes from tumor-bearing EP2^–/– and wild-type mice were prepared 21 days after MC26 inoculation, at which time we observed the maximum tumor-growth difference. Cells were labeled with PE-conjugated anti–I-A^d_, CD11c Ab’s, anti-CD4, and anti-CD8 Ab’s. Cells were then washed and analyzed using a FACScan flow cytometer. The total number of positive cells per mouse is shown. Each data point is from three mice.

Decreased tumor size and increased host survival in EP2^–/– mice. (a) Tumor growth of MC26 was significantly attenuated in EP2^–/– host mice. A cell suspension containing 5 × 10⁵ MC26 cells in 100 μl PBS was injected subcutaneously into the right flank of EP2^–/– (white bars) and wild-type mice (black bars) in the BALB/c background. Tumor volume was determined by direct measurement of tumor dimensions, using calipers. Results are presented as the mean tumor volume ± SEM (n = 34 for both EP2^–/– and wild-type mice). (b) Survival plot of EP2^–/– and wild-type mice after 3LL tail vein injection. Cell suspensions of 3LL containing 5 ×10⁵ cells in 100 μl PBS were injected into the tail vein of EP2^–/– (dashed line) and wild-type mice (solid line) on the C57BL/6 background (n = 11 for both EP2^–/– and wild-type mice). The time of death, or when mice became moribund, was recorded and plotted as percentage of survival.

Inhibition of expression of DC markers by PGE₂. (a) Characterization of EP2 receptor expression in bone marrow progenitors of wild-type and EP2^–/– mice. Total RNA was extracted from bone marrow progenitors, and cDNA was amplified by RT-PCR. Primers specific for the EP2 receptor were used. Lane 1, DNA marker; lane 2, wild type; lane 3, EP2^–/–; lane 4 and 5, no reverse transcriptase control for wild type and EP2^–/–, respectively. (b) DC differentiation to CD11c⁺, I-Ad⁺, CD86⁺, and CD40⁺ cells was significantly inhibited after PGE₂ treatment. Bone marrow progenitors from EP2^–/– (white bars) and wild-type (black bars) mice were cultured in GM-CSF/IL-4 medium with increasing concentrations of PGE₂. At day 9, FACScan flow cytometer was used to analyze the phenotype of DCs. The percentage of expression is normalized relative to untreated cells from wild-type mice. Each surface marker was plotted on a bar graph as shown in b. Graphs depict: (c) CD11c; (d) CD86; (e) MHC class II; and (f) CD40. Untreated wild-type cells (black bars), wild-type treated with 100 nM PGE₂ (gray bars), untreated EP2^–/– cells (white bars), EP2^–/– cells treated with 100 nM PGE₂ (dark gray bars). The PGE₂ dose response for CD11c expression is a representative experiment from two independent experiments with similar results. All others are from three to four independent experiments. For each graph there is a statistically significant difference between wild-type/no PGE₂ and wild-type/PGE₂ (P < 0.05); there are no statistically significant differences between EP2^–/–/no PGE₂ and EP2^–/–/PGE₂ (P > 0.05).