Fig. 2: PGE2–EP2/EP4 signalling restricts IL-2 signalling in TILs by deregulating the IL-2R complex.
From: PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function
a, Relative TIL count following treatment with PGE2 at various doses for 5 days (n = 5). FC, fold change. b, Relative TIL count following treatment for 72 h with PGE2 at different doses of IL-2 (n = 6). Ctrl, control. c, Relative CD8+ TIL count following treatment for 72 h with PGE2, EP2/EP4 antagonists (EP2/4), or combined treatment (n = 4). d, Surface expression of IL-2Rα, IL-2Rβ and IL-2Rγc in CD8+ TILs treated with PGE2 and EP2/EP4 antagonists for 72 h (n = 4). MFI, mean fluorescence intensity. e, Relative IL-2Rγc expression in CD8+ TILs treated with PGE2 for 2 h, or treated with PGE2 for 2 h and then re-exposed to medium without PGE2 for 70 h (n = 3). f, IL-2Rγc expression in unstimulated T cells treated with PGE2, the calcium chelator BAPTA, the cAMP antagonist Rp-8-CPT, ionomycin or combined treatment for 2 h (n = 4). g,h, Flow cytometry image of IL-2Rα, IL-2Rβ and IL-2Rγc expression (g; representative of four biological replicates) and colocalization of IL-2Rβ and IL-2Rγc in CD8+ TILs (h) upon 24 h treatment with PGE2, assessed by ImageStream (n = 4). A 7 μm scale bar is shown at bottom left of each row. i, FRET analysis of IL-2Rβγc in TILs treated with PGE2 for 24 h (n = 6). j, Relative mRNA expression of indicated genes in unstimulated T cells, RA T cells and TILs (n = 4). k, IL-2 signalling in RA T cells treated with PGE2 for 48 h and subsequently stimulated with IL-2 or IL-2v for 15 min (representative of 3 biological replicates). l, pS6 levels in CD8+ TILs treated for 2 h with PGE2 and subsequently stimulated for 30 min with IL-2, anti-CD3 or anti-CD3 plus anti-CD28 (anti-CD3/CD28) (n = 3). Data are mean ± s.d. Paired two-tailed t-test (h,i); one-way ANOVA with Dunnett’s post hoc test for multiple comparisons (a–f,j,l). Independent biological samples were used; exact numbers of biological replicates are listed in each panel. pJAK3, pS6, pAKT, STAT1, STAT3, pSTAT3, JAK1, pJAK1 and STAT5 were run on separate gels for blotting.
