Extended Data Fig. 3. PGE₂-EP2/EP4 signalling restricts IL-2 mediated TIL proliferation by deregulating IL-2R complexes.

a, Frequency of CFSE^high and b, Ki67⁺TILs (% CD8⁺) treated with PGE₂ for 5d in high dose IL-2 (6000IU/ml) (n = 3). c, Relative CD4⁺ count (Fold change to CTRL) of TILs treated for 72 h with PGE₂ +/− EP2/EP4 antagonists (n = 4). d, Representative flow cytometry plots of IL-2Rα/β/γ_c chains surface expression on CD8⁺ TILs treated or not with PGE₂ at 1 or 5uM. e, IL-2Rγ_c, IL-2Rα and IL-2Rβ surface expression in CD4⁺ TILs treated with PGE₂ +/− EP2/EP4 antagonists for 72 hrs (n = 4). f, Relative mRNA expression (Fold change to CTRL) of IL-2RA, IL-2RB and IL-2RG in TILs treated with PGE₂ +/− EP2/EP4 antagonists (n = 4). g, Relative IL-2Rγ_c expression (Fold change to CTRL) in CD4⁺ TILs treated with PGE₂ for 2 h, or for 2h and then re-exposed to media without PGE₂ for 70 h (n = 3). h, Relative surface and total IL-2Rγ_c expression on CD8⁺ TILs (Fold change to CTRL) after 2 h or 72 h PGE₂ treatment (n = 3). i, Time-lapse assessment (left) and representative image (right) of calcium mobilization in TILs upon PGE₂ (representative from 2 biological replicates). j, Colocalization of IL-2Rα/β/γ_c chains by ImageStream in CD8⁺ and k, CD4⁺ TILs treated with PGE₂(n = 4). l, Colocalization of IL-2Rβ and IL-2Rγ_c by confocal microscopy in TILs treated with 24 h PGE₂ (representative from 4 biological replicates). m, Representative dSTORM image of IL-2Rβ and IL-2Rγ_c levels in TILs treated with 24 h PGE₂ (representative of 2 biological replicates). n, Proximity ligation assay (PLA) images and quantification of IL-2Rβ/γ_c in TILs upon 24 h PGE₂ (representative of 3 biological replicates). o, Relative cell count (Fold change to CTRL) of unstimulated or repeatedly activated left) CD4⁺ and right) CD8⁺ T cells upon 72 h PGE₂ at different doses (n = 3). p, Relative frequency (Fold change to CTRL) of Ki67⁺(%CD8⁺) repeatedly activated T cells after PGE₂ for 5d (n = 2). q, Relative cell count (Fold change to CTRL IL-2 low) of repeatedly activated CD8⁺ T cells treated with PGE₂ in the presence of IL-2 or IL-15 for 48 h (n = 3). r, Relative IL-2Rα, IL-2Rβ and IL-2Rγ_c expression in unstimulated and repeatedly activated top) CD4⁺ and bottom) CD8⁺ T cells upon increasing doses of PGE₂ (Fold change to CTRL) (n = 3). s, Densitometry quantification of pJAK3 levels in repeatedly activated T cells from experiment in Fig. 2k (n = 3). Samples derived from the same experiments and gels/blots were processed in parallel. t, pS6 dose response curves in TILs after 30 min stimulation with IL-2 or u, anti-CD3 (n = 3). v, Representative flow cytometry plots of pS6 expression in TILs stimulated with IL-2 or IL-2 and PGE₂. Data are presented as the mean ± S.D. Statistical comparisons were performed using paired (j,k) or unpaired two-tailed t test (l,n,p) or one-way ANOVA (a,b,c,e,f,g,h,o,q,r,s) with Dunnett post-hoc test for multiple comparisons. Independent biological samples were used with exact numbers of biological replicates listed in each panel. p ≥ 0.05, not significant (ns).