Fig. 5: Blockade of the PGE₂–EP2/EP4 axis increases TIL expansion, fitness and tumour reactivity.

a, Relative number of pre-REP TILs from different solid tumours treated with IL-2, IL-2 plus PGE₂, IL-2 plus EP2/EP4 antagonists or IL-2 plus ketorolac (COXi) during the first 48 h of culture (n = 13). b, Relative number of REP TILs in the COXi group from different solid tumours (n = 8). c, Uniform manifold approximation and projection (UMAP) projection of 34-parameter CyTOF data showing sub-clustering of control and COXi REP TILs from three patients with melanoma (n = 3). d, Violin plots showing expression of indicated proteins detected by CyTOF in five different clusters in control and COXi REP TILs. e, Fold change frequency of CD8⁺ REP TILs per cluster in control and COXi groups (n = 3). f,g, Frequency of TCF1⁺CD39⁻ (f) and TOX⁺TCF1⁻ (g) TILs as a percentage of CD8⁺ TILs in control and COXi REP TILs (n = 7). h, Fold change of mitochondrial DNA copy number in REP COXi TILs (n = 5). i,j, Relative cell count of tumour-reactive CD8⁺ (n = 3) (i) and CD4⁺ (j) TILs at the end of REP step (n = 3). Tumour reactivity was assessed via 41BB surface expression upon co-culture with autologous tumours. k, Fold change in frequency of TNF⁺, IFNγ⁺ or TNF⁺IFNγ⁺ CD8⁺ REP TILs as a percentage of CD8⁺ TILs upon overnight co-culture with autologous tumour cells (n = 3). l,m, Tumour growth kinetics (l) and survival curve (m) for mice treated with COXi and control REP TILs in a Winn assay transfer (n = 6 mice per group). n,o, Frequency as a percentage of total cells (n) and representative images (o) of intratumoral human CD45⁺ cells upon treatment of COXi or control REP TILs (n = 3 or 4 mice per group). Scale bars, 100 μm. Data are mean ± s.d. One-way ANOVA (a,e,k,n) or two-way ANOVA with Dunnett’s post hoc test for multiple comparisons (l,m); Cohen’s D test (d) (Supplementary Table 5); or paired (f–h) or unpaired (b,i,j) two-tailed t-test. Independent biological samples were used; exact numbers of biological replicates are listed in each panel.