Extended Data Fig. 6: PGE₂-EP2/EP4 axis blockade increases TIL expansion, fitness and tumour-reactivity.

a, Schematic representation of conventional TIL expansion protocol. TILs are expanded from tumours fragments with IL-2 6000IU/ml for 14-28d (pre-REP phase) and then with IL-2 3000IU/ml, anti-CD3 and feeder cells for 14d (REP phase). b, Correlation between best overall clinical response at 3-months and “PGE₂ signature”, “Eicosanoid ligand binding receptor” and “Prostanoid ligand receptors” signature scores in CD8⁺ TILs from REP-TIL product of melanoma patients enrolled in phase I ACT-TIL therapy trial (n = 13). CR: Complete Response, PR: Partial Response, SD: Stable Disease, PD: Progressive Disease. c, Time-course of PGE₂ concentration (n = 8) and d, PGE₂ concentration at day 7 in the supernatant of expanding pre-REP TILs derived from breast (n = 21), melanoma (n = 12), ovarian (n = 3), and lung (n = 5) tumours. e, Baseline PGE₂ concentration in the supernatant of 48 h pre-REP cultures treated with PGE₂, EP2/4 blockade or Ketorolac/COXi (n = 4). f, Kinetics of expansion of pre-REP TILs treated with IL-2 (6000IU/ml), IL-2 + PGE₂, or IL-2 + Ketorolac/COXi at initiation of the culture (n = 2). g, Frequency of IL-2Rγ_c^high CD8⁺ TILs (%CD8⁺) at day 7 of expansion in presence of different doses of Ketorolac/COXi (n = 2). h, Heatmap representation of TCF7, Myb, PGC1A, PTGER2, PTGER4 mRNA expression in pre-REP TILs expanded with or without Ketorolac/COXi (n = 3). i, Frequency of CD8⁺ TILs per cluster between CTRL and Ketorolac/COXi expanded TILs from mass cytometry (n = 3). j, Heatmap representation of 32 CyTOF markers expression in each cluster. Frequency of CD8⁺ TILs per cluster are depicted at the bottom. k, Representative flow cytometry plots of TOX/TCF1 and TCF1/CD39 of CD8⁺ PBLs or TILs. l, Representative flow cytometry plot of TCF1/CD39 CTRL and COXi-CD8⁺TILs. m, Relative TMRM/mitotracker green ratio (Fold change to CTRL) (n = 3) and n, Lipid peroxidation quantification (n = 3) and o, Relative Oxidized (GSSG)/reduced (GSH) glutathione ratio (Fold change to CTRL) in pre-REP CD8⁺ Ketorolac/COXi TILs (n = 3). p, Representative flow cytometry plots and p, Phenotypic characterization of multimer stained MART-1 tumour-reactive CD8⁺ TILs expanded with IL-2 or IL-2+Ketorolac/COXi (n = 1). q, Schematic representation of autologous tumour cells and TILs co-culture assay. r, Representative flow cytometry plot of 41BB⁺ CD8⁺ TILs in absence (TILs alone) or presence (TILs + Tumour) of tumour cells. s, Relative frequency of tumour-reactive CD4⁺ TILs and t, CD8⁺ TILs at REP in the Ketorolac/COXi (Fold change to CTRL). Tumour-reactive T cells were assessed via 41BB surface staining expression upon co-culture with autologous tumour by flow cytometry staining (n = 3). u, Representative flow cytometry plot of TNFα⁺IFNγ⁺ CD8⁺ TILs in absence (TILs alone) or presence (TILs + Tumour) of tumour cells. v, Relative frequency (Fold change to CTRL) of tumour-reactive Ketorolac/COXi CD8⁺ TILs assessed by IFNγ⁺ expression upon co-culture with autologous tumour line in two out of the three melanoma patients tested (last patient had no IFNγ detected) (n = 2). w, TCRβ repertoire analysis of REP TILs expanded with IL-2 or IL-2+Ketorolac/COXi (n = 3). x, Frequency of CD103⁺ (n = 3) or y, PD1⁺ (%CD8⁺) intratumoural TILs in tumours from mice treated with CTRL or Ketorolac/COXi-expanded REP TILs (n = 3). Data are presented as the mean ± S.D. Statistical comparisons were performed using paired (n) unpaired two-tailed t test (m,o,s,t) or one-way ANOVA (b,e,h,x,y) with Dunnett post-hoc test for multiple comparisons. p ≥ 0.05, not significant (ns). Independent biological samples/patients were used with exact numbers of biological replicates listed in each panel. Panels a and p were created with BioRender.com.