Extended Data Fig. 5: Gzmb^crePtger2^−/−Ptger4^fl/fl mice exhibit Ptger4 knockout in TCF1⁺CD8⁺ TILs; increased proliferation of TCF1⁺CD8⁺ TILs and their progeny in T cell-specific EP₂/EP₄ double deficient mice.

a, Gene tracks showing the average scRNA-seq read coverage on Ptger2 and Ptger4 loci in CD8⁺ TILs from BRAF^V600E melanoma tumours in WT, Ptger2^−/−Ptger4^fl/fl, Cd4^crePtger2^−/−Ptger4^fl/fl and Gzmb^crePtger2^−/−Ptger4^fl/fl mice across all replicates. Dashed boxes highlight knockout(KO)-specific target regions. b, Quantification of Ptger2 and Ptger4 read coverage for KO-specific target regions based on a. n = 4. c,d, Gene tracks showing the scRNA-seq read coverage on Ptger2 and Ptger4 loci in c, TCF1⁺CD8⁺ TILs and d, TIM-3⁺ TIL progeny from WT, Ptger2^−/−Ptger4^fl/fl, Cd4^crePtger2^−/−Ptger4^fl/fl and Gzmb^crePtger2^−/−Ptger4^fl/fl mice. Data for one representative replicate with comparable cell numbers is shown. e,f, WT or Gzmb^crePtger2^−/−Ptger4^fl/fl TCF1⁺ stem-like and TCF1⁻ effector CD8⁺ T cells from in vitro T cell cultures were stimulated or not with anti-CD3/CD28 for 24 h. e, Representative flow cytometry plots showing TCF1, CD62L and GZMB protein expression. f, Ptger4 mRNA expression as measured by knockout-sensitive RT-PCR (n = 2). g, Expression of a CD8⁺ T cell proliferation signature across the distinct populations of CD8⁺ TILs from Ptger2^−/−Ptger4^fl/fl, Cd4^crePtger2^−/−Ptger4^fl/fl and Gzmb^crePtger2^−/−Ptger4^fl/fl mice based on scRNA-seq. h, Analysis of Ki-67 protein expression in TCF1⁺ and TIM-3⁺ CD8⁺ TILs 11 days after inoculation of Ptger2^−/−Ptger4^fl/fl, Cd4^crePtger2^−/−Ptger4^fl/fl and Gzmb^crePtger2^−/−Ptger4^fl/fl mice with 2 × 10⁶ control BRAF^V600E melanoma cells. n = 7. i, Expression of a gene signature for T cell effector function, analysed as in g. Data in a-e,g,i are from one experiment. Data in b,h are pooled from two (b) or three (h) independent experiments and depicted as mean ± s.e.m. Data in f are depicted as mean ± s.d. from one experiment. P values are from one-way ANOVA with Tukey’s multiple-comparison test. P ≥ 0.05, not significant (NS).

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