Extended Data Fig. 4: Proliferation and survival analyses of REGNASE-1-null CD8⁺ T cells in tumour immunity.

a, List of the top-10 significantly (FDR < 0.05) upregulated and downregulated pathways in TIL Regnase-1-sgRNA-transduced OT-I cells, as revealed by performing GSEA using Hallmark gene sets. Specifically, control-sgRNA- (n = 4) and Regnase-1-sgRNA (n = 5)-transduced OT-I cells were mixed and transferred into tumour-bearing mice, and tumour-infiltrating OT-I cells were isolated at day 7 for transcriptional profiling by RNA-seq. b, GSEA enrichment plots of TIL Regnase-1-sgRNA-transduced OT-I cells using cell-cycling-associated gene sets, including E2F targets (top), G2M checkpoint (middle) and mitotic spindle (bottom). c–g, OT-I cells transduced with control sgRNA (mCherry⁺) and Regnase-1 sgRNA (ametrine⁺) were mixed and transferred into tumour-bearing mice, and tumour-infiltrating OT-I cells were analysed at day 7 (d–g) (n = 6 mice) and day 14 (c) (n = 5 mice) by flow cytometry for Ki-67 expression (c, left; e, left), BrdU incorporation (d, top; pulse for 18 h), active caspase-3 expression (f, left), Ser139 phosphorylation of histone variant H2A.X (g, top), and quantification of MFI of Ki-67 (c, right; e, right), frequency of BrdU⁺ cells (d, bottom), frequency of active caspase-3⁺ cells (f, right) and the frequency of the Ser139-phosphorylated histone variant H2A.X⁺ cells (g, bottom). Numbers in graphs indicate the MFI of Ki-67 (c, left; e, left). Numbers in plots indicate the frequencies of BrdU⁺ cells (d, top), active caspase-3⁺ cells (f, left) and Ser139-phosphorylated histone variant H2A.X⁺ cells (g, top). h, List of the top-15 significantly (FDR < 0.05) upregulated and top-4 significantly downregulated pathways in PLN Regnase-1-sgRNA-transduced OT-I cells, as revealed by performing GSEA using Hallmark gene sets. Specifically, control-sgRNA- (n = 4) and Regnase-1-sgRNA (n = 5)-transduced OT-I cells were mixed and transferred into tumour-bearing mice, and PLN OT-I cells were isolated at day 7 for transcriptional profiling by RNA-seq. i, j, OT-I cells transduced with control sgRNA (mCherry⁺) and Regnase-1 sgRNA (ametrine⁺) were mixed and transferred into tumour-bearing mice, and OT-I cells in the spleen were analysed at day 7 (i, j) (n = 6 mice) by flow cytometry for BrdU incorporation (i, top; pulse for 18 h) and active caspase-3 expression (j, top), and quantification of frequencies of BrdU⁺ cells (i, bottom) and active caspase-3⁺ cells (j, bottom). Numbers in plots indicate the frequencies of BrdU⁺ cells (i, top) and active caspase-3⁺ cells (j, top). Mean ± s.e.m. (c–g, i, j). *P < 0.05, **P < 0.01, ***P < 0.001. Kolmogorov–Smirnov test followed by Benjamini–Hochberg correction (a, b, h) or two-tailed unpaired Student’s t-test (c–g, i, j). Data are representative of two (c) independent experiments, or pooled from two (d–g, i, j) independent experiments.

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