Extended Data Fig. 5: Characterization of sorted tumor-specific PKM2^WT and PKM2^KO T cells from tumors from mice treated with IgG control or anti-PD-1.

a–l, Activated OT-I+Thy1.1 + PKM2^WT (NTC-2, red) or PKM2^KO (Pkm2-8, blue) CD8 + T cells distinguished by Thy1.1 zygosity were adoptively co-transferred into lymphodepleted C57Bl/6 mice 7 days after orthotopic implantation of HKP1-ova-GFP tumors. 3 doses of IgG control or anti-PD-1 were administered on days 10, 14, and 17 after tumor implantation. T cells were sorted from tumors based on Thy1.1 zygosity, phenotyped by flow cytometry, and bulk RNA sequenced. a, Gating strategy for identifying adoptively co-transferred PKM2^WT (Thy1.1 homozygous, red) and PKM2^KO (Thy1.1 heterozygous, blue) T cells sorted back from tumors. b, Histograms of PKM2 (left) and PKM1 (right) expression in activated CD8 + T cells electroporated with guides targeting PKM2 (Pkm2-8, blue) or non-targeting control (NTC-2, red). c, Bioluminescence imaging (BLI) group averages (top) and curves from individual mice (bottom) to measure tumor burden in mice treated with IgG control (orange) or anti-PD-1 (purple) from harvests on days 12 (left) and 16 (right) post-tumor implantation. d-e, Flow cytometric analysis of PKM2^WT (NTC-2, red) and PKM2^KO (Pkm2-8, blue) CD8 + T cells for percentage of donor pool (d) and for CD44 + CD62L+ percentage (e) with indicated treatments and harvest timepoints. f-l, Gene set enrichment analyses examining: signatures for effector and memory cells at days 12 (left two) and 16 (right two) (f), progenitor and terminal exhaustion signatures at days 12 (g) and 16 (h), and hallmark signatures at days 12 (i) and 16 (j) in PKM2^KO compared with PKM2^WT; and hallmark signatures in anti-PD-1-treated mice compared with IgG at days 12 (k) and 16 (l). Numbers: (c) n = 6 biological replicates per group. (d-l) n = 3 biological replicates per group. Statistics: (d-e) paired two-tailed t-tests; (f-l) GSEA by fgsea. Data in (c) are presented as mean ± standard deviation. Abbreviations: BV711, Brilliant Violet 711; BV785, Brilliant Violet 785; SSC-A, Side Scatter-Area; FSC-A, Forward Scatter-Area; FSC-H, Forward Scatter-Height; FSC-W, Forward Scatter-Width; SSC-H, Side Scatter-Height; SSC-W, Side Scatter-Width; DAPI, 4’,6-Diamidino-2-phenylindole; FITC, Fluorescein isothiocyanate; PE, Phycoerythrin.

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