Extended Data Fig. 4: Anti-PD-1 treatment results in rapid tumor control and phenotypic alterations in PKM2 wild-type (PKM2^WT) and PKM2 knockout (PKM2^KO) T cells.

a-c, Adoptive transfers of either PKM2^WT (red) or PKM2^KO (blue) activated OT-I+Thy1.1 + CD8 + T cells were performed into lymphodepleted C57Bl/6 mice 7 days after HKP1-ova-GFP implantation, with anti-PD-1 or IgG at days 10 and 14, and mice harvested at day 16 post-tumor-implantation. a, Bioluminescence imaging as counts (left) or fold of day 6 luminescence (right). b,c, Counts (c) and population quantification (d) of adoptively transferred T cells gated on TCF1 expression in draining lymph nodes (dLN) (left) or tumors (right). d–r, Adoptive co-transfers of activated PKM2^WT (red) or PKM2^KO (blue) OT-I+Thy1.1 + CD8 + T cells distinguished by Thy1.1 zygosity were performed into lymphodepleted C57Bl/6 mice 7 days after HKP1-ova-GFP implantation, with anti-PD-1 or IgG at days 10 and 14, and mice harvested at days 13 and 15 post-tumor-implantation. d, Bioluminescence imaging quantification in anti-PD-1- (purple) or IgG-treated (orange) mice. e-n, Quantification of populations of PKM2^WT and PKM2^KO CD8 + T cells from tumors: frequencies as percentages of donor (e), CD44/CD62L population proportions (g), Klrg1/CD127 population proportions (i), TCF1 gated populations (k), and IFNγ + TNFα+ proportions after restimulation (m). Representative contour plots from day 13 for each analysis (f,h,j,l,n). o–r, Quantification of Ki67 (o) and IL-2 (q) mean fluorescence intensity, normalized to average fluorescence from activated control samples, in PKM2^WT and PKM2^KO CD8 + T cells from tumors, with Ki67 further gated on TCF1 expression. Representative histograms for Ki67 expression from day 13 (p) and IL-2 expression from day 15 (r), with TCF1 hi (solid contours) and TCF1 lo (dotted lines) populations for Ki67. Numbers: Biological replicates per group: (a-c) n = 10; (d–r) n = 11-13, aggregate of two experiments. Statistics: (a-c) Two-way ANOVA, Tukey’s multiple comparisons; (e,k,o,r) Paired and unpaired two-tailed t-tests; (g,i,m) Multiple paired and unpaired two-tailed t-tests, Holm-Šídák multiple comparisons. Data in (a-e,g,i,k,m,o,q) are presented as mean ± standard deviation. Abbreviations: BV711, Brilliant Violet 711; BV785, Brilliant Violet 785; AF488, Alexa Fluor 488; PECy7, Phycoerythrin-Cyanine7; BV421, Brilliant Violet 421; PE, Phycoerythrin; APC, Allophycocyanin; PCP5.5, Peridinin Chlorophyll-A Protein-Cyanine5.5; FITC, Fluorescein isothiocyanate.

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