Extended Data Fig. 3: Reduced proliferation, sustained elevated TCF1 expression with PKM2 knockout (PKM2^KO).

a-g, Activated OT-I+Thy1.1 + T cells electroporated with guides targeting PKM2 (sgPKM2) or non-targeting controls (sgNTC), labeled with CellTrace Violet, then co-cultured with HKP1-ova-GFP until 4 days post-initial stimulation. a, Representative contour plots for Annexin V (Ann V) and CellTrace Violet expression in either control (red) or PKM2^KO T cells (blue). b-c, Quantification of percent viable T cells (b) and CellTrace Violet mean fluorescence intensity (MFI) (c) in sgNTC (red) and sgPKM2 (blue) T cells. d, Representative contour plots for CD44 and CD62L expression in either control (red) or PKM2^KO (blue) T cells. e-g, Quantification of: CD44 + CD62L+ as percent of Annexin V- Thy1.1+ singlets (e); Annexin V- cells as percent of CD44 + CD62L- or CD44 + CD62L+ Thy1.1+ singlets (f); and CellTrace Violet MFI in viable CD44 + CD62L- or CD44 + CD62L+ Thy1.1+ singlets (g) in sgNTC (red) and sgPKM2 (blue) T cells. h-j, Activated OT-I+Thy1.1 + T cells electroporated with guides targeting PKM2 (sgPKM2) or non-targeting controls (sgNTC), co-cultured with HKP1-ova-GFP for 6-9 days post-initial stimulation. h-i, Representative contour plots for Tox and TCF1 staining in control (red) and PKM2^KO (blue) T cells at 6 (h) and 9 (i) days post-initial stimulation. j, Quantification of TCF1 lo and TCF1 hi populations from (h-i). k-n, Activated pmel-1+Thy1.1 + T cells electroporated with guides targeting PKM2 (sgPKM2) or non-targeting controls (sgNTC), co-cultured with B16F10-ova-GFP until 6 days post-initial stimulation. k-m, Representative contour plots for Tox and TCF1 (k), CD44 and CD62L (l), and TIM3 and SlamF6 (m) in control (red) or PKM2^KO (blue) T cells. n, Quantification of TCF1 hi, CD62L + , and SlamF6+ populations from (k-m). Numbers: (a-g) n = 6 biological replicates per group, (h-j) n = 2–3 biological replicates per guide, 4-6 biological replicates per group, (k-n) n = 6 biological replicates per group. Statistics: (b,c,e) Unpaired two-tailed t-tests; (f,g), Two-way ANOVA, Šídák multiple comparisons; (j,n) Multiple unpaired two-tailed t-tests, Holm-Šídák multiple comparisons. Data in (b-c,e-g,j,n) are presented as mean ± standard deviation. Abbreviations: Ann V, Annexin V; APC, Allophycocyanin; FITC, Fluorescein isothiocyanate; PECy7, Phycoerythrin-Cyanine7; PE, Phycoerythrin; PB, Pacific Blue; APCCy7, Allophycocyanin-Cyanine7; AF647, Alexa Fluor 647.

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