Extended Data Fig. 2: Expression and presentation of Ova257-264 in ova-GFP-expressing cell lines, characterization of antigen-specific T cell phenotypes elicited by in vitro co-culture with antigen-expressing tumor cells, and pyruvate kinase isoform expression characteristics in CD8 + T cells.
a,b, Contour plots displaying PD-L1 and MHC I-Ova257-264 (left) and GFP and MHC I-Ova257-264 (right) in HKP1-ova-GFP cells (a) or B16F10-ova-GFP cells (b) with (blue) or without (red) overnight stimulation with 20 ng/mL IFNγ. c, Schematic for co-culture experiment. d, Histograms of IFNγ and GzmB expression on days 3-10 of the experiment. e, Mean fluorescence intensities of checkpoint proteins evaluated in naïve T cells and on days 5-9 of the experiment. f, Representative contour plots displaying Tox and TCF1 expression in naïve T cells (blue) and co-cultured T cells (red) at days 3-14 of the experiment. g, PKM1 and PKM2 isoform counts from bulk RNA Sequencing analysis of lung-infiltrating CD8 + T cells at different times and tumor burdens post-implantation. h, Histograms of fluorescence of PKM1 (solid) or isotype control (dotted) of naïve OT-I+Thy1.1 + T cells (blue) or from co-culture with HKP1-ova-GFP cells (red) at days 3-14 post-initial stimulation. i, Histogram of CD4 expression in CD4 + T cells with shRNA hairpins targeting CD4 (blue) or not transduced (red). j, Histogram of PKM2 expression in CD8 + T cells with shRNA hairpins targeting PKM (blue) or CD4 (red). k-l, Histograms of PKM2 (e) and PKM1 (f) expression in CD8 + T cells with sgRNA guides targeting PKM2 (blue) or non-targeting controls (red). m, Western blots for Total PKM, PKM2, PKM1, alpha-tubulin, and Histone H3 in protein lysates from intact or fractionated cells with either non-targeting control (PKM2wt) or PKM2-specific (PKM2ko) guides. Numbers: (d) n = 1 biological replicate per timepoint, experiment repeated twice; (e-f) n = 2 biological replicates per timepoint, experiment repeated twice. (g) Biological replicates: Naïve n = 3, Day 7 n = 6, Low n = 6, High n = 5. (m) n = 1 biological replicate fractionated, experiment repeated twice. Statistics: (g) Two-way ANOVA, Sidak’s multiple comparisons. Data in (g) are presented as mean ± standard deviation. Abbreviations: APC, Allophycocyanin; PECy7, Phycoerythrin-Cyanine7; PE, Phycoerythrin; PB, Pacific Blue; FITC, Fluorescein isothiocyanate; PCP5.5, Peridinin Chlorophyll-A Protein-Cyanine5.5.
