Extended Data Fig. 1: Glut2 expression by immune cells and its effects on T cell metabolism.

(a) Gating strategy for the identification of immune cell subsets (applies to all Figures and extended data Figures). (b) Expression of Glut2 by ex vivo mouse naïve (CD44low) and memory (CD44high) CD8+ and CD4+ T cells was analysed by flow cytometry. Histograms and mean data from a representative experiment (±SD; n = 3, N = 3) are shown. (c) Representative confocal microscopy images of DAPI, Glut2 and CD8 expression by antibody-activated CD8 + T cells. (d) Representative histograms of Glut2 expression by the indicated immune cell subsets ex-vivo (n = 2, N = 2). (e-f) Mouse naïve T cells were activated with plate-bound anti-CD3 (1 µg/ml) and anti-CD28 (5 µg/ml) monoclonal antibodies for 1-7 days before analysing surface Glut2 (e) and Glut1 (f) expression by flow cytometry. Histograms and mean data from a representative experiment (±SD; n = 3, N = 3) are shown. (g) T cells were purified from Glut2+ and Glut2- mice and Glut2 transcription was measured by RT-PCR. Gene expression was normalized to housekeeping gene and control Glut2+ set as 1 (±SD; n = 3, N = 3). (h) The ability of CD44low T cells to take up the fluorescent glucose analogue 6-NBDG were analysed by flow cytometry. Histograms and mean data from a representative experiment (±SD; n = 3, N = 3) are shown. (i-j) Energy metabolism was assessed by extracellular acidification rate (ECAR, mpH/min) in (i) activated CD8 + T cells, (j) activated CD4+ T cells treated with Glut1 inhibitor (STF-31, 1.25 µM) or Glut2 and Glut1 dual inhibitor (Phloretin, 75 µM) or vehicle. Bar graphs show mean glycolysis and glycolytic capacity (±SD; n = 4-6, N = 2). (k) Transcription of the indicated genes in 2-day activated Glut2+ and Glut2- CD4+ T cells was measured by RT-PCR. Gene expression was normalized to tubulin and control Glut2+ set as 1. (±SD; n = 4-6, N = 2). b, h, unpaired, two-tailed Student’s t-test; e-f, i-j, One-way ANOVA; g, k, Kruskal-Wallis test.

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