Extended Data Fig. 5: Sucralose does not impede glucose metabolism, TCR-independent proliferation, IL2-STAT5 signalling, and activation markers, but reduces membrane order.

a) Flow cytometry analysis of 2NBDG-glucose uptake of T cells activated for 24 h with αCD3 and αCD28 in T cell media (TCM) with (Scrl) or without (Ctrl) 0.5mM sucralose. Graph shows the increase in mean fluorescence intensity (MFI) over time. N = 3 technical replicates except for sucralose at 5 min n = 2. (b–c) CD4⁺ (b) and CD8⁺ (c) T cells activated for 48 h in control media or in presence of 0.5 mM Scrl followed by stable isotope tracing analysis of U-[¹³C]-glucose. Mass isotopomer distribution (MID) of ¹³C₆-glucose-derived carbon into pyruvate, lactate, and malate as indicated. N = 4 (Ctrl), n = 3 (Scrl) for CD4⁺ T cells, and n = 5 (Ctrl) and n = 4 (Scrl) for CD8⁺ T cells. Dots represent technical replicates. Values below 10⁴ were considered zero. d) PCA from RNAseq of CD4⁺ T cells activated with αCD3 and αCD28 for 24 h and 48 h in control medium or medium supplemented with 0.5 mM of either NaS or Scrl. Dots represent technical replicates. e) Top 20 enrichment pathways identified using DAVID. Pathways are ordered by p-values from most significant (top, dark red) to less significant (bottom, grey). f) IL2 (100 ng/ml)-induced proliferation of VPD450-stained CD8⁺ T cells in the presence or absence of 0.5 mM sucralose for 4 days. (g–h) Expression of the activation markers CD44, CD69, PD1 and CD25 in CD8⁺ T cells (g) and CD4⁺ T cells (h) activated for 24 h with αCD3 (2 μg ml⁻¹) and αCD28 (1 μg ml⁻¹) in the presence or absence of 0.5 mM Scrl. n = 3 technical replicates. i) Western blot analysis probing for phospho-STAT5^(Tyr694) and STAT5 as a loading control from protein lysates of 24 h-activated T cells in the presence of control media or 0.5 mM Scrl. Each lane is a pool of T cells collected from a single well. j) Concentration of IL2 detected by enzyme linked immunosorbent assay (ELISA) from supernatant of T cells activated for 24 h with αCD3/CD28. Each dot represents a single well of technical replicates (n = 4/condition). k) Cell proliferation histogram overlay of VPD450-loaded T cells activated with αCD3/CD28, with or without 20 μg -ml⁻¹ of IL-2, and either in control medium (grey) or in presence of 0.5 mM Scrl (blue). Data presented with mean value ± s.e.m. (a, b, c, g, h) or ± s.d. (j) Statistical significance was tested using, mixed-effects model (REML) with Sidak’s multiple comparison test (a); 2-way ANOVA (b, c), unpaired (g–j) 2-tailed student’s t-test. Data are representative of 3 independent experiments (a, f–k).

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