Fig. 2: Sucralose decreases intracellular calcium flux downstream of the TCR.

a, Schematic of the TCR signalling cascade. ER, endoplasmic reticulum. b, Western blot of phosphorylated and total PLCγ1 in anti-CD3-stimulated T cell lysates. c, Liquid chromatography–mass spectrometry (LC–MS) quantification of Scrl in whole-cell lysate, cytosolic fraction and membrane fraction of Jurkat T cells exposed to 0.5 mM Scrl. Whole-cell lysate of Jurkat T cells grown in T cell medium with (WC Scrl) or without (WC TCM) 0.5 mM Scrl are shown as controls. n = 4 independent preparations. d, Cryogenic OrbiSIMS analysis of Scrl-treated mouse T cells shows the intensity–depth profile above background (grey shaded area) for Scrl [CClNa₂O]⁺ and lipid cell marker [C₁₆H₁₁0₅]⁺ fragments. Inset, ion intensity map for the [C₂HO]⁺ cell marker (m/z = 40.99), illustrating the 8 cells quantified (circled). TIC, total ion count. e, The percentage of CD4⁺ T cells with intermediate (left) and low (right) membrane order activated in the presence or absence of Scrl. n = 17 biological replicates. f, Representative 3D reconstruction (z-stacks) from naive T cells cultured with or without Scrl and activated with anti-CD3. Scale bars, 2 µm. g, Average volume of PLCγ1 clusters. n = 3 average volumes of at least 3 cells per image in separate fields. h, Representative flow cytometry plot for calcium flux using INDO1 in T cells activated with anti-CD3 and streptavidin. i, The percentage of T cells undergoing calcium flux. j, Representative flow cytometry plot for intracellular calcium flux with INDO1 in the presence of 1 mM EDTA. k, The percentage of T cells undergoing intracellular calcium flux. n = 3 technical replicates per condition. l,m, T cells were activated with anti-CD3/CD28 in the presence of DMSO or ionomycin (Iono) (125 ng ml⁻¹) with or without 0.5 mM Scrl. l, The percentage of proliferating T cells. n = 3 technical replicates/condition. m, Intracellular cytokine staining for IFNγ and TBET. n = 5 technical replicates per condition. Data are mean ± s.e.m. (d) or mean ± s.d. (c,g,i,k,l,m). Significance was tested using unpaired (g,i,k,l) or paired (e) two-tailed Student’s t-test; one-way ANOVA with Tukey’s multiple comparison test (m). Data are representative of two (f,g) or three (b,h–m) independent experiments.

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