Extended Data Fig. 1: Sucralose detection in mice and its effect on macrophage, B cell and T cell responses.

a). Sucralose peak detection by LC-MS in the plasma of mice fed with water or 0.72 mg ml⁻¹ of sucralose for 2 weeks. b) Box plots of frequencies of B220⁺ B cells, CD8⁺ T cells, CD4⁺ T cells, CD4⁺FOXP3⁺ T cells (T_reg), CD11b⁺ pan myeloid population, CD11b⁺NK1.1⁺ cells (NK cells), CD11c⁺ dendritic cells (DCs), monocytes (CD11b⁺Ly6C⁺) and neutrophils (CD11b⁺Ly6G⁺) within mesenteric lymph nodes, peripheral lymph nodes, and the spleen of mice fed with water), 0.17 mgml⁻¹ Scrl, 0.72 mg ml⁻¹ Scrl, and 0.72 mg ml⁻¹ sodium saccharin (NaS) (n = 6 per treatment) for over 12 weeks. As assessed by flow cytometry. Min-max box plot: centre lines show median, box limits are 1^st and 3^rd quartiles, with whiskers indicating the min and max value. (c-f) C57BL/6J mice fed with water (n = 10) or 0.72 mg ml⁻¹ Scrl (n = 8) and immunized with sheep red blood cells (sRBC). Each dot represents a biological replicate. c) Total splenocyte numbers at day 7 post sRBC immunization. d) Percentage of splenic B220⁺ B cells 7 days post SRBC immunization. e) Representative density plot of gated B220⁺ B cells and the frequency of germinal centre (GC) B cells (GL7⁺CD95⁺) at day 7 post immunization. f) Quantification of the percentage of GC B cells as depicted in Extended Data Fig. 1e, of the splenic B220⁺ B cell population (left) and as a percentage of total splenocytes (right). g) Pairwise comparison of pro-IL1β⁺TNF⁺ (n = 7/condition), TNF⁺IL6⁺ (n = 4/condition) and TNF⁺IL12p70⁺ (n = 3/condition) production assessed by flow cytometry in bone marrow derived macrophages (BMDMs) stimulated with lipopolysaccharide (LPS) either in control media or in presence of 0.5 mM of Scrl. Paired dots indicate biologically independent samples. h) IL1β plasma concentration from water (n = 7) or 0.72 mg ml⁻¹ sucralose (n = 7) fed C57BL/6J mice for 2 weeks prior to LPS challenge (0.1 mg kg⁻¹). (i–j) Generation of alternatively activated macrophages (CD11b⁺F4/80⁺CD301⁺CD206⁺) in C57BL/6J mice intraperitoneally injected with IL4 complex (IL4c) or PBS fed water (or 0.72 mg ml⁻¹ Scrl . Each dot represents a single mouse. N = 5 biological replicates per group. i) Frequencies. j) Absolute numbers. k) Homeostatic proliferation at day 3 of naïve CFSE-loaded CD8⁺ and CD4⁺ T cells injected to Rag2^−/− recipients fed water (n = 6) or 0.17 mg ml⁻¹ Scrl (n = 5) for two weeks and until the end of the experiment. Means represent ± s.e.m for biological replicates (c, d, f, g, h, i, j, k). Statistical significance was determined using 2-tailed unpaired (c, d, f, h, k) or paired (g) Student’s t test; one-way ANOVA with Tukey’s multiple comparison test (i, j), or ordinary one-way ANOVA for identical immune populations/condition (b). Data are representative of 2 (h–i) or 3 (c–f, k) independent experiments.

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