Fig. 3: E-motif promotes aging partly through adaptive immune activation.

a, Schematic representation of CyTOF analysis performed after 24 weeks of continuous E-motif treatment. b, Left, different cell clusters based on t-SNE dimensionality reduction, with arrows indicating clusters with obviously changed proportions; right, quantification of cell proportions, with red boxes indicating obviously changed clusters (n = 5 per group). NK, natural killer; DC, dendritic cell. c, SPADE analysis and scatter plot illustrating the proportions of cell clusters (n = 5 per group). d, Flow cytometry validation of the T/B cell proportions (T cells: n = 8, B cells: n = 7; T cells: E-motif versus vehicle P = 0.0056, B cells: E-motif versus vehicle P = 0.0022). e, Illustrative representation of the tail vein injection of E-motif in Rag or Rag + T/B mice for healthspan and lifespan examinations. f, Survival curves of Rag and Rag + T/B mice treated with or without E-motif (Rag + vehicle: n = 8, Rag + E-motif: n = 8, Rag + T/B + vehicle: n = 6, Rag + T/B + E-motif: n = 7; Rag + T/B + E-motif versus Rag + T/B + vehicle: P = 0.0021 by log-rank test and P = 0.0032 by Gehan–Breslow–Wilcoxon test, Rag + E-motif versus Rag + vehicle: P = 0.2437 by log-rank test and P = 0.2671 by Gehan–Breslow–Wilcoxon test). g, Body weight changes in mice over time (n = 6 per group over time; Rag + T/B + E-motif versus Rag + T/B + vehicle: at 20 weeks P = 0.0074, at 24 weeks P = 0.0016, at 28 weeks and after P < 0.0001). h, Proportion of fat tissue relative to body weight in mice at the experimental endpoint (age 32 weeks) (n = 7 per group; Rag + T/B + E-motif versus Rag + T/B + vehicle P = 0.0008, Rag + E-motif versus Rag + vehicle P = 0.2992). i, Schematic representation of serum extraction from the peripheral blood of mice. j,k, Serum concentrations of markers of liver function (AST and ALT) (j) and inflammatory factors (IL-1, TNF and MCP-1) (k) (n = 6 per group; ALT: Rag + T/B + E-motif versus Rag + T/B + vehicle P = 0.0044, Rag + E-motif versus Rag + vehicle P = 0.3191; AST: Rag + T/B + E-motif versus Rag + T/B + vehicle P = 0.0007, Rag + E-motif versus Rag + vehicle P = 0.3888; IL-1: Rag + T/B + E-motif versus Rag + T/B + vehicle P = 0.0008, Rag + E-motif versus Rag + vehicle P = 0.4325; TNF: Rag + T/B + E-motif versus Rag + T/B + vehicle P = 0.0034, Rag + E-motif versus Rag + vehicle P = 0.3478; MCP-1: Rag + T/B + E-motif versus Rag + T/B + vehicle P = 0.0011, Rag + E-motif versus Rag + vehicle P = 0.5740). l, Violin plots demonstrating elevated expression of inflammaging-related genes (Cd8a, Trp53, Ifng and Gzmk) in the E-motif group (n = 6 per group). m, UMAP plot showing the clustering and distribution of all T cell subsets after vehicle or E-motif treatment for 24 weeks (left), with pie charts displaying the proportions of T cell subsets in the vehicle and E-motif groups (right) (n = 6 per group). T_FH, T follicular helper. n, Bar plot of the relative abundance of T cell subsets between the vehicle and E-motif groups (n = 6 per group). NS, no significance; **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical tests: log-rank test and Gehan–Breslow–Wilcoxon test (f); two-way ANOVA (g); unpaired two-tailed Student’s t test (equal variance) or unpaired two-tailed t test with Welch’s correction (unequal variance) with a 95% confidence interval (d, h, j–k). Data are presented as mean values ± s.d. (d, g–h, j–k). Illustrations in panels a and e were created using FigDraw.

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