Extended Data Fig. 1: The dynamic changes of thymocytes with aging.
(a-b) Uniform and manifold approximation and Projection (UMAP) visualization of thymocyte clusters (a), based on the differentially expressed genes (DEGs) visualized in violin plots (b). Colors represent the annotation of cell identity. (c-d) Representative flow cytometry data of CD45+ and CD45− cells in thymus across age groups (c). Statistics of the percentages of CD45+ and CD45− cells in live cells, along with the total cell number in thymi across Groups I to III (d) (n = 9 for Group III; n = 7 for Groups I and II). P values (from left to right): ns, P = 0.8451; **P = 0.0025; **P = 0.0094; ns, P = 0.8428; **P = 0.0025; **P = 0.0096; ****P < 0.0001; ****P < 0.0001; ns, P = 0.5519; **P = 0.0020; ***P = 0.0003; ns, P = 0.8186. (e) Representative flow cytometry data of T, CD19+ B and CD33+ myeloid cells in thymus across age groups. All cells were firstly gated on CD45+ cells. T cells are divided into double negative (DN), double positive (DP), CD4 single positive (SP) and CD8 SP cells, gated on CD45+CD19−CD33− cells. (f) Statistics of cell numbers of DN, DP, CD4 SP, CD8 SP, CD19+ B cells and CD33+ myeloid cells in CD45+ cells in thymi across Groups I to III (n = 9 for Group III; n = 7 for Groups I and II). P values (row-wise: from left to right): ***P = 0.0002; ****P < 0.0001; ns, P = 0.6188; ****P < 0.0001; ****P < 0.0001; ns, P = 0.5985; ****P < 0.0001; ****P < 0.0001; ns, P = 0.4760; ***P = 0.0003; ****P < 0.0001; ns, P = 0.6310; **P = 0.0015; ***P = 0.0005; ns, P = 0.9676; *P = 0.0183; **P = 0.0081; ns, P = 0.9814. (g-h) Flow cytometry gating strategies (g) and representative data (h) of NK cells, ILC in thymi across Groups I to III. All cells are gated on Lin−CD45+ cells. NK, CD56+; ILC1, CD56−CD161+ CD117−CRTH2−; ILC2, CD56−CD161+ CRTH2+; ILC3, CD56−CD161+ CD117+CRTH2−. (i) Statistics of the percentages of ILCs and NK in Lin−CD45+ cells in thymus with aging (n = 7 for Group II; n = 9 for Groups I and III). P values (from left to right): ns, P = 0.5107; ***P = 0.0006; *P = 0.0178; ns, P = 0.9917; *P = 0.0302; ns, P = 0.0570; ns, P = 0.5592; ***P = 0.0003; **P = 0.0084; ns, P = 0.3243; *P = 0.0143; ns, P = 0.3498; ns, P = 0.5336; ***P = 0.0008; *P = 0.0203. All data represent biological replicates. Data are presented as mean ± standard error of mean (s.e.m.). Statistical analysis was performed using Bonferroni-adjusted two-tailed Wilcoxon rank sum test (b) or One-way ANOVA with Tukey’s multiple comparisons test (d, f, i).
