FIGURE 2.
CD8+ T cells with high fSA‐βGal signal intensities are senescent. (a) Representative dot plot illustrating gates used to sort and isolate CD8+ T cells by FACS from a donor in their 20s and 60s, as indicated, based on low, inter(med)iate, and high fSA‐βGal signal intensities. (b) Representative Cell Trace Violet histograms of CD8+ T cells, sorted as in (a), following anti‐CD3 and anti‐CD28 stimulation for 5 days from a donor in their 20s and 60s as indicated. Bar graph, quantification of more than two cell divisions of CD8+ T cells from 6 healthy donors (3 donors in their 20s, 3 donors in their 60s) following anti‐CD3 and anti‐CD8 stimulation for 5 days. *p = 0.0002; **p < 0.0001. (c) RT‐qPCR expression profiles for indicated senescence‐associated genes in CD8+ T cells at indicated fSA‐ßGal levels in donors in their 20s (blue) and donors in their 60s (red). Average and SEM of five (CDKN1A and CDKN2A) and three (IL6) independent experiments. Statistical significance was determined by a two‐tailed unpaired t test, with Y‐low as the reference value. *p < 0.05; **p < 0.001; Y: younger donors in their 20s, O: Older donors in their 60s. (d) Immunofluorescence analysis of sorted CD8+ T cells from a donor in their 20s and 60s, as indicated, using antibodies against 53BP1 (green) and p16INK4a (red). Outline of cell nuclei is indicated with white border. Scale bar: 10 μm. (e) Quantification of the percentage of CD8+ T cells positive for 53BP1 foci at each fSA‐ßGal level in younger (n = 8) and older donors (n = 8) combined. Whiskers depict mean ± SEM. Statistical significance was determined by a one‐way ANOVA. **p = 0.001. (f) Sorted CD8+ T cells with high fSA‐βGal signal intensities were simultaneously immunostained using antibodies against 53BP1 (green) and analyzed by FISH to detect telomeres (red). Blue: DAPI. Enlarged versions of the numbered DNA damage foci showing co‐localization with telomeres are shown in the right micrographs. Scale bar: 10 μm. (g) Quantification of mean TIF per cell in sorted CD8+ T cells, as indicated, in donors in their 20s (n = 5) and 60s (n = 5) combined. Whiskers depict mean ± SEM. Statistical significance was calculated by a one‐way ANOVA. *p = 0.0280