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. 2022 Jul 1;13:3799. doi: 10.1038/s41467-022-31135-4

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Blood and spleens from young (3 months) and aged (24 months) wild-type mice fed a chow diet were collected. Filipin (a, b) and choleratoxin B (c, d) stainings on blood T cells. a Representative flow cytometry plots of filipin staining on CD4⁺ (p < 0.000001) and CD8⁺ (p < 0.000001) T cells gated as in Supplementary Fig. 1e and (b) quantification. c Representative flow cytometry plots of choleratoxin B staining on CD4⁺ and CD8⁺ (p = 0.00002) T cells gated as in Supplementary Fig. 2c and (d) quantification. b, d n = 10 young and n = 10 aged wild-type mice. e Splenic T cells were isolated and total cholesterol (p = 0.013) was measured by Gas Chromatography – Mass Spectrometry. n = 5 young and n = 6 aged wild-type mice. f Total (p < 0.000001), CD4⁺ (p < 0.000001), and CD8⁺ (p = 0.0026) T cells as a percentage of total blood leukocytes. T_naive (p < 0.000001) and T_mem/eff (p < 0.000001) cells as a percentage of CD4⁺ (g) and T_naive (p < 0.000001), T_mem/eff (p < 0.000001), and T_CM (p = 0^.00016) cells as a percentage of CD8⁺ T cells (h) in blood. f–h n = 10 young and n = 10 aged wild-type mice. i–m Splenic total, CD4⁺, and CD8⁺ T cells were isolated. i–k T cells were labeled with CFSE and stimulated with αCD3/αCD28 beads. CFSE dilution was measured by flow cytometry at 72 h after stimulation. i Representative CFSE dilutions gated as in Supplementary Fig. 11c and quantification of the number of divisions of (j) CD4⁺ (division 0, p = 0.030; 3, p = 0.035; 4, p = 0.023; 5, p = 0.026) and (k) CD8⁺ T cells. n = 4 young and n = 4 aged wild-type mice. l–m CD4⁺ and CD8⁺ T cells were stimulated with αCD3/IL-2 for 12 h with concomitant staining for cleaved caspase 3/7. I Representative flow cytometry plots of cleaved caspase 3/7 in CD4⁺ (p = 0.004) and CD8⁺ (p = 0.009) T cells gated as in Supplementary Fig. 12d and (m) quantification. n = 6 young and n = 6 aged wild-type mice. For all panels, error bars represent SEM. Biologically independent samples were included. p value was determined by unpaired two-tailed Student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.