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. 2024 Apr 15;4(5):727–744. doi: 10.1038/s43587-024-00613-3

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a–c, Beeswarm Milo plots showing the distribution of log₂-transformed fold change in cell abundance with age across neighborhoods of cells in the microenvironment. AdvFB, adventitial fibroblasts; EnFB, endoneurial fibroblasts; PnFB, perineural fibroblasts.d, Co-immunofluorescence of CD3 and laminin on fresh-frozen sections. Bar plot showing number of CD3⁺ cells per field (four young versus six aged donors). Scale bar, 10 µm. P: unpaired two-tailed t-test. *P < 0.05. e, Subset of four markers from a 15-plex RareCyte protein panel indicating proximity between CD4⁺ T cells and CD31⁺ vessels (two young versus two aged donors). Scale bar, 40 µm. f, Co-immunofluorescence of NKG7 and laminin on fresh-frozen sections. Bar plots show the number of NKG7⁺ cells per mm² (three young versus four aged donors). Scale bar, 10 µm. P: unpaired two-tailed t-test. *P < 0.05. g,h, RNAscope (g) and bar plot (h) showing number of LYVE1 cells per field on FFPE sections (two young versus two aged donors). Scale bar, 50 µm. P: one-way ANOVA test. ***P < 0.001. i,j, Co-immunofluorescence of ACTA2 and laminin on fresh-frozen sections (i) and bar plot illustrating proportion of MFs with 0 (none), 1, 2, 3 or more ACTA2⁺ cells surrounding them (j) (three young versus three aged donors). Scale bar, 50 µm. P: unpaired two-tailed t-test. *P < 0.05. k, Dot plot illustrating aging changes of chemokine and interleukin genes. Significant genes were defined based on direction of change, proportion of cells >0.05 and LTSR (significance) > 0.9. l, Co-immunofluorescence of ACTA2 and CCL2 on FFPE sections. Bar plot shows percentage of CCL2⁺ACTA2⁺ cells (two young versus two aged donors). Scale bar, 50 µm. P value: one-way ANOVA test. ***P < 0.001. m, CellPhoneDB analysis of cell–cell interactions mediated via CCL2 produced by various cell types in the microenvironment. Emitter (ligand) cells: leftmost; receiver (receptor) cells: rightmost. FB, fibroblast. All data in d,f,h,j and l are mean ± s.e.m. with individual data points shown. See the Source Data for exact P values.

Source data