Extended Data Fig. 2: Aging-induced decline in TRM is unrelated with cell proliferation, apoptosis and trafficking.

a, Scheme showing mixed young/aged mice-derived splenic CD8⁺ T cells transfer into Rag1^–/– mice for further analysis. b, Flow cytometric analysis of TRM cells in CD8⁺ T cell from lung, liver and kidney of Rag1^–/– mice (n = 4 mice/group). c,d, Flow cytometric analysis of annexin V expression and the percentage of Ki-67⁺ cells in CD8⁺ T cells of Rag1^−/− mice were adoptively transferred with young or aged-derived splenic CD8⁺ T cell and s.c. inoculated with MB49 tumor cells (n = 5 mice/group). e, Experimental scheme of Rag1-KO mice that were s.c. inoculated with MB49 tumor cells, and then intratumor injected with mixed young/aged mice-derived splenic CD8⁺ T cells as indicated. f,g, Flow cytometric analysis of the percentage of TRM/TEM/TCM cells in CD8⁺ T cells from mice as described in E (n = 5 mice/group). h-j, Tumor growth (h), Flow cytometric analysis (i) and the corresponding statistical analysis (j) of the frequencies and absolute number of tumor infiltrating CD8⁺CD69⁺CD103⁺ TRM cells of young/aged mice that were s.c. inoculated with MB49 tumor cells (n = 5 mice/group). k,l, Experimental scheme (k) and tumor growth (l) of Rag1^–/– mice that were transferred with young (2-month-old) and aged (18-month-old)-derived splenic CD8⁺ T cells, and then intratumorally injected with PBS or TRM cells (n = 6 mice/group). The transferred CD8⁺CD69⁺CD103⁺TRM cells were sorted directly from tumor tissue of young mice that were inoculated with MB49 tumor. Bar graphs are presented as mean ± s.e.m. A two-tailed Student’s t-test was performed for comparisons. The data are representative of two (a,b,e-l) or three independent experiments (c, d).

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