Fig. 3: Accumulation of senescent immune cells in aged SLOs.

A Left Panel: tSNE plot illustrating subclusters of cell subsets in young and old LNs; Right Panel: Density distribution plot of aging scores. B Flow cytometric analysis of p21 and p16 levels in CD45⁺ leucocyte populations in young and old LNs (n = 5 mice/group). C Histological sections of LNs with immunostaining for B cells (B220, green) and measurement of p21 MFI of B220⁺ B cell area in young and old LNs. Scale bars represent 20 μm (n = 5 mice/group). D Histological sections of LNs with immunostaining for T cells (CD3, red) and measurement of p21 MFI of CD3⁺ T cell area in young and old LNs. Scale bars represent 20 μm (n = 5 mice/group). E Flow cytometric analysis of ROS levels in CD19⁺ B cell and CD3⁺ T cell populations in young and old LNs (n = 5 mice/group). F Flow cytometric analysis of p21 levels in naïve CD4⁺ T cells, naïve CD8⁺ T cells, CD4⁺ CTLs, CD8⁺ CTLs, Tex cells, Tfh cells and Th1 cells populations in young and old LNs (n = 5 mice/group). G Flow cytometric analysis of p21 and p16 levels in CD45⁺ leucocyte populations in young and old spleen (n = 5 mice/group). H Histological sections of spleen with immunostaining for B cells (B220, green) and measurement of p21 MFI of B220⁺ B cell area in young and old spleen. Scale bars represent 10 μm (n = 5 mice/group). I Histological sections of spleen with immunostaining for T cells (CD3, red) and measurement of p21 MFI of CD3⁺ T cell area. Scale bars represent 10 μm (n = 5 mice/group). J Flow cytometric analysis of ROS levels in B cell and T cell populations in young and old spleen (n = 5 mice/group). K Flow cytometric analysis of p21 levels in naïve CD4⁺ T cells, naïve CD8⁺ T cells, Treg cells, Tfh cells and Tex cells in young and old spleen (n = 5 mice/group). P-values were calculated between two groups using an unpaired t-test. ns not significant, *P < 0.05; **P < 0.01; ***P < 0.001. BC B cell, TC T cell, NK natural killer cell, cDC classical dendritic cell, Mono monocyte, Macro macrophage, pDC plasmacytoid dendritic cell, Neu neutrophil.