Abstract
Cellular senescence is an intriguing phenomenon in which cells enter cell cycle arrest and obtain peculiar properties upon encountering genotoxic stressors. Cellular senescence of cultured cells has been widely investigated, but studies on in vivo senescent cells are still scarce. Importantly, no study has provided a panoramic view of in vivo cellular senescence of immune cells. In this study, through single-cell RNA-seq of multiple aged organs, we identified several subsets of immune cells we named “Senescence-Prone Immune Cell Subsets”, which are prone to becoming senescent.
To elucidate the nature of in vivo senescent immune cells, we created a single-cell atlas of senescent immune cells. We conducted single-cell RNA-seq (scRNA-seq) using aged p16-reporter mice (p16-CreERT2-tdTomato mice), which report the expression of p16: a cyclin-dependent kinase inhibitor protein that characterizes senescent cells. We analyzed seven organs: spleen, bone marrow, lymph node, lung, colon, liver, and kidney. Surprisingly, the p16-high cells were specifically enriched in several immune cell types, including plasma cells, γδT cells, ILC1-like NK cells, and M2-like macrophages. Importantly, many of these cell types harbored abundant p16-high cells commonly in multiple organs. We named these subsets collectively as “Senescence-Prone Immune Cell Subsets” (sene-prone immune cell subsets).
To observe how sene-prone subsets respond to organismal aging, we compared aged and young mice and analyzed the dynamic alterations of the sene-prone subsets. Although the abundance of p16-high cells within each subset during aging was case-dependent, all the sene-prone subsets similarly expanded relative to the counterpart population in aged mice across multiple organs. Additionally, to elucidate the effect of the immune environment on this expansion, we performed cell transfer experiments, in which CD45.1+ immune cells were transferred intravenously to young or aged recipient mice and then re-isolated one month later for FACS analysis. In cells transferred to aged mice, sene-prone subsets were enriched. Hence, the aged environment favors proliferation and/or survival of sene-prone subsets.
Next, to confirm that sene-prone subsets are actually prone to cellular senescence, we stimulated sene-prone cell types and non-prone counterpart cell types in vitro. Immune stimulation (e.g. TCR stimulation by anti-CD3/CD28 antibodies in γδT vs. αβT) induced p16 expression in sene-prone cell types, but not in non-prone cell types. Thus, these cell subsets are more likely to become p16-high due to their cell-intrinsic nature and age-related chronic inflammatory environment.
To clarify the biology behind the senescence-proneness in these sene-prone subsets, we conducted bulk RNA-seq analyses comparing sene-prone vs. non-prone immune cell types. We found that ER stress response pathway was overall enhanced in sene-prone subsets in all comparisons. This phenomenon was accompanied by higher mitochondrial quantity in some cell types, which implied increased reactive oxygen species production contributing to the intrinsic difference in these sene-prone subsets.
In summary, we identified groups of immune cells with a new feature named “Senescence-Prone Immune Cell Subsets”, which are more prone to becoming p16-high senescent cells compared with the counterpart populations. These cell types showed a proportional increase in aged mice compared with young mice, and this expansion was driven by external factors from the aged environment and the internal characteristics of these subsets. The general phenotype of these subsets was newly identified related to immune aging, which may have physiological significance in age-related diseases including cancer and autoimmunity.
