Senescence-associated β-galactosidase activity and other markers of senescence are present in human peripheral blood CD8+ cells during healthy aging

Abstract

Abstract Aging is associated with a decline in the immune system, termed “immunosenescence”. Cellular senescence is defined by persistent cellular growth arrest and overall loss of function. If immune cells undergo cellular senescence in vivo, and if it has a role in immunosenesence, remains controversial due to the lack of specific markers to identify these cells. Human CD8+ T cells from younger and older donors were isolated from blood and labeled with a fluorescent substrate for senescence-associated b-galactosidase (SA-βGal). CD8+ T cells were sorted based on SA-βGal and analyzed by qRT-PCR, RNA sequencing, and microscopy. We found a higher percentage of CD8+ T cells with high SA-βGal activity in the older cohort. SA-βGal high CD8+ T cells displayed increased p21, p16INK4a, and inflammatory cytokine transcripts. They also had greater DNA damage response foci and p16INK4a protein and had deficient proliferation. SA-βGal increased as T cells matured, which was consistent regardless of age. Intriguingly, a portion of effector cells did not exhibit a senescent phenotype and maintained low SA-βGal. RNA sequencing of CD8+ T cells revealed cells with low, medium, and high SA-βGal had distinct profiles. In addition, SA-βGal high CD8+ T cells had larger mitochondrial mass than cells with low SA-βGal as measured by MitoID staining. This may indicate differences in metabolism, which may contribute to senescence. The presence of p16INK4a, DNA damage, and inflammatory cytokines and deficient proliferation demonstrate that SA-βGal is a strong indicator of cellular senescence in CD8+. T cells. SA-βGal can be leveraged to further identify senescent immune cells and elucidate cellular senescence’s role in immunosenesence.