Senescence-associated β-galactosidase activity and other markers of senescence are present in human peripheral blood mononuclear 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 whether cellular senescence has a role in immunosenesence remains controversial due to the lack of specific markers to reliably identify these cells in blood. Freshly isolated human peripheral blood mononuclear cells from younger and older donors were fluorescently labeled for senescence-associated β-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 cells with high SA-βGal in the older vs. younger cohort. This increase was apparent in NK cells, CD4+ and CD8+ T cells. CD8+ T cells with high SA-βGal displayed increased transcripts of p21, p16INK4a, and inflammatory cytokines. They also had greater DNA damage response foci and p16INK4a protein. SA-βGal increased as T cells matured, which was consistent regardless of age. Intriguingly, a portion of T effector cells did not exhibit a senescent phenotype and maintained low SA-βGal. RNA sequencing of CD8+ T cells revealed alterations in gene expression were associated with SA-βGal and cells with low, medium, and high SA-βGal shared similar profiles, independent of age. The presence of p16INK4a, DNA damage, and inflammatory cytokines demonstrate that SA-βGal is a strong indicator of cellular senescence. SA-βGal can be leveraged to further identify senescent immune cells and elucidate cellular senescence’s role in immunosenesence. Understanding this connection in healthy aging will further our understanding of premature aging seen chronic diseases.