SA-β-Gal stratification reveals senescence-associated heterogeneity and differential cell death susceptibility in human CD8 effector T cells

Introduction Cellular senescence in human T cells remains incompletely defined, particularly with respect to how senescence-associated states influence susceptibility to cell death. Methods We used flow-cytometric SPiDER-based detection of senescence-associated β-galactosidase (SA-β-Gal) activity to profile human CD8+ T-cell subsets from young and old donors. We stratified effector memory (CD8 EM ) and effector memory re-expressing CD45RA (CD8 EMRA ) subsets by the lowest (bottom 25%) and highest (top 25%) quartiles of SA-β-Gal signal. Results CD8 EM and CD8 EMRA cells displayed the greatest within-subset heterogeneity in SA-β-Gal activity. The SA-β-Gal low and high populations occupied distinct regions in UMAP projections and were reproducibly enriched in different FlowSOM metaclusters, indicating that SA-β-Gal activity captures structured phenotypic states within differentiated CD8+ T-cell compartments. These states differed in senescence-associated markers, including CD27, CD28, CD57, and CX3CR1, as well as granzyme-associated programs involving Gzm A, B and K, whereas chronological age had only a limited effect once subsets were analyzed separately. Functionally, nutrient-limited conditions unmasked marked differences in cell death susceptibility between these populations. Following PMA/ionomycin stimulation, SA-β-Gal high CD8 EM and CD8 EMRA cells underwent accelerated stimulation-induced cell death, showed increased caspase-3/7 activation, and exhibited lower post-treatment γH2AX signal relative to SA-β-Gal low counterparts. In contrast, in the absence of stimulation-induced energetic demand, SA-β-Gal high cells displayed greater resistance to cell death. Discussion Together, SA-β-Gal stratification resolves structured heterogeneity within human CD8 effector T cells and supports a context-dependent model in which senescence-associated effector states become selectively vulnerable to stimulation-induced death under metabolic stress.