T cells with effector-memory function selectively accumulate in the CD8 T-cell subset during advanced aging. (A) Representative FCM histograms of TNF-α ICS in splenic T cells on 6-hour in vitro anti-CD3 stimulation. Cells were surface-stained with CD8β and CD4 mAbs, and gates were set on CD4⁺ or CD8⁺ cells as indicated. Background levels of TNF-α–producing cells in nonstimulated control cultures were below detection. A neonate (1 day old), young adult (7 years old), and an aging (23 years old) animal were chosen to illustrate significant age-related changes in TNF-α response, which served as a readout of effector-memory T-cell function. Percentage of TNF-α⁺ cells was ∼10-fold higher in the adult compared with the neonate animal. Although both the adult and the old subject had comparable levels of TNF-α⁺ cells in the CD4 subset, there was an evidently higher percentage of TNF-α⁺ CD8 T cells in the aging animal compared with the young adult animal. (B) Bar graphs represent mean ± SEM values for percentage of TNF-α⁺ cells from animals pooled in 4 age groups, in CD8 (left panel) and CD4 (right panel) subsets as indicated. Statistical significance (P value) was determined by using the ANOVA test. White bar indicates neonate (1 day old); light gray, juvenile (5 years old); dark gray, adult (5-10 years old); and black, old (aged 15 years or more).