The association of CD28^– T-cell phenotype with in vitro effector-memory functional response differs between CD4 and CD8 subsets. (A) Representative FCM dot plots of ICS for TNF-α in CD28^– versus CD28⁺ populations. The percentages of total CD4 or CD8 T cells with CD28^– and CD28⁺ phenotype are given in parentheses, followed by the proportion of the respective population that is positive for TNF-α. In both young adult (6 years old) and old (23 years old) animals, there was a larger proportion of TNF-α⁺ effector-memory cells within CD28^– than CD28⁺ population. Within the CD4⁺CD28^– T cells, the distribution of TNF-α⁺ cells was comparable between the adult and the old animal; by contrast, in CD8⁺CD28^– T cells, the proportion of TNF-α⁺ cells was significantly higher in the old animal. This observation held for the entire cohort, as shown in panel B. (B) Bar graphs represent mean ± SEM values for percentages of TNF-α⁺ T cells with CD28^– or CD28⁺ phenotype from animals pooled in 4 age groups. Statistical significance (P value) was determined as in Figure 3. Shading key is the same as in Figure 3.