(A) Outline for mixed AT/rechallenge experiments. Unless noted otherwise, CD8⁺ T cell–enriched populations containing 2 × 10³ congenic young (Y) and old (O) D^bNP₃₉₆⁺CD8⁺ T_M each were transferred i.v. into congenic recipients prior to i.p. LCMV challenge and analysis of II° CD8⁺ T_E expansions on day 8. (B) Relative frequencies of II° D^bNP₃₉₆⁺CD8⁺ T_E among all donor CD8⁺ T cells; donor age (time after I° LCMV challenge), factor of differential II° expansion and statistical significance are indicated. (C) Respective frequencies of NP₃₉₆-specific II° CD8⁺ T_E in young and old CD8⁺ donor T cell compartments as assessed by tetramer or IFN-γ staining. (D) Mixed AT/rechallenge experiments were conducted with CFSE-labeled donor cells to calculate proliferation indices; corresponding data points from individual recipients are connected by a line. (E) Expansion kinetics of II° CD8⁺ T_E in peripheral blood (dot plots gated on all donor CD8⁺ T cells). (F–H) AT/rechallenge experiments were performed with CFSE-labeled p14⁺ T_M. (I) II° D^bNP₃₉₆⁺CD8⁺ T_E responses analyzed on day 5. To visualize earlier stages of the II° response in panels D–I, escalating numbers of CD8⁺ T cell–enriched donor populations were transferred containing (a) 2 × 10⁵, (b) 4 × 10⁴ or (c) 5 × 10³ young and old D^bNP₃₉₆⁺CD8⁺ T_M each, or (d) 1 × 10⁶ p14⁺ T_M. Data are the mean ± 1 SEM or feature individual data points with n ≥ 3 mice for multiple independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 by 1 way ANOVA or Student’s t test. BM, bone marrow; MedLN, mediastinal lymph nodes; MLN, mesenteric lymph nodes; PBMC, peripheral blood mononuclear cells; PC, peritoneal cavity.