Representative plots of CD4+ T-cells bound to (A and B) E641:I-Ab, (C and D) OVA:I-Ab, and (E and F) MCC:I-Ek tetramers in adult (top) and old (bottom) mice. Absolute number of CD4+ CD44lo (left Y-axis) and CD4+ CD44hi (right Y-axis) T-cells bound to (G) E641:I-Ab, (H) OVA:I-Ab, or (I) MCC:I-Ek tetramers only enumerated after dump tetramer analysis (‘Materials and methods’), or those binding (J) E641:I-Ab + OVA:I-Ab, (K) OVA:I-Ab + MCC:I-Ek, or (L) E64:I-Ab + MCC:I-Ek tetramers in combination enumerated after both dump and two-color tetramer analysis (‘Materials and methods’). Bars indicate median (*p < 0.05, **p < 0.005, ***p < 0.0001, ns = non-significant; Mann–Whitney). Fold change (Δ) in means between adult and old is shown. Results are from three experiments with 4 mice/group.