Differential antigen dose requirements for early CD8⁺ and CD4⁺ T cell activation. (A and B) Mice were injected with ∼0.75 × 10⁶ OT-I and OT-II T cells and 1 d later immunized with the indicated doses of OVA. 12 h later, draining LNs were examined for the expression of CD69 and CD62L on transferred T cells by flow cytometry (A), with the percentage activated T cells (CD69⁺CD62L⁻) quantified across individual animals (n = 4; B). (C) The same experimental procedure as in Fig. 3 A, examining the relative OT-I and OT-II T cell cluster formation after low-dose (2 µg) OVA immunization. Data were normalized to T cell cluster formation after high-dose (10 µg) OVA immunization. (D) Extended dataset of experiment presented in Fig. 3, examining the relative distribution of OT-I and OT-II T cell clusters with respect to distance to the nearest LS after low-dose (2 µg) or high-dose (10 µg) antigen administration. Error bars are mean ± SEM. (E and F) Mice were injected with ∼0.5 × 10⁶ P14 and Smarta T cells and immunized with the indicated doses of GP. 12 h after immunization, draining LNs were examined for expression of CD69 and CD62L on transferred T cells by flow cytometry (E), with the percentage activated T cells quantified across different animals (F). Data represent at least two independent experiments. Numbers in flow plots represent frequencies. Error bars are mean ± SD, unless otherwise indicated. ***, P ≤ 0.001; *, P ≤ 0.05.