Detection of in vivo natural counterpart of Tm2 during recall response. (A-B) On days 9, 16, and 30 after immunization (as described in Figure 6), various cytokine-producing cells in CD44^hi CD62L⁻ CD45.1⁺ CD4⁺ OT-2 cells from splenocytes were analyzed by intracellular staining after ex vivo antigenic restimulation with OVA_323-339. Numbers in the plots indicate the percentage of various cytokine-producing cells among gated CD44^hi CD62L⁻ CD45.1⁺ CD4⁺ OT-2 cells (A) and these are summarized in (B). Each group contained 3 mice. (C) On day 30 after immunization, mice with or without adoptive transfer of diffDCs were rechallenged with 2 × 10⁶ OVA_323-339-loading mature DCs via intravenous injection. The percentage of peripheral blood and splenic CD45.1⁺ CD4⁺ OT-2 memory T cells among total CD4⁺ T cells was assessed by flow cytometry in the mice before and 4 days after rechallenge. Memory CD4 T-cell expansion folds were defined as the ratio of the percentage of CD45.1⁺ CD4⁺ OT-2 memory T cells among total CD4⁺ T cells 4 days after and before rechallenge. Each group contained 5 mice. *P < .05, “OVA/CFA+diffDC” group compared with “OVA/CFA” group. Similar results were obtained in at least 3 independent experiments.