Heterogeneity in early CD4 and CD8 T cell responses across the lymphatic chain. (A–E) Mice were adoptively transferred with CFSE-labeled CD45.1⁺ OT-II CD4 T cells, and 1 day later, immunized with OVA + CpG. Starting 2 days after immunization, mice were also treated with FTY720. IC-LNs were harvested 4 days after immunization for analysis by flow cytometry. (n = 3–5, four experiments). (A) Cellular frequencies and representative CFSE dilution plots for CD44⁺ OT-II CD4 T cells are shown. (B–E) Expression pattern of the indicated transcription factors and surface markers by the activated OT-II CD4 T cells. (F) Expression levels of β7 and CCR9 by the activated OT-I CD8 T cells across IC-LNs following immunization, also compared with the endogenous CD44⁺ CD8 T cells isolated from mesenteric LNs (n = 4–5, two experiments). (G and H) Analysis of cellularity and Ki67 and T-BET expression, as well as IFNγ production following ex vivo restimulation for CD44⁺ OT-I T cells 4 days after footpad immunization with either 1 or 0.1 µg OVA in formulation with 20 µg CpG (n = 5, two experiments). Interconnected dots represent individual lymphatic chains per mouse. Data from multiple pooled experiments are denoted by different symbols within the same group. Data were analyzed using paired Student’s t test. ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns).