Site-specific heterogeneity in early CD8 T cell responses to immunization across the lymphatic chain. (A and B) Experimental design as described in Fig. 1, G–I (same data set as Fig. 1, G–I). (A) Representative image depicting T-BET and TCF1 expression by the activated OT-I CD8 T cells. T-BET and TCF1 signal was first masked within activated (IRF4⁺CD45.1⁺) OT-I T cells for visual clarity. Scale bar equals 50 µm, n = 5, three experiments. (B) Representative histo-cytometry plots and quantification of percent T-BET⁺ or TCF1⁺ of activated OT-I T cells. (C–H) CFSE-labeled naïve CD45.1⁺ OT-I cells were transferred into CD45.2⁺ B6 mice, and 1 day later, the mice were immunized with OVA + CpG. Starting 2 days after immunization, mice were treated daily with FTY720. IC-LNs were harvested 3–4 days after immunization for analysis by flow cytometry (n = 4–5, four experiments). (C) Representative flow plots and quantification of mean cellularity of activated (Ki67⁺CD44⁺) OT-I T cells across multiple experiments are shown. Dots represent individual experimental means (n ≥ 3 mice per experimental groups, six experiments). (D) Representative CFSE dilution plot for activated OT-I T cells across IC-LNs. OT-I T cells from PBS-immunized mice were used as a negative control. Summary graph show quantification of Ki67 gMFI within Ki67⁺ OT-I T cells. (E–H) Representative flow plots and quantification showing the indicated surface marker or transcription factor staining and cytokine production after ex vivo restimulation by the activated OT-I T cells or naïve (CD44⁻) endogenous CD8 T cells. (I and J) Mice were adoptively transferred with CD45.1⁺ OT-I CD8 T cells and 1 day later, i.m. immunized with OVA + CpG. Starting 2 days after immunization, mice were treated with FTY720. IC-LNs were harvested 4 days after immunization for analysis by flow cytometry (n = 4 mice, two experiments). (I) Quantification of cellularity of activated OT-I T cells and quantification of Ki67 gMFI within Ki67⁺ OT-I T cells. (J) Representative flow plots and quantification of indicated marker expression and IFNγ production following restimulation by activated OT-I T cells or Ki67⁻ endogenous CD8 T cells. Interconnected dots represent individual lymphatic chains per mouse. Data from multiple pooled experiments are denoted by different symbols within the same group. Graphs show mean ± SD and were analyzed using paired Student’s t test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). gMFI, geometric mean fluorescence intensity.