Site-specific generation of heterogeneity in endogenous CD8 T cell responses to immunization. (A–E) B6 mice were immunized in the footpad with OVA + CpG, FTY720 treated on days 2 and 3, and the IC-LNs were analyzed for endogenous CD8 T cell responses using Kb-OVA tetramers (Kb-SIINFEKL) 4 days after immunization (n = 4–5, four experiments). (A) Representative plots demonstrating detection of OVA-tetramer⁺ CD8 T cells across IC-LNs and non-draining LN (ndLN). Cells were first gated on CD3⁺CD8⁺CD4⁻CD44⁺Ki67⁺ live singlets. (B) Quantification of percent and total cell number of activated OVA-tetramer⁺ CD8 T cells across LNs. (C–E) Representative flow plots and quantification of the indicated transcription factor or surface marker expressed by the activated OVA-tetramer⁺ CD8 T cells. (F–H) Mice were immunized with OVA in formulation with alum/LPS, as well as treated with FTY720 starting day 2. 4 days after immunization, the (G) total number of activated OVA-tetramer⁺ CD8 T cells and (H) expression of the indicated markers by OVA-tetramer⁺ cells was quantified (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 < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns).