Generation of anamnestic responses by CD8 T cells derived from proximal and distal IC-LNs. (A–C) 4 days after immunization of B6 mice transferred with OT-I T cells, proximal and distal IC-LNs were harvested into separate single-cell suspensions, labeled with CFSE, and quantified. A total of 10⁵ OT-I cells from either IC-LN type was retransferred into separate cohorts of naïve B6 mice. 1 day later, spleen and pooled cutaneous LNs from individual mice were analyzed by flow cytometry (n = 3–4, two experiments). (A) Schematic of experimental design. (B) Quantification of the total number of proximal and distal IC-LN–derived OT-I T cells. (C) Quantification of CFSE gMFI and expression of Ki67 by the retransferred OT-I T cells. (D–F) Congenically disparate (CD45.1⁺/CD45.1⁺.2⁺) OT-I T cells were primed in separate B6 recipients, isolated from proximal and distal IC-LNs, and then retransferred at a 1:1 ratio into new CD45.2⁺ B6 hosts, which were either untreated (naïve) or immunized with OVA + CpG in the footpad at the time of retransfer (2′ immunized). 30 days after retransfer, mice were i.v. administered OVA + CpG to elicit recall. 3 days later, pooled LNs, spleen, and lung were harvested for analysis by flow cytometry (n = 5, three experiments). (D) Schematic of experimental design. (E) Frequency of responding transferred OT-I T cells that are either of distal or proximal IC-LN origin. (F) Expression patterns of the indicated cell markers by OT-I T cells isolated from the spleen following recall (n = 5, two experiments). 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 for comparison within the same mice or unpaired for comparisons between mice. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Wilcoxon signed-ranked test was used to compare new ratios of distal to proximal cells to a hypothetical value of 1 (ratio at time of transfer). gMFI, geometric mean fluorescence intensity.