Antigen gradients and inflammatory microenvironments across lymphatic chains. (A) Representative plot showing gating of migratory (Mig) and resident (Res) DCs, as well as the quantification of OVA-AF488 uptake 2 h after immunization (n = 4, two experiments). (B) Quantification of OVA-AF488 uptake by migratory and resident DCs 24 h after immunization (n = 3, two experiments). (C) Representative flow plots demonstrating IL-12p40 expression by DCs (n = 4, two experiments). (D and E) Naïve CD45.1⁺ OT-I T cells were transferred into naïve CD45.2⁺ mice, which were 1 day later immunized in the footpad with OVA + CpG. 48 h after immunization, proximal- and distal-draining IC-LNs were harvested for analysis by confocal microscopy (n = 5, two experiments). (D) Representative images of indicated markers across the IC-LNs. Quantification of pS6 and IRF4 expression by the activated (IRF4⁺) OT-I T cells and CXCL9 production within the LN and T cell zone (scale bar equals 40 µm). (E) Representative images of indicated markers across the IC-LNs and quantification of cell–cell spatial correlation analysis of activated (IRF4⁺) CD45.1⁺ OT-I T cells in relation to DC1 (Clec9a⁺SIRPα⁻CD11c⁺CD64⁻ cell objects) and DC2 (SIRPα⁺Clec9a⁻CD11c⁺CD64⁻ cell objects) (scale bar equals 40 µm). Interconnected dots represent individual lymphatic chains per mouse. Panel E shows individual draining LNs and mean. All data were analyzed using paired Student’s t test. ****P < 0.0001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns).