Generation of antigen and inflammation gradients across interconnected lymphatic chains. (A) B6 mice were injected with Evans blue in the footpad, and 30 min later, LNs were harvested to assess drainage (n = 3, three experiments). (B) 2 h after footpad immunization with OVA-AF488 in formulation with CpG, IC-LNs and non-draining contralateral inguinal LNs (ndLN) were harvested to assess antigen uptake by DCs, defined as CD11c⁺MHC2⁺CD64⁻B220⁻Dump^neg (CD3, NK1.1, Ly6G, and Ter119) cells (n = 4, three experiments). (C–F) IC-LNs were harvested 24 h after immunization (n = 5, two experiments). (C) Quantification of antigen uptake by DC1 (XCR1⁺CD11b⁻ DCs) and DC2 (CD11b⁺XCR1⁻ DCs) 24 h after immunization. (D) Representative concatenated flow plots and quantifications of CD80 versus CD86 expression by migratory (Mig) and resident (Res) DCs. (E) Quantification of CD80/86 expression and IL-12p40 production by DC1 and DC2 24 h after immunization. (F) Representative flow plots and quantification of monocytes across IC-LNs. Monocytes were defined as Ly6C⁺CD64⁺B220⁻Dump^neg cells. Quantification of percent monocytes, which are IL12p40⁺. (G–I) Naïve CD45.1⁺ OT-I were transferred into naïve CD45.2⁺ mice, which were 1 day later immunized in the footpad with OVA + CpG. 2 days after immunization, proximal and distal IC-LNs were harvested for analysis by confocal microscopy (n = 5, three experiments). (G) Quantification of monocyte representation in the imaged proximal and distal IC-LN tissue sections. (H) Representative images of the indicated markers across the IC-LNs (scale bar equals 50 µm). (I) Spatial distribution maps demonstrating the positioning of indicated myeloid cells and activated (IRF4⁺) CD45.1⁺ OT-I T cells. (J) Quantification of cell-cell spatial correlations of activated OT-I CD8 T cells with the indicated myeloid cell populations across different IC-LNs. Interconnected dots represent individual lymphatic chains per mouse. 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).