Fig 2: (A) Structure of p(GluNAc) conjugated to an antigen lysyl side chain amine via a self-immolative linker. (B, C) Accumulation of OVA647 or OVA647-p(GluNAc) in the dLNs and livers 15 h after s.c. or i.v. injection. (B) Representative NIR images of dLNs and liver. (C) Average NIR intensities of dLNs and livers. (D, E) Mice were injected with saline or 5 µg of OVA647 or OVA647-p(GluNAc) s.c. and the draining popliteal LNs were isolated and imaged at various timepoints between 1-72 h post-injection n (dLN)= 2 for saline and 4 for OVA647 or OVA647-p(GluNAc) at each timepoint. (D) Representative NIR images of dLNs. (E) Quantification of OVA accumulation in dLNs, expressed as µg (left) or % of initial injected dose (right). (F, G) Flow cytometry analysis of LN cells that took up OVA647-p(GluNAc) 15 h after s.c. injection of 20 µg of OVA647-p(GluNAc). DC, dendritic cell; pDC, plasmacytoid dendritic cell; MAC, macrophage; SCS, subcapsular sinus; med, medullary; TZ MAC, T cell zone macrophage; LEC, lymphatic endothelial cell (CD45-CD31+gp38+); BEC, blood endothelial cell (CD45-CD31+gp38-); FRC, fibroblastic reticular cell (CD45-CD31-gp38+CD21/35-); FDC, follicular dendritic cell (CD45-CD31-gp38+CD21/35+); DNSC, double negative stromal cell (CD45-CD31-gp38-). (F) Percent of each cell subset that is OVA+. (G) OVA647 MFI of each OVA+ cell subset. (H–J) Representative whole mount confocal images of immunostained dLNs after s.c. injection of OVA647-p(GluNAc) (red). (H) LNs stained versus CD11c (green), CD11b (blue) and Collagen IV (white), 8 h post-injection (p.i.). CD11c+CD11b+ DCs are shown in aqua and co-localized OVA-p(GluNAc) is shown in magenta. Scale bar ranges from 20-100 µm and is indicated in each panel. (I) LNs stained versus CD11c (green) and CD8 (blue) 18 h p.i. CD11c+CD8+ double positive DCs are shown in aqua and co-localized OVA-p(GluNAc) is shown in magenta. Scale bar ranges from 5-50 µm and is indicated in each panel. (J) LNs stained versus CD169 (green, top) and Lyve1 (blue, bottom). Co-localized OVA-p(GluNAc) with CD169+ macrophages is shown in yellow (top), and OVA-p(GluNAc) co-localized with the lymphatics is shown in magenta (bottom). Scale bar ranges from 5-50 µm and is indicated in each panel. Data represent mean ± SD. *p = 0.05, **p = 0.01, ***p = 0.001, ns is not significant by one-way ANOVA using Tukey’s post hoc test in (C), and two-way ANOVA using Sidak’s post hoc test in (E).

Fig 3: Macrophages and Batf3+ DCs cooperatively stimulate CD8 T cell response.(A) WT B6 mice were injected with control or clodronate-loaded liposomes. 5 days later, mice were subsequently immunized with Ova and AV. Ova-specific CD8 T cell response at day 7 post-boost in different tissues. *p<0.05, **p<0.01 (t-test). (B and C) Mice got intraperitoneal injection with 400 ng of diphtheria toxin per mouse, and two days later, they were subsequently immunized by Ova plus AV. (B) The depletion of LN-resident macrophages was measured in CD169-DTR mice two days after the DT injection. *p<0.05 (t-test). (C) Ova-specific CD8 T cell responses in WT B6 and CD169-DTR mice at day 7 post-boost. *p<0.05 (t-test). (D) Ova-specific CD8 T cell response in WT B6 and Batf3 KO mice. *p<0.05, **p<0.01 (t-test). Data are representative of two independent experiments (mean and s.e.m.).