Fig 2: Adjuvant induced antigen presence in draining lymph nodes is affected in immunized low dose clodronate treated mice and CD169 knockout mice. Mean fluorescent intensity (MFI) of antigen (OVA) in draining lymph nodes 24 h post vaccination of either OVA-A594, OVA-A594+PorB, OVA-A594+CpG, or OVA-A594+Alum was measured via ImageJ. Wild type control injections are shown in the bars with no pattern. Low dose clodronate treated animals are shown in the striped bars. CD169 knockout mice are shown in checkered bars. MFI of OVA was quantified by the ImageJ measurement tool after the subtraction of the background. n = 8–13. Statistics were calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig 3: PorB-induced antigen association with dendritic cells (DCs) and follicular dendritic cells (FDCs) is diminished in low dose clodronate treated mice and CD169 knockout mice. These graphs display ovalbumin (OVA)-A594 association with DC and FDC and unassociated OVA within draining lymph node 24 h post subcutaneous immunizations of indicated adjuvants with OVA in WT mice, low dose clodronate mice of CD169 knockout mice. Injection of OVA without adjuvants was used as a control. MFI of OVA=594 associated with DC (A), FDC (B), or unassociated OVA (C) was assessed using the JaCoP plugin within ImageJ and determining the Mander’s correlation coefficient after subtracting the background and using an unsharp mask filter. The correlation coefficient is the percentage of OVA associated with either FDCs or DCs. This percentage was then multiplied by the total MFI of OVA within the lymph node ( Figure 2 ) to determine MFI of OVA associated with DCs or FDCs. Wild type control injections are shown in the bars with no pattern. Low dose clodronate treated animals are shown in the striped bars. CD169 knockout animals are shown in checkered bars. Statistics were calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. n = 5–7 *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 4: Adjuvant induced increase of antigen deposition on follicular dendritic cells (FDCs) is diminished in low dose clodronate treated mice and CD169 knockout mice. Representative immunohistochemistry images from draining lymph nodes from wildtype, clodronate treated mice, and CD169 knockout mice 24 h post subcutaneous immunizations of ovalbumin (OVA) with PorB (A), CpG (B), or Alum (C) and 48 h post clodronate treatment. FDC is shown in green. OVA-A594 is shown in red. Images were taken at 63x objective using a Leica SP5 microscope. Typical areas of co-localization are emphasized with yellow arrows. Scale bar represents 20 µM. One out of six representative experiments is shown. (D) Quantification of colocalization between fluorescently labeled OVA-A594 with FDC within draining lymph nodes 24 h post subcutaneous injections. Colocalization was assessed using Pearson Correlation coefficients calculated within JaCoP plugin in ImageJ after background subtraction and unsharp mask filter. n = 8–12/group. Wild type control injections are shown in the bars with no pattern. Low dose clodronate treated animals are shown in the stripped bars. CD169 knockout animals are shown in checkered bars. Statistics were calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.

Fig 5: Antigen-specific immunoglobulin (IgG) is significantly decreased in immunized low dose clodronate treated mice and CD169 knockout mice (A) Total anti-ovalbumin (OVA)-IgG (B) anti-OVA-IgG1 and (C) anti-OVA-IgG2c levels were measure by enzyme-linked immunosorbent assay (ELISA) following immunizations with PBS, OVA alone, or OVA + PorB, OVA + CpG, or OVA + Alum. Wildtype (open bars), wildtype treated low dose clodronate (striped bars), or CD169 knockout mice (checkered bars) were immunized three times at 2-week intervals. The results shown are from sera collected 2 weeks after third immunizations. n = 7–14 per group. Statistics were calculated by ordinary one-way ANOVA with Sidak’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001 ****p < 0.0001.