Figure 3.
Figure 3. CD180 targeting induces affinity maturation, EF responses, GC formation, and immunological memory. (A) Sera from WT (C57BL/6) or CD40 KO mice immunized with 100 µg NP-αCD180 or 10 µg NP-αDCIR2 were analyzed for affinity to NP on days 5 and 7 p.i. (B) WT mice were inoculated with 100 µg NP-αCD180 alone or with the indicated adjuvant (50 µg CpG-A, 50 µg CpG-B, 20 µg R848, or 4 µg LPS) and then bled at days 7 and 28; sera were analyzed for affinity against NP. Controls included mice inoculated with NP-iso, NP-iso + alum, or NP-αDCIR2. (C) WT mice were inoculated with 50 µg NP-αCD180 alone or with the indicated adjuvants as in B and bled at days 7, 14, 21 and 28; sera were analyzed for levels of NP-specific IgM (left) or IgG (right) Abs. Data are presented as mean ± SEM. A representative experiment of three experiments each for A–C is shown. (D–F) 2 × 105 NP-binding B cells from Ly5.1+ B1-8hi mice were adoptively transferred to Ly5.2+ WT recipients on day −1. On day 0, the mice were inoculated with 100 µg of either NP-αCD180 or NP-isotype, and spleens were harvested at either day 4 or 7 for flow cytometric analysis. The number of NP-specific B220+ B cells (D) and NP-specific PNA+GL7+ GC B cells (E) per spleen were determined by sequential gating on Ly5.1+ NP-binding B cells. (F) The number of B220lo CD138+ AFCs per spleen is plotted. Data are representative of two experiments for D–F. (G) 8-µM frozen spleen sections from mice immunized with NP-CGG + alum or NP-αCD180 either 4 or 7 d previously were stained with anti-B220-eFluor450 (blue), PNA-FITC (red), and NP-PE (green). Data are representative of multiple sections analyzed from two to three mice per time point. Arrows indicate GCs. Bars, 100 µM. (H) Mean number of PNA+ GCs per follicle from mice in G (day 7 time point). Each dot represents one animal from which four individual sections were analyzed. Data in G and H depict a representative experiment of two independent experiments. (I) WT and CD40 KO mice were primed as indicated, rested for 10 wk, and then given a secondary challenge i.p. with 20 µg Ag or PBS. Spleens were harvested at day 4 after boost and analyzed for NP-specific AFCs by ELISPOT. The combined results from two independent experiments using three mice/group are shown. Each dot represents an individual animal. Horizontal bars indicate mean. Statistical values compare groups with mice primed with NP-αCD180 and challenged with NP-isotype. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

CD180 targeting induces affinity maturation, EF responses, GC formation, and immunological memory. (A) Sera from WT (C57BL/6) or CD40 KO mice immunized with 100 µg NP-αCD180 or 10 µg NP-αDCIR2 were analyzed for affinity to NP on days 5 and 7 p.i. (B) WT mice were inoculated with 100 µg NP-αCD180 alone or with the indicated adjuvant (50 µg CpG-A, 50 µg CpG-B, 20 µg R848, or 4 µg LPS) and then bled at days 7 and 28; sera were analyzed for affinity against NP. Controls included mice inoculated with NP-iso, NP-iso + alum, or NP-αDCIR2. (C) WT mice were inoculated with 50 µg NP-αCD180 alone or with the indicated adjuvants as in B and bled at days 7, 14, 21 and 28; sera were analyzed for levels of NP-specific IgM (left) or IgG (right) Abs. Data are presented as mean ± SEM. A representative experiment of three experiments each for A–C is shown. (D–F) 2 × 105 NP-binding B cells from Ly5.1+ B1-8hi mice were adoptively transferred to Ly5.2+ WT recipients on day −1. On day 0, the mice were inoculated with 100 µg of either NP-αCD180 or NP-isotype, and spleens were harvested at either day 4 or 7 for flow cytometric analysis. The number of NP-specific B220+ B cells (D) and NP-specific PNA+GL7+ GC B cells (E) per spleen were determined by sequential gating on Ly5.1+ NP-binding B cells. (F) The number of B220lo CD138+ AFCs per spleen is plotted. Data are representative of two experiments for D–F. (G) 8-µM frozen spleen sections from mice immunized with NP-CGG + alum or NP-αCD180 either 4 or 7 d previously were stained with anti-B220-eFluor450 (blue), PNA-FITC (red), and NP-PE (green). Data are representative of multiple sections analyzed from two to three mice per time point. Arrows indicate GCs. Bars, 100 µM. (H) Mean number of PNA+ GCs per follicle from mice in G (day 7 time point). Each dot represents one animal from which four individual sections were analyzed. Data in G and H depict a representative experiment of two independent experiments. (I) WT and CD40 KO mice were primed as indicated, rested for 10 wk, and then given a secondary challenge i.p. with 20 µg Ag or PBS. Spleens were harvested at day 4 after boost and analyzed for NP-specific AFCs by ELISPOT. The combined results from two independent experiments using three mice/group are shown. Each dot represents an individual animal. Horizontal bars indicate mean. Statistical values compare groups with mice primed with NP-αCD180 and challenged with NP-isotype. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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