Ligation of CD11c during vaccination promotes germinal centre induction and robust humoral responses without adjuvant

Abstract

In this study, we investigated the mouse dendritic cell (DC) receptor, complement receptor 4 (CR4; CD11c/CD18), as an immunotarget for triggering humoral immunity. Comparison of antibody titres generated against a panel of 13 anti-antigen-presenting cell receptor monoclonal antibodies, with or without conjugated ovalbumin (OVA), revealed uniquely rapid and robust responses following CR4 targeting, with antibody titres approaching 1 : 100 000 7 days after a single dose of antigen. Furthermore, using just 100 ng OVA conjugated to anti-CD11c Fab', we generated anti-OVA titres greater than those produced by a 100-fold higher dose of OVA in complete Freund's adjuvant at day 28. These anti-OVA antibody titres were sustained and could be boosted further with targeted OVA on day 21. Investigations to explain this vaccine potency showed that, in addition to targeting splenic DC, anti-CDl1c antibodies delivered a powerful adjuvant effect and could boost humoral immunity against OVA even when the OVA was targeted to other molecules on DC, such as major histocompatibility complex class II, CD11a and CD11b. However, interestingly, this adjuvant effect was lost if OVA was targeted to other cells such as B cells via CD21 or CD19. The adjuvant effect was mediated through a marked enhancement of both germinal centre and extrafollicular plasma cell formation in responding spleens. These results demonstrate that anti-CD11c monoclonal antibody can both target antigen and act as a powerful adjuvant for rapid and sustained antibody responses. They also point to an interesting role for CR4 on DC in triggering B cells during humoral immunity.

Figure 1

Targeting to CR4 induces high titre, rapid antibody responses. Mice were immunized intravenously with 2·5 μg immunoglobulin G (IgG) directed against the indicated antigen-presenting cell surface molecules. All IgG were rat unless indicated as hamster (Ham). Control IgG were raised against the BCL₁ idiotype. Serum anti-rat or anti-hamster antibody titres were determined 7 and 28–35 days later. Results for individual animals are shown. Bars represent mean values. For day 7 titres *P < 0·001 versus **. For day 28–35 titres, *P < 0·01 and **P < 0·0001 versus CD11c (Ham) and CD18.

Figure 2

Targeting to CR4 can induce high-titre antibody responses against covalently associated antigen. (a) Mice were immunized intravenously with 2·5 μg of [Fab′×OVA] directed against the indicated receptors. Serum titres of anti-ovalbumin (OVA) immunoglobulin G (IgG) were determined 7 days later. Results for individual animals are plotted and are typical of more than 10 independent experiments. Bars represent mean. *P < 0·001 versus **. (b) Duplicate mice were immunized with 2·5 μg [anti-CD11c×OVA] by four different routes, as indicated. Serum anti-OVA IgG titres were determined at the indicated time-points and are plotted as mean and range. Results represent one of two independent experiments. (c) Bone-marrow-derived dendritic cells were incubated with different concentrations of the indicated [Fab′×OVA] conjugates. Binding was assessed by flow cytometry using fluorescein isothiocyanate-conjugated rabbit anti-OVA for detection. Results represent mean fluorescence intensity (MFI)/maximal MFI × 100 and show mean and range for duplicate samples; data from one of three independent experiments.

Figure 3

Targeting to CD11c increases the sensitivity of antibody responses greater than 100-fold compared with complete Freund's adjuvant (CFA). (a) Mice were immunized with various doses of ovalbumin (OVA) administered intravenously as soluble protein or as [Fab′×OVA] conjugate targeted to the indicated receptors, or subcutaneously as an OVA/CFA emulsion. Serum anti-OVA immunoglobulin G (IgG) titres were determined on day 7 for mice immunized with [Fab′×OVA] or soluble OVA, or on day 28 for mice immunized with OVA/CFA. Individual points represent data (mean and range) for two to five mice. (b) Mice were immunized on day 0 with 2·5 μg [anti-CD11c×OVA] then boosted (triangles) or not (squares) on day 21 (arrow) with a further 2·5 μg [anti-CD11c×OVA]. Serum anti-OVA IgG titres were determined at the indicated time-points and are presented as mean and range for two mice. Results are representative of three independent experiments. (c) C3H/HeN [wild-type (WT)] and C3H/HeJ [that have a non-functional toll-like receptor 4; knockout (KO)] mice were immunized with 2·5 μg [anti-CD11c×OVA]. Results for individual animals are plotted and represent one of two independent experiments. Bars represent mean. Non-targeted [Fab′×OVA] produced anti-OVA titres of 50 in both mouse strains.

Figure 4

Differential targeting of antigen-presenting cells by anti-CD11c, anti-MHCII and anti-CD21 [Fab′×OVA]. (a,b) Mice were injected intravenously with 10 μg [Fab′×OVA] against the indicated receptors. Two and 24 hr later, spleens were removed and analysed by immunofluorescence microscopy with antibodies against ovalbumin (OVA) and the indicated cell markers. Inset is an enlarged image of the boxed area to show co-localization of CD19 and OVA. (c) Spleens from mice immunized with non-targeted [Fab′×OVA] (Control) or [anti-CD11c×OVA] were analysed by flow cytometry 2 hr after injection using fluorescein isothiocyanate-conjugated anti-OVA. Numbers show % of cells that were OVA⁺. (d) OVA⁺ cells from mice immunized with [anti-CD11c×OVA] in (c) were stained with anti-CD11b, anti-CD8 and anti-MHCII as indicated and analysed by flow cytometry. Results represent one of four experiments.

Figure 5

Anti-CD11c promotes antibody responses to dendritic cell-targeted antigen. (a) Day 7 serum anti-ovalbumin (OVA) immunoglobulin G (IgG) levels in mice immunized with 2·5 μg [Fab′×OVA] against the indicated receptors in the absence or presence of 2·5 μg N418 IgG. Results for individual animals with each conjugate are shown and are typical of > 10 experiments; P values for individual conjugates are shown. NS = no significant difference. (b) Mice were immunized with 2·5 μg [anti-CD11a×OVA] in the absence or presence of a 2·5 μg dose of the indicated antibody. HL3 = hamster anti-CD11c, 223H7 = rat anti-CD11c, Fab′₂ = N418. Day 7 serum anti-OVA concentrations for individual animals are shown. With the exception of HL3, where P = 0·065, P < 0·05 for all conditions versus control. Similar results were obtained for MHCII-targeted OVA (not shown).

Figure 6

Targeting CD11c induces extensive germinal centre (GC) responses. (a) C57BL/6 mice were immunized intravenously with 2·5 μg [Fab′×OVA] either non-targeted (Irr) or targeted to CD11c. Seven days later spleens were isolated and sectioned for immunohistology. (a) Sections were stained for GC using peanut agglutinin (PNA; blue) and anti-immunoglobulin D (IgD) (brown) to identify B-cell follicles. The graph shows the percentage of spleen section that is GC for all immunized mice (P < 0·01 for Irr versus CD11c). (b) Serial spleen sections were stained for ovalbumin (OVA) -binding cells (blue) and either IgM (brown; top panels) or IgG1 (brown; bottom panels). Cells specific for OVA and isotype develop as black or blue/black cells. The top graph shows the frequency/mm² of OVA-specific cells from both groups of mice and the bottom graph shows the percentage OVA-specific cells that were IgG1⁺. F = follicle. P < 0·01 for Irr versus CD11c in each case.