1). 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. studies have shown that targeting of antigen to cell surface receptors on APC through conjugation to anti-receptor monoclonal antibodies (mAb), can promote antibody responses to low doses of antigen, often in the absence of additional adjuvant.12C15 The x/CD11c integrin subunit is expressed on all conventional DC subsets in mice as a component of complement receptor 4 (CR4; CD11c/CD18).16C18 A number of studies19C22 have shown that targeting antigen to CD11c through conjugation to the hamster anti-mouse CD11c mAb, N418, can promote rapid, high-titre antibody responses Amebocyte Lysate test kit (Pyrotell; AMS Biotechnology Ltd, Abingdon, UK). All conjugates were endotoxin low ( 05 ng endotoxin/mg of conjugate). Immunization Mice were immunized as detailed for L-Ornithine individual experiments. Unless otherwise stated, immunization was i.v. using the tail vein in a total volume of 200 l saline. For immunization with complete Freund’s adjuvant (CFA; BD Biosciences), antigen was added to a 50% CFA/50% saline solution, emulsified and 2 100 l was injected subcutaneously (s.c.) into the left and right flanks. ELISA Serum antibody titres were determined by ELISA. For anti-IgG responses, 96-well plates were coated with monoclonal IgGs of appropriate species/subclass, incubated with serially diluted serum samples and developed using horseradish peroxidase (HRP) -conjugated rat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, Newmarket, UK) and 005. Results Targeting antigen to CR4 promotes uniquely rapid, high titre antibody responses First we compared the ability of a panel of rat and hamster mAb directed against a series of APC receptors to stimulate primary anti-rat and anti-hamster antibody responses in mice after injection of a single 25-g dose. Results revealed uniquely rapid and large responses after targeting CR4 (Fig. 1). Hamster anti-CD11c (N418), rat anti-CD11c or rat anti-CD18 produced anti-hamster and anti-rat titres as high as 1 : 100 000 (mean 1 : 24 000, 1 : 17 000 and 1 : 25 000, respectively) by day 7 that remained high for at least 28 days (Fig. 1). In contrast, control, non-targeted IgG produced minimal detectable responses. Titres against mAb directed L-Ornithine against all other APC receptors were 10-fold lower than for anti-CR4 L-Ornithine at day 7 (Fig. 1 top; 0001 in each case) and, with the exception of anti-CD40, were still significantly lower at 28 days (Fig. 1 bottom). Open in a separate window Figure 1 Targeting to CR4 induces high titre, rapid antibody responses. Mice were immunized intravenously with 25 g 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 28C35 days later. Results for individual animals are shown. Bars represent mean values. For day 7 titres * 0001 versus **. For day 28C35 titres, * 001 and ** 00001 versus CD11c (Ham) and CD18. Antibody responses to [FabOVA] conjugates We next compared the ability of several of the different mAb to stimulate antibody responses against a covalently linked heterologous antigen. For this, [FabOVA] conjugates were used. Each conjugate consisted of the model antigen, OVA, chemically L-Ornithine linked to three anti-receptor Fab fragments. Ace2 This approach allowed a comparison of how the response to a single protein antigen was influenced by targeting different DC receptors in L-Ornithine the absence of any influence of Fc : Fc receptor interactions. Injection of 25 g [anti-CD11cOVA] induced the highest anti-OVA titres.