This would indicate that macrophages were the main cells targeted by QS-21 within the first hours post-injection. robust antigen-specific cellular and humoral adaptive responses (reviewed in Gar?on that is important for CD8 T cells and IgG2c antibody responses18. The goal of this study was to identify mechanisms involved in the activation of the immune system by the QS-21 component of AS01. We identified CD169+ resident macrophages of the lymph node draining the injection site as the main cells targeted by QS-21 when formulated into liposomes. Results QS-21 incorporated in liposomes induces robust adaptive immune responses to co-administered antigens QS-21 was incorporated into cholesterol-containing liposomes, similarly to the clinical AS01 formulation9. To assess the adjuvanticity of this QS-21 formulation, mice were immunised intramuscularly against two Rabbit polyclonal to AGBL2 model antigens (Ag): HBs, a clinically relevant Ag used in 5-BrdU the were further analysed by confocal microscopy. The draining (iliac) lymph nodes were recovered 30?min and 3?h following injection and visualised by confocal microscopy. Bodipy-labelled QS-21 accumulated in the subcapsular sinus (SCS) as early as 30?min following injection (Fig. 3A) and colocalised at both time points with CD11b+ CD169+ cells, which are macrophages known for their ability to capture lymph-borne particles21. As previously demonstrated in the context of lymph-borne infections22,23, immunisation with QS-21 led to a rapid loss of lymph node macrophages, the CD11b+ CD169+ F4/80? SCS subpopulation being more affected than CD11b+ CD169+ F4/80+ medullary sinus macrophages (Fig. 3B,C). Open in a separate window Figure 3 QS-21-bodipy rapidly accumulates in the draining lymph node where it colocalises with CD11b+ CD169+ macrophages of the subcapsular sinus.(A) Mice were injected i.m. with liposomes containing bodipy-labelled QS-21. The draining lymph nodes were recovered 30?min and 3?h post injection and stained with anti-CD11b, anti-CD169 and anti-B220 antibodies and analysed by confocal microscopy. (B) Flow cytometry analysis of subcapsular sinus (CD169+ F4/80?) and medullary sinus (CD169+ F4/80+) macrophages in mice 24?h following PBS or QS-21 injection. (C) Quantification of the loss of LN-resident macrophages detected by flow cytometry. Results from two independent experiments. Each point represents one 5-BrdU mouse. Statistical significance was determined by a non-parametric Mann-Whitney test. Lymph Node Resident 5-BrdU macrophages are critical for innate and effector responses to antigens adjuvanted 5-BrdU with QS-21 LN-resident macrophages have been shown to initiate the response to and limit the dissemination of various pathogens and particles24,25,26. To determine the role of resident macrophages in the response to QS-21, these cells were depleted by i.m. injection of clodronate-containing liposomes (CL)27 6 days prior to the first immunisation. Mice were immunised following the protocol described in Fig. 1A. Six days 5-BrdU post CL injection, depletion was limited to CD169+ macrophages of the DLN, with no significant effect on other innate cell populations (Supplementary Fig. S2). Macrophage depletion resulted in the abrogation of monocyte, neutrophil and dendritic cell recruitment to the DLN 24?h post-immunisation (Fig. 4A). Clodronate treatment suppressed QS-21-induced upregulation of CD80 and CD86 by dendritic cells (DCs), indicating that macrophages are required for their phenotypic maturation (Fig. 4B). The adaptive response was also affected by CL-treatment, as depletion of LN-macrophages led to a strong decrease in the frequency of HBs-specific CD4 (Fig. 4C) and HBs- and OVA-specific CD8 (Fig. 4E) T cells. Polyfunctional T cells (i.e. cells simultaneously producing a combination of effector cytokines) can be crucial determinants for the efficacy of vaccines28. CD4 T-cell polyfunctionality was quantified following a previously described index29. Remaining CD4 T cells from CL-treated animals showed decreased polyfunctionality when compared to controls (Fig. 4D). Finally, the HBs-specific IgG1, IgG2c and total IgG titres were strongly decreased after CL-mediated macrophage depletion, with little effect on the antibody responses induced by alum-adjuvanted antigens, indicating that the effects of macrophage depletion are.