Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B‐cell responses

Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169+ macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood‐borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169+ macrophages in mice resulted in strong, isotype‐switched, high‐affinity Ab production and the preferential induction and long‐term persistence of Ag‐specific GC B cells and follicular Th cells. In agreement with these observations, CD169+ macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B‐cell responses. Our results identify CD169+ macrophages as promoters of high‐affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses.


Immunofluorescence
Spleen fragments were embedded in O.C.T. (Tissue-Tek, Sakura) and 5 µm cryosections were fixed in dehydrated acetone for 5 min. and air-dried. Sections were blocked with 10% mouse serum in PBS for 15 min. prior to staining. Sections were stained with antibodies listed above and analyzed using a Leica DM6000 microscope.

OVA ELISA
High binding 96 well plates (Nunc Maxisorp) were coated with serial dilutions (1:3) starting with 1 µg/ml mAb:OVA in PBS for 1 hr at 37 °C and blocked with 1% BSA in PBS. Detection was achieved using 1:1000 diluted polyclonal rabbit anti-OVA in 1% BSA in PBS followed by antirabbit Ig conjugated HRP (Jackson Immune Research), both incubated for 1 hr. In between all steps plates were was intensively with PBS containing Tween.

Ex vivo CD8 + T cell restimulation assay
Splenocytes from mice 9 days after i.v. immunization with 1 µg mAb:OVA together with 25 µg poly(I:C) and 25 µg αCD40 with or without treatment with clodronate liposomes 7-8 days prior to immunization were restimulated in vitro for 5 hrs with MHC class I restricted OVA 257-264 peptide (100 ng/ml) in the presence of GolgiPlug (BD Biosciences). Cells were analyzed for intracellular cytokine expression by flow cytometry. minutes and detected with anti-rat IgG-Alexa 488 Ab (green) or rabbit anti-OVA followed by anti-rabbit IgG-Alexa 488 Ab (green). CD11c + DCs were detected by anti-CD11c staining (red).
Shown are representative pictures of a single experiment using 1 mouse. Background staining with cIg:OVA was hardly present. (B) Splenocytes were incubated with 10 ug/ml cIg, αDEC205, cIg:OVA, or αDEC205:OVA for 30 minutes. Bound cIg, αDEC205, cIg:OVA and αDEC205:OVA were detected by incubation with anti-rat IgG-Alexa 488 or by polyclonal rabbit anti-OVA followed by anti-rabbit IgG-Alexa 488. Shown are representative histograms gated on CD11c + CD8 + DCs from two independent experiments using one mouse. Grey histogram is cIg binding, black lines represent αDEC205 binding. (C) mAb:OVA complexes were coated on Maxisorb plates in serial dilution (start 1 ug/ml, 1:3 titrations) and evaluated for the presence of OVA by ELISA. Depicted is the mean ± SEM of the OD450 of a representative experiment from three independent experiments using three replicates/group. Bonferroni's correction. P-value indicator ** refers to P < 0.005.  CD4+ T cells were defined as CD4+ and IFNγ producing was evaluated against CD11a expression.
Shown are representative dot plots of six experiments using four to seven mice/group.

Fig. S5: Flow cytometry of OT-II T cells. (A) Gating strategy of OT-II T cells. Cells were first
gated on the basis of forward and side scatter, live cells were identified as FVD-. Single cells were gated by pulse width. Autofluorescent cells were gated out using an empty channel from the violet laser. CD4+ T cells were defined as CD4+, and OT-II cells were defined as CD45.1+. Shown are representative dot plots of four experiments using three to five mice/group.  B6 mice were immunized with 1 µg of indicated mAb:OVA complexes together with 25 µg αCD40 and poly(I:C) and analyzed for OVA localization by flow cytometry 30 min after injection. CD169 + AF and non-AF cells were gated as described in Fig. S6. Shown is the geometric mean fluorescence intensity (mean ± SEM) of anti-OVA staining on AF CD169 + and non-AF CD169 + cells. Data are from a single experiment with three mice/group. Similar data was obtained in an independent experiment using 1 µg of indicated mAb:OVA complexes without adjuvants which was analyzed 5 min after immunization (data not shown).