Low-affinity B cells transport viral particles from the lung to the spleen to initiate antibody responses

Abstract

The lung is an important entry site for pathogens; its exposure to antigens results in systemic as well as local IgA and IgG antibodies. Here we show that intranasal administration of virus-like particles (VLPs) results in splenic B-cell responses with strong local germinal-center formation. Surprisingly, VLPs were not transported from the lung to the spleen in a free form but by B cells. The interaction between VLPs and B cells was initiated in the lung and occurred independently of complement receptor 2 and Fcγ receptors, but was dependent upon B-cell receptors. Thus, B cells passing through the lungs bind VLPs via their B-cell receptors and deliver them to local B cells within the splenic B-cell follicle. This process is fundamentally different from delivery of blood or lymph borne particulate antigens, which are transported into B cell follicles by binding to complement receptors on B cells.

Fig. 1.

Generation of VLP-specific splenic B-cell responses following intranasal immunization. (A) VLP-specific IgG titers in serum of C57BL/6 mice immunized with 50 μg of VLP either intranasally (i.n.) (●) or subcutaneously (s.c.) (■) determined by ELISA. Geometric mean titers ± SEM (n = 6) are shown. (B) Numbers of AFC secreting VLP IgG were determined by ELISPOT in the spleen of either intranaslly (●) or subcutaneously (■) immunized mice. Mean values ± SEM (n = 3) are given. (C) Immunofluorescence micrographs of spleen sections stained 12 d after intranasal (Left), subcutaneous (Center), or intranvenous (i.v.) (Right) immunization for PNA (red) identifying GCs and VLP-specific B cells (green). (Original magnification: 40×.) Note that VLP-specific B cells are found in GCs (PNA⁺) or in large aggregates (PNA⁻) as plasmablasts outside B-cell FO. (D) Enumeration (Left) and mensuration (Right) of VLP-specific GCs by histological analysis. Mean values ± SEM (n = 6) are given. The data shown are representative of two independent experiments.

Fig. 2.

Detection of antigen in spleen and serum after immunization with VLPs. Immunohistochemistry of spleen sections stained for VLP antigen from mice immunized either with 50 μg (A) or with 1 mg (B) of VLP either subcutaneously (Left) or intranasally (Center) or from naïve (Right) mice. Arrows indicate VLP stain in the MZ (Upper Left), RP (Lower Left), and FO compartments (Upper Left and Lower Center). (Original magnification: 40×.) (C) VLP concentrations in serum of mice 2 and 7 h after intranasal (gray diamonds), subcutaneous (black squares), or intravenous (red triangles) immunization with 100 μg of VLPs. Mean concentrations for three mice are given ± SEM. The data shown are representative of two independent experiments.

Fig. 3.

Detection of VLPs in association with B cells. (A) Mice were immunized either subcutaneously (Left) or intranasally. (Center) with 100 μg of Alexa 488-conjugated VLP or left untreated (right). Analysis of interaction with Alexa 488-VLP in blood was determined 4 h later by flow cytometry. Mean percentages of VLP-binding B cells are indicated (n = 3). (B) Lung sections were histologically analyzed 4 h postintranasal immunization with 100 μg of Alexa 488-conjugated VLP. Red, CD19 staining B cells; green, Alexa 488-conjugated VLPs; blue, CD31 staining blood vessels. (Scale bar, 80 μm.) The data shown are representative of three independent experiments.

Fig. 4.

B-cell responses induced by lung B cells capturing VLPs. (A) VLP-specific IgG titers in serum of C57BL/6 mice immunized intranasally with 100 μg of VLP or in mice transfused with lung-B cells from intranasally immunized mice were determined by ELISA. VLP-specific IgG titers from naïve and from mice receiving lung B cells isolated from naïve mice are shown as control. Lung B cells were isolated 4 h after intranasal immunization. Geometric mean titers + SEM (n = 5) are given. (B) VLP-specific IgG titers in serum of C57BL/6-Igh^b transfused with lung B cells from C57BL/6-Igh^a mice immunized intranasally were determined by ELISA 12 d after B-cell transfer. VLP-specific IgG titers from C57BL/6-Igh^b and C57BL/6-Igh^a immunized with 100 μg of VLPs intranasally are shown as control. (C) Staining of splenocytes on day 12 of C57BL/6 Ly5.1 mice receiving lung B-cells either from intranasally immunized (Lower) or untreated (Upper) congenic Ly5.2 mice. Analysis was performed by gating on isotype-switched B cells (CD19⁺, IgM, IgD, CD4, CD8, CD3, CD11c, CD11b, and Gr-1^–). (D) Localization of VLPs in inguinal nondraining lymph nodes 1 d after intranasal immunization. Mice were immunized intranasally with 50 μg of VLPs-Alexa 488 and the presence of VLPs was assessed in inguinal lymph node by flow cytometry 24 h later. The percentage of live leukocytes in association with VLP Alexa 488 is shown. Inguinal lymph nodes of subcutanesoulsy immunized is shown as control. (E) CD62L expression level was determined on VLP-bearing blood B cells 4 h after intrnasal immunization by flow cytometry. Data shown are representative of two independent experiments.

Fig. 5.

Determination of receptors involved in VLPs binding to B cells after intranasal immunization. Flow cytometry analysis of binding of Alexa 488-conjugated VLPs by B cells in the blood of (A) naïve, C57BL/6 wild-type, FcγRIIB^−/−, and Cr2^−/− mice or (B) in C57BL/6 WT and (VI10)xYEN mice. Mean percentages of VLP-binding B cells are indicated (n = 3). (C) Histological detection of VLPs within splenic B-cell FO of WT and (VI10)xYEN mice 24 h after intranasal administration of 1 mg of VLPs. (Original magnification: 40×.) (D) Binding of Alexa 488-conjugated VLPs by B cells in mixed bone-marrow chimeras containing a polyclonal repertoire of natural antibodies as well as VSV-specific and polyclonal BCRs 4 h after intranasal immunization with 100 μg of VLPs. The data shown are representative of two independent experiments. C57BL/6 mice received 100 µg AP205-VLP-Alexa 647 and 100 µg Qβ-VLP-Alexa 488 intranasally. Four hours later the blood was analyzed for B220⁺ B cells that were positive for either AP205-VLP or Qβ-VLP (E) and mediastinal lymph nodes were analyzed for CD11c⁺ CD11b⁺ DCs cells, which were positive for AP205-VLP and Qβ-VLP (F).