Antigen phagocytosis by B cells is required for a potent humoral response

Follicular B cells phagocytose particulate antigens in vitro through a RhoG‐dependent mechanism. Flow cytometry plots of purified FO B cells incubated for 1 h at 0°C or 37°C with 1 μm fluorescent beads coated with a goat anti‐mouse anti‐IgM antibody and stained afterwards extracellularly on ice with an anti‐goat IgG 488. Gate shows the B cells with internalized beads (negative for the anti‐goat IgG staining).

Flow cytometry plots of purified FO B cells incubated with 1 and 3 μm Y/G fluorescent beads coated with anti‐IgM for 2 h at 37°C. Cells were afterwards stained directly on ice with an anti‐goat IgG 647 (plots on the left) or after fixation and permeabilization (plots on the right).

Flow cytometry plots of WT B cells non‐treated or treated with Latrunculin A and incubated for 1 h with 1 and 3 μm fluorescent beads coated with anti‐IgM antibody. Subsequently, cells were stained extracellularly with anti‐goat 488. Gates indicate those B cells with internalized beads. Bar plots on the bottom show the phagocytic index of Rhog^−/− or WT B cells non‐treated or treated with Cytochalasin D (1 μg/ml), Latrunculin A (20 μg/ml) or PP2 (20 μM) after 1 h incubation at 0°C or at 37°C with 1 (left graph) or 3 μm beads (right graph) coated with anti‐IgM. Data represents means and SEM (n = 3). *P < 0.05; **P < 0.005; (unpaired Student's t test).

Confocal microscopy images of WT and Rhog^−/− B cells after 1 h incubation at 37°C with 3 μm beads coated with anti‐IgM. Stainings for IgM, F‐actin and beads are shown in red, green and blue, respectively.

Overlayed histograms showing Cell Trace Violet (CTV) dilution of OT CD4⁺ T cells incubated for 4 days with B1‐8^hi B cells, WT or or Rhog^−/−, previously incubated with either 1 μm beads coated with NIP‐OVA (3:1 ratio beads:B cell) or 100 ng/ml soluble NIP‐OVA.

Bar plots show quantitative data on OT2 T cell proliferation calculated according to the number of cell divisions, as shown in (A), or a proliferation index (Materials and Methods). Data represent means ± SEM (n = 3). n.s P > 0.05; ***P < 0.0005 (unpaired Student's t test).

Confocal microscopy images obtained with a 10×, 25×, and 63× objective from spleen sections of WT mice 5 h post‐intraperitoneal (ip) immunization with 1 μm fluorescent beads coated with NIP‐OVA. The left images (10× objective) show beads (gray) distribution along a spleen slice where MOMA1 staining (red) determines the outer/inside part of the follicles and DAPI (blue) the nucleus. Upper right image shows both the marginal zone (MZ) and follicular area (FO): DAPI (blue); MOMA1 (green); 1 μm bead (gray). The arrow points to a bead located in the FO zone. The below image shows an amplification of a follicular B cell with a 1 μm bead: B220 (red); 1 μm bead (gray).

Phagocytosis of 1 μm fluorescent beads covalently bound to NIP‐OVA by splenic B cells from WT or Rhog^−/− B1‐8^hi mice was assessed after 5 h post‐IP immunization through extracellular staining with an anti‐ovalbumin antibody. Cytometry plots show the identification of NP‐reactive B cells (CD19⁺ B220⁺) with attached or intracellular beads (NP⁺ fluorescent beads⁺). Anti‐OVA staining distinguishes between the attached and the already phagocytosed beads. Quantification charts represent the means ± SEM of phagocytic NP‐reactive (NP⁺) and non‐reactive (NP⁻) B cells in WT (black) and Rhog^−/− (red) mice.

Follicular (CD21⁺ CD23⁺) and MZ (CD21⁺ CD23⁻) B cells were identified in WT and Rhog^−/− mice immunized as in (A), and their B‐cell phagocytic ability was measured also by anti‐OVA staining (CD19⁺ beads⁺ anti‐Ova⁻). The bar graph shows the percentage of phagocytic follicular and MZ B cells in WT B1‐8^hi, Rhog^−/− B1‐8^hi, and non‐transgenic WT (non‐Tg) mice as a control. Data represent means ± SEM (n = 3).

WT B1‐8^hi B cells were incubated in vitro with fluorescent 1 μm beads coated with NIP‐OVA at 0°C for 1 h and subsequently left unstained or stained with an anti‐OVA antibody. B cells with extracellular membrane‐attached beads that are not internalized are positive for the fluorescent beads and the anti‐ova staining (red histogram), while the control without primary antibody is in grey.

Phagocytosis of 1 μm fluorescent beads covalently bound to NIP‐OVA by splenic macrophages from WT or Rhog^−/− B1‐8^hi and WT non‐transgenic mice was assessed after 5 h post‐IP immunization using extracellular staining with an anti‐ovalbumin antibody. The cytometry plot shows staining with the anti‐OVA antibody in the CD11b⁺ F4/80⁺ macrophage population. The graph on the right shows the percentage of phagocytic splenic macrophages in WT and Rhog^−/− mice. Data represents means and SEM (n = 3). n.s, not significant (unpaired Student's t test).

Confocal image of spleen sections of WT mice 5 days post‐immunization with NIP‐OVA bound to 1 μm fluorescent beads. IgD (red); GL7 (green); 1 μm fluorescent beads (gray). White arrows point FO B cells (IgD⁺) with beads. Representative image of 3 GCs per spleen section and per immunized mouse (n = 6 mice).

Analysis of germinal center B cells (CD95⁺ GL7⁺) in WT and Rhog^−/− mice 7 days post‐immunization with 1 μm beads covalently coated with NIP‐OVA. The bar graph shows the mean percentage ± SEM of CD95⁺ GL7⁺ B cells (n = 3). **P < 0.005 (unpaired Student's t‐test).

WT and Rhog^−/− B1‐8^hi CD45.2 B cells were adoptively transferred to congenic CD45.1 receptor mice immunized with 1 μm beads covalently bound to NIP‐OVA. The flow cytometry panel illustrates germinal center B cells (CD95⁺ GL7⁺) within the transferred WT (upper panel) or Rhog^−/− (lower panel) B cells (CD45.2⁺ CD45.1⁻). Quantification charts show the percentage of transferred B cells (CD45.2⁺ B220⁺) and GC B cells (CD95⁺ GL7⁺). Data represent means ± SEM (n = 3). **P < 0.005 (unpaired Student's t‐test).

Quantification chart of the percentage of germinal center B cells within the WT and Rhog^−/− B1‐8^hi CD45.2 B cells adoptively transferred to congenic CD45.1 receptor mice, as in (C), and immunized with 3 μm beads covalently bound to NIP‐OVA. Data represent means ± SEM (n = 6). *P < 0.05 (unpaired Student's t‐test).

WT and Rhog^−/− mice were immunized with 1 and 3 μm beads covalently bound with NIP‐OVA. Sera were collected after 14 days and high‐affinity NP(7) and low‐affinity NP(41)‐specific IgM (upper graphs) and IgG1 (lower graphs) were measured by ELISA. Graphs show means ± SEM (n = 4) as well as the ratios of absorbance for NP(7) vs. NP(41) binding. n.s. P > 0.05; *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005 (unpaired Student's t‐test).

Confocal microscopy image of antigen aggregates generated with NIP‐BSA‐FITC complexed with alum.

Confocal microscopy image and orthogonal views of WT and Rhog^−/− B1‐8^hi B cells after 2 h of incubation at 37°C with antigen aggregates generated with NIP‐BSA‐FITC complexed with alum. B220 (red), DAPI (blue), and NIP‐BSA‐FITC (green). Completely phagocytosed alum aggregate is indicated with an arrow, and non‐phagocytosed aggregate is indicated with an asterisk.

Flow cytometry plots of WT‐ and RhoG‐deficient B1‐8^hi B cells incubated for 2 h with antigen aggregates generated with NIP‐BSA‐FITC complexed with alum and stained afterward extracellularly with an anti‐FITC 647 antibody to distinguish those B cells with only internalized aggregates from those still attached to the membrane. B cells positive for NIP‐BSA‐FITC aggregates were analyzed for lack of anti‐FITC staining. The graph below the plots shows the percentage of WT (black) and Rhog^−/− (red) B cells with phagocytosed NIP‐BSA‐FICT aggregates. Data represent means ± SEM (n = 3). **P < 0.005 (unpaired Student's t‐test).

WT and Rhog^−/− mice were immunized with NIP‐OVA complexed with alum or NIP‐OVA diluted in PBS and supplemented with LPS. Sera from WT and Rhog^−/− mice were collected at different time points (7 and 14 days) and high‐affinity NP(7) and low‐affinity NP(41)‐specific IgM and IgG1 were measured by ELISA. IgM data are shown only at day 14 after immunization. Graphs show means ± SEM (n = 4) as well as the ratios of absorbance for NP(7) vs. NP(41) binding.

Analysis of germinal center B cells (CD95⁺ GL7⁺) on WT (black; gray) and Rhog^−/− (red; pink) mice 7 days post‐immunization with NIP‐OVA complexed with alum, NIP‐OVA diluted in PBS, or NIP‐OVA diluted in PBS and supplemented with 50 μg of LPS.

Quantification chart of the mean ± SEM of the percentage of GC (CD95⁺ GL7⁺), NP⁺‐specific B cells (NP⁺ B220⁺), and IgG1 class‐switched (IgG1⁺ IgD⁻) B cells of mice immunized as in (B) (n = 5).

WT and Rhog^−/− B1‐8^hi CD45.2 purified B cells were adoptively transferred to CD45.1 receptor mice immunized with NIP‐OVA complexed with alum. Flow cytometry plots show germinal center (CD95⁺ GL7⁺) B cells in the transferred B cells (B220⁺ CD45.2⁺). Quantification charts represent the mean ± SEM of the percentage of GC (GL7⁺CD95⁺), NP‐specific (NP⁺ B220⁺), and IgG1 class‐switched (IgG1⁺ IgD⁻) B cells (n = 3).