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

Ana Martínez‐Riaño, Elena R Bovolenta, Pilar Mendoza, Clara L Oeste, María Jesús Martín‐Bermejo, Paola Bovolenta, View ORCID ProfileMartin Turner, View ORCID ProfileNuria Martínez‐Martín, View ORCID ProfileBalbino Alarcón
DOI 10.15252/embr.201846016 | Published online 09.07.2018
EMBO reports (2018) e46016
Ana Martínez‐Riaño
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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Elena R Bovolenta
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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Pilar Mendoza
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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Clara L Oeste
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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María Jesús Martín‐Bermejo
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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Paola Bovolenta
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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Martin Turner
Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK
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Nuria Martínez‐Martín
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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Balbino Alarcón
Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
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Author Affiliations

  1. Ana Martínez‐Riaño1,
  2. Elena R Bovolenta1,
  3. Pilar Mendoza1,
  4. Clara L Oeste1,
  5. María Jesús Martín‐Bermejo1,
  6. Paola Bovolenta1,
  7. Martin Turner2,
  8. Nuria Martínez‐Martín (nmartinez{at}cbm.csic.es)*,1 and
  9. Balbino Alarcón (balarcon{at}cbm.csic.es)*,1
  1. 1Centro de Biologia Molecular Severo Ochoa, CSIC‐UAM, Madrid, Spain
  2. 2Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK
  1. ↵* Corresponding author. Tel: +34911964721; Fax: +34911964420; E‐mail: nmartinez{at}cbm.csic.es
    Corresponding author. Tel: +34911964555; Fax: +34911964420; E‐mail: balarcon{at}cbm.csic.es
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  • Figure 1.
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    Figure 1. Follicular B cells phagocytose particulates antigens in vitro through a RhoG‐dependent mechanism

    1. Confocal section of follicular B cells in the process of phagocytosing 1 and 3 μm beads coated with anti‐IgM. Purified follicular B cells were incubated with 1 or 3 μm fluorescent beads coated with a goat anti‐mouse anti‐IgM for 1 h at 37°C and afterward stained with an anti‐goat 488 antibody on ice to distinguish cells with attached or already internalized beads. Beads are shown in green, the extracellular staining with anti‐goat IgG in red, and the cortical actin cytoskeleton in blue. Completely phagocytosed beads, negative for anti‐goat IgG, are indicated with an arrow, and non‐phagocytosed beads are indicated with an asterisk.

    2. Flow cytometry plots of WT‐ and RhoG‐deficient B cells incubated for 1 h with 1 μm fluorescent beads coated with anti‐IgM antibody and stained afterward extracellularly with anti‐goat 488, as in (A). The phagocytic index was calculated according to the stepwise increase in the beads’ mean fluorescence intensity and lack of anti‐goat 488 staining on B cells with beads. The graphs below the plots show the phagocytic index of WT and Rhog−/− B cells incubated with 1 or 3 μm beads. Data represent means ± SEM (n = 3).

    3. Phagocytic index for WT B cells incubated for 1 h with 1, 3, and 10 μm beads coated with anti‐IgM. Data represent means ± SEM (n = 3).

    4. Confocal section and orthogonal images of follicular WT and Rhog−/− B cells in the process of phagocytosing 3 μm beads coated with anti‐IgM as in (A). Beads are shown in blue, the extracellular staining with anti‐goat IgG in red, and B220 in green. Completely phagocytosed beads, negative for anti‐goat IgG, are indicated with an arrow, and non‐phagocytosed beads are indicated with an asterisk.

    5. Antigen presentation of Eα peptide on MHC‐II measured by flow cytometry. Splenic follicular B cells from WT mice were incubated with beads coated with Eα peptide (orange), with Eα + anti‐IgM (black), or uncoated (gray) for 2 h. The bar graph shows the means ± SEM of Eα‐MHC‐II MFI (n = 3).

    6. Proliferation profiles of OT2 T cells after 3 days of culture with WT (black) or Rhog−/− (red) B cells stimulated with 1 μm beads coated with anti‐IgM and ovalbumin. The right bar graph shows the OT2 proliferation index upon different anti‐IgM+ovalbumin‐coated bead:B‐cell ratios. As controls, beads were incubated only with anti‐IgM or with ovalbumin. Data represent means ± SEM (n = 3).

    Data information: *P < 0.05; **P < 0.005; ***P < 0.0005 (unpaired Student's t‐test).

  • Figure EV1.
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    Figure EV1. Follicular B cells phagocytose particulate antigens in vitro through an actin‐ and RhoG‐dependent mechanism

    1. 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).

    2. 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).

    3. 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).

    4. 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.

  • Figure EV2.
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    Figure EV2. Rhog−/− B cells are defective in the acquisition and presentation of antigen to T cells when antigen is given bound to beads but not in soluble form

    1. Overlayed histograms showing Cell Trace Violet (CTV) dilution of OT CD4+ T cells incubated for 4 days with B1‐8hi 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.

    2. 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).

  • Figure 2.
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    Figure 2. Antigen‐specific splenic B cells phagocytose antigens in vivo through a BCR‐driven process also dependent on RhoG

    1. 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).

    2. Phagocytosis of 1 μm fluorescent beads covalently bound to NIP‐OVA by splenic B cells from WT or Rhog−/− B1‐8hi 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.

    3. 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 B1‐8hi, Rhog−/− B1‐8hi, and non‐transgenic WT (non‐Tg) mice as a control. Data represent means ± SEM (n = 3).

    Data information: *P < 0.05 (unpaired Student's t‐test).

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    Figure EV3. Antigen‐specific splenic B cells phagocytose antigens in vivo through a BCR‐driven process also dependent on RhoG

    1. WT B1‐8hi 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.

    2. Phagocytosis of 1 μm fluorescent beads covalently bound to NIP‐OVA by splenic macrophages from WT or Rhog−/− B1‐8hi 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).

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    Figure 3. Antigen phagocytosis by B cells is important for the germinal center response

    1. 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).

    2. 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).

    3. WT and Rhog−/− B1‐8hi 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).

    4. Quantification chart of the percentage of germinal center B cells within the WT and Rhog−/− B1‐8hi 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).

    5. 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).

  • Figure 4.
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    Figure 4. Alum–antigen aggregates are phagocytosed by B cells

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

    2. Confocal microscopy image and orthogonal views of WT and Rhog−/− B1‐8hi 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.

    3. Flow cytometry plots of WT‐ and RhoG‐deficient B1‐8hi 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).

  • Figure 5.
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    Figure 5. Alum‐based vaccination induces a potent humoral response dependent on RhoG GTPase

    1. 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.

    2. 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.

    3. 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).

    4. WT and Rhog−/− B1‐8hi 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).

    Data information: *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005; *****P < 0.000005 (unpaired Student's t‐test).

  • Figure EV4.
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    Figure EV4. Rhog−/− B cells show normal proliferation and plasma cell differentiation upon TLR and BCR soluble stimulation but not to a particulate BCR‐stimulation

    • A. In vitro plasma cell differentiation (CD138+) obtained after 3 days of stimulation of Cell Trace Violet‐stained WT and Rhog−/− B cells with 1 μg/ml CpG or 1 μg/ml LPS in the presence of IL‐4 and IL‐5. Bar graphs show the means ± SEM of plasma cell (PC) percentages (CD138+ IgD−) and the proliferation index (n = 3).

    • B, C CTV‐B cells from WT and Rhog−/− mice were stimulated for 3 days with anti‐IgM (5 μg/ml) + CpG (1 μg/ml), soluble anti‐IgM (3, 10, 30 μg/ml) (B), or bead‐bound anti‐IgM (4:1, 10:1, 15:1 ratio beads:B cell) in the presence of IL‐4 and IL‐5 (C). Histogram plots of CTV‐ B cells from WT and Rhog−/− mice after the different stimulations. Quantification charts show the proliferation index (n = 3). Data shows means ± SEM.

    Data information: n.s P > 0.05; *P < 0.05; **P < 0.005 (unpaired Student's t test).

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Volume 20, Issue 2
01 February 2019
EMBO reports: 20 (2)
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