Research ArticleB CELLS

Intrinsic properties of human germinal center B cells set antigen affinity thresholds

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Science Immunology  30 Nov 2018:
Vol. 3, Issue 29, eaau6598
DOI: 10.1126/sciimmunol.aau6598
  • Fig. 1 GC B cells engage antigen through BCRs concentrated in F-actin and ezrin-rich pod-like structures.

    (A) DIC, IRM, and merged images of immune synapses of live naïve B cells placed on antigen-containing PLB and live LZ GC B cells placed on antigen-containing PLB or PLB with no antigen. (B) Kymographs of DIC images of naïve and LZ GC B cell immune synapses on antigen-containing PLB. (C) Membrane movement in the immune synapses of live naïve, LZ GC, and DZ GC B cells with time on antigen-containing PLB imaged by IRM. (D) STED super-resolution images of F-actin formed in immune synapses of naïve B cells and GC B cells placed on PLB that contained antigen. (E) Colocalization of F-actin and BCR in immune synapses of naïve and LZ GC B cells imaged by confocal microscopy on PLB that contained antigen. (F) Left: Confocal microscopy images of immune synapses of naïve B cells and LZ GC B cells on antigen-coated PLB stained with Alexa Fluor 488 phalloidin for F-actin (green) and antibodies specific for ezrin (red). Quantification of the MFI of ezrin (top right panel) and colocalization of ezrin with F-actin (bottom right panel) in the immune synapse of confocal images. (G) Bottom and orthogonal views of F-actin (green) and BCR (red) in naïve and LZ GC B cells imaged by confocal microscopy on antigen-containing PLB. (H) Side and top views of naïve and LZ GC B cells imaged by scanning electron microscopy on PLB without or with antigen. (I) Colocalization of pod-like structures and antigens in immune synapse of LZ GC B cells imaged by TIRFM on antigen-containing PLB. Scale bars, 5 μm. **P ≤ 0.01 and ****P ≤ 0.0001 (unpaired t test). Data are representative of two experiments [mean and SEM (F)].

  • Fig. 2 Human GC B cells signal in response to and exert pulling forces on membrane-bound antigens through pod-like structures.

    (A) Immune synapses of naïve B cells and GC B cells imaged by TIRFM on PLB containing F(ab′)2 anti-λ/κ. (B) MFI of pPI3K and pSHP-1 and colocalization of BCR with pPI3K or pSHP-1 in the contact area of naïve and GC B cells placed on antigen (Ag)–containing PLBs for 7 or 25 min. (C) Top: Atto 647N FI (red) and Atto 550 FI (green) merged images. Bottom: Sensor opening ratio (the ratio of Atto 647N FI to Atto 550 FI). (D) Quantification of sensor opening for 9-pN sensor and control sensor. (E) Colocalization of sensor opening locations and contact sites to the membrane with time imaged by TIRFM and IRM. Scale bars, 5 μm. *P ≤ 0.05 and ****P ≤ 0.0001 (unpaired t test). Data are representative of two experiments [mean and SEM (B and D)].

  • Fig. 3 Human GC B cells have a high-affinity threshold for antigen.

    Naïve and LZ GC B cells were placed on PLB that did not contain antigen (resting) or placed on PLB that contained either high- or low-affinity anti-κ mAbs. (A) TIRFM images were obtained from cells stained for the BCR and pCD79A. (B and C) Quantification of the BCR (B) and pCD79A (C) in the contact area of the B cells with the PLB in TIRFM images. (D to F) Quantification of TIRF images of cells stained with pSyk (D), pBLNK (E), and pPLCγ2 (F) in the contact area with the PLB of naïve and LZ GC B cells activated by high- and low-affinity antigen for 7 or 25 min. (G) DIC and IRM images of naïve and LZ GC B cells imaged by TIRFM on PLB containing either high- or low-affinity anti-κ mAbs. The still images were taken between 3 and 12 min of movie S6. (H) Kymographs of IRM images of naïve and LZ GC B cell immune synapse on low- or high-affinity antigen-containing PLB. Red arrowheads on low-affinity naïve B cell are extended membrane ruffles and on high-affinity LZ GC B cell are pod-like structures that established stable contacts with antigen-containing PLB. (I) Quantitative analysis of pod-like structure movement in DIC images (number of tracks: n = 347 for high affinity early, n = 887 for high affinity late, n = 241 for low affinity early, and n = 432 for low affinity late). Scale bars, 5 μm. ns > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001 (unpaired t test). Data are representative of two experiments [mean and SEM (B to F)].

  • Fig. 4 Extraction and trafficking of membrane-associated antigen distinguishes GC B cells.

    (A) Antigen extraction by naïve and GC B cells placed for 2 hours on PMS that contained either high- or low-affinity anti-κ mAbs. Antigen-positive B cell populations were quantified by flow cytometry. (B) Transport of extracted antigens in naïve and LZ GC B cells placed on antigen-containing PMS. F-actin (green) and antigen (red). (C) Distance of MTOC in naïve and LZ GC B cells to antigen-containing PMS. F-actin (green), γ-tubulin (cyan), and antigen (red). (D) Localization and quantification of SNX9 or SNX18 in naïve and LZ GC B cells 45 min after activation on antigen-containing PMS imaged by confocal microscopy. F-actin (green), SNX9 and SNX18 (cyan), and antigen (red). MFI of SNX9 and SNX18 per each z-stack from the cell bottom to the top (n = 25 per group). (E) Localization of SNX9 or SNX18 with MTOC in naïve and LZ GC B cells 45 min after activation on antigen-containing PMS. α-Tubulin (green), SNX9 and SNX18 (cyan), and antigen (red). (F) Localization pattern of LAMP-1 in naïve and LZ GC B cells (left) 30 min after activation on antigen-containing PMS. 3D colocalization of antigen with LAMP-1 in naïve and LZ GC B cells (right). LAMP-1 (cyan) and antigen (red). (G) Trafficking of extracted antigens to acidic intracellular compartments in naïve and LZ GC B cells with time on antigen-containing PMS. (H) pHrodo FI of naïve B cells and LZ GC B cells (bottom left). Antigen and pHrodo FIs for naïve and LZ GC B cells (top left). Percentage of pHrodo-positive naïve B cells (top right) and LZ GC B cells (bottom right) for B cells that have acquired similar amount of antigen. ns > 0.05, **P ≤ 0.01, and ****P ≤ 0.0001 (unpaired t test). Data are representative of two (B to F) or three experiments (A, G, and H) [mean and SEM (A, C, D, and F)].

  • Fig. 5 Human GC B cells express IRF-4 in response to high-affinity antigen in combination with Tfh cell help.

    (A and B) Relative mRNA expression level of IRF-4 in LZ GC (A) and naïve B (B) cells after activation with antigens on PLB and/or Tfh-like stimuli composed of anti-CD40 mAb, IL-21, and IL-4. ns > 0.05 and *P ≤ 0.05 (paired t test) [mean and SEM (A and B)].

  • Fig. 6 The differential expression of key surface markers may further contribute to affinity discrimination by human GC B cells.

    (A) Heat map: Comparisons of differential surface molecule expression. (B and C) Surface levels of CD29, CD49d, and VLA-4 active epitope in naïve and LZ GC B cells. (D) Binding ability of naïve and LZ GC B cells to VCAM-1–coated beads. ****P ≤ 0.0001 (unpaired t test). Data are from one of five individuals (B) or from representative of three experiments (D) [mean and SEM (C and D)].

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/29/eaau6598/DC1

    Methods

    Fig. S1. Fluorescence-activated cell sorting gating strategy for naïve, memory, LZ GC, and DZ GC B cells.

    Fig. S2. Quantification of dynamic movement of unique pod-like structures formed in the immune synapse of LZ GC B cell and expression level of BCR and ezrin.

    Fig. S3. Antigen-induced BCR signaling in naïve B cells and GC B cells.

    Fig. S4. Design of force sensor and detection of pod-like structures.

    Fig. S5. Polarization of SNX9 and SNX18 in naïve and LZ GC B cells after activation on antigen-containing PMS.

    Fig. S6. The differential expression of key surface markers.

    Fig. S7. Surface expression of LFA-1 and specificity of VLA-4 binding.

    Movie S1. Distinctive membrane dynamics in the immune synapse of live naïve and LZ GC B cells placed on antigen-containing PLB were visualized by DIC and IRM.

    Movie S2. The dynamics of membrane movement in the immune synapse upon activation were visualized by IRM.

    Movie S3. Time-lapse imaging of CD19 and BCR in the immune synapse of naïve B cells and LZ GC B cells.

    Movie S4. Pulling forces in the immune synapse of live naïve and LZ GC B cells were monitored with time using DNA-based force sensors.

    Movie S5. Colocalization of the cell contact sites and the locations of pulling force in LZ GC B cell.

    Movie S6. The dynamics of membrane movement in the immune synapse of naïve and LZ GC B cell upon activation by high- or low-affinity antigen were visualized by DIC and IRM.

    Movie S7. Motile LZ GC B cells were observed upon activation by low-affinity antigen.

    Movie S8. Distinct antigen transport pattern shown in human LZ GC B cells on PMS presenting surrogate antigens as compared with naïve B cells.

    Movie S9. Localization of MTOC with SNX9 or SNX18 after activation in naïve and LZ GC B cells.

    Movie S10. Localization of LAMP-1 after activation in naïve and LZ GC B cells.

    Table S1. Raw data file (Excel file).

  • Supplementary Materials

    The PDF file includes:

    • Methods
    • Fig. S1. Fluorescence-activated cell sorting gating strategy for naïve, memory, LZ GC, and DZ GC B cells.
    • Fig. S2. Quantification of dynamic movement of unique pod-like structures formed in the immune synapse of LZ GC B cell and expression level of BCR and ezrin.
    • Fig. S3. Antigen-induced BCR signaling in naïve B cells and GC B cells.
    • Fig. S4. Design of force sensor and detection of pod-like structures.
    • Fig. S5. Polarization of SNX9 and SNX18 in naïve and LZ GC B cells after activation on antigen-containing PMS.
    • Fig. S6. The differential expression of key surface markers.
    • Fig. S7. Surface expression of LFA-1 and specificity of VLA-4 binding.
    • Legends for movies S1 to S10
    • Legend for table S1

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Distinctive membrane dynamics in the immune synapse of live naïve and LZ GC B cells placed on antigen-containing PLB were visualized by DIC and IRM.
    • Movie S2 (.mov format). The dynamics of membrane movement in the immune synapse upon activation were visualized by IRM.
    • Movie S3 (.mov format). Time-lapse imaging of CD19 and BCR in the immune synapse of naïve B cells and LZ GC B cells.
    • Movie S4 (.mov format). Pulling forces in the immune synapse of live naïve and LZ GC B cells were monitored with time using DNA-based force sensors.
    • Movie S5 (.mov format). Colocalization of the cell contact sites and the locations of pulling force in LZ GC B cell.
    • Movie S6 (.mov format). The dynamics of membrane movement in the immune synapse of naïve and LZ GC B cell upon activation by high- or low-affinity antigen were visualized by DIC and IRM.
    • Movie S7 (.mov format). Motile LZ GC B cells were observed upon activation by low-affinity antigen.
    • Movie S8 (.mov format). Distinct antigen transport pattern shown in human LZ GC B cells on PMS presenting surrogate antigens as compared with naïve B cells.
    • Movie S9 (.mov format). Localization of MTOC with SNX9 or SNX18 after activation in naïve and LZ GC B cells.
    • Movie S10 (.mov format). Localization of LAMP-1 after activation in naïve and LZ GC B cells.
    • Table S1. Raw data file (Excel file).

    Files in this Data Supplement:

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