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Long-term retention of antigens in germinal centers is controlled by the spatial organization of the follicular dendritic cell network

Nature Immunology volume 24, pages 1281–1294 (2023)Cite this article

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An Author Correction to this article was published on 20 October 2023

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Abstract

Germinal centers (GCs) require sustained availability of antigens to promote antibody affinity maturation against pathogens and vaccines. A key source of antigens for GC B cells are immune complexes (ICs) displayed on follicular dendritic cells (FDCs). Here we show that FDC spatial organization regulates antigen dynamics in the GC. We identify heterogeneity within the FDC network. While the entire light zone (LZ) FDC network captures ICs initially, only the central cells of the network function as the antigen reservoir, where different antigens arriving from subsequent immunizations colocalize. Mechanistically, central LZ FDCs constitutively express subtly higher CR2 membrane densities than peripheral LZ FDCs, which strongly increases the IC retention half-life. Even though repeated immunizations gradually saturate central FDCs, B cell responses remain efficient because new antigens partially displace old ones. These results reveal the principles shaping antigen display on FDCs during the GC reaction.

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Fig. 1: Long-term dynamics of antigen localization in B cell follicles.
Fig. 2: CD40L signals induce FDC network expansion and activation but do not regulate antigen centralization or retention.
Fig. 3: Central and peripheral LZ FDCs express different levels of IC-binding receptors.
Fig. 4: Single-cell transcriptomics differentiates two LZ FDC clusters with different functional activity.
Fig. 5: Central and peripheral LZ FDCs show a similarly low ability to degrade antigens.
Fig. 6: Membrane levels of CR2 dictate the half-life of antigen retention on peripheral and central FDCs.
Fig. 7: Antigen display by FDCs regulates the B cell response while successive immunizations partially saturate central FDCs.

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Data availability

Ensembl GRCm38 was used as the reference genome to build the index. The mouse scRNAseq data are available in GEO under accession no GSE213254. Source data are provided with this paper.

Code availability

Matlab script for FDC segmentation is available at https://github.com/ptolar/FDCseqmentation. Code used to analyze scRNAseq is available at https://github.com/FrancisCrickInstitute/Cxcl13_project.

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Acknowledgements

We thank D. Calado for the Cr2-KO (Cr2tm1Hmo) mice and B. Haynes for the YU-gp120 pcDNA3.1 plasmid. We thank M. Howarth for the SpyTag plasmids and J. Eisenman for the C3dg pET13b plasmid. We thank L. Wasim and S. Hernández for critical reading of the paper. This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2006), the UK Medical Research Council (CC2006) and the Wellcome Trust (CC2006), and by the UK Medical Research Council (grant MR/X009254/1). S.W. is supported by the National Science Foundation (NSF) Grant MCB-2225947 and an NSF CAREER Award PHY-2146581. For the purpose of open access, the author has applied a CC BY public copyright license to any author accepted manuscript version arising from this submission. We thank the Francis Crick Institute Animal facility, Light Microscopy, Flow Cytometry and Advanced Sequencing Technology Platforms.

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Authors and Affiliations

  1. Immune Receptor Activation Laboratory, The Francis Crick Institute, London, UK

    Ana Martínez-Riaño, Sophie Minoughan, Antonio Casal & Pavel Tolar

  2. Division of Infection and Immunity, Institute of Immunity and Transplantation, University College London, London, UK

    Ana Martínez-Riaño & Pavel Tolar

  3. Department of Physics and Astronomy, University of California Los Angeles, Los Angeles, CA, USA

    Shenshen Wang

  4. Bioinformatics and Biostatistics Science Technology Platform, The Francis Crick Institute, London, UK

    Stefan Boeing

  5. Department of Physics, King’s College London, London, UK

    Katelyn M. Spillane

  6. Randall Centre for Cell and Molecular Biophysics, King’s College London, London, UK

    Katelyn M. Spillane

  7. Institute of Immunobiology, Medical Research Center, Kantonsspital St. Gallen, St. Gallen, Switzerland

    Burkhard Ludewig

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Contributions

A.M.-R. designed and performed the experiments and analyzed the data. S.W. developed and analyzed the mathematical model of IC-FDC dissociation. S.B. analyzed the scRNAseq data. S.M. measured the CR2-C3dg binding rates. A.C. produced the YU-gp120-SpyTag protein. K.M.S. provided advice. B.L. provided the CXCL13-TdTomato mice and advice. P.T. designed the experiments and supervised the research. A.M.-R. and P.T. prepared the paper.

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Correspondence to Pavel Tolar.

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Nature Immunology thanks Michael Carroll and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available. Primary Handling Editor: L. A. Dempsey in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Antigens centralize on the FDC network.

a) Maximum intensity projection of confocal images of clarified LNs of mice immunized with IC-PE (magenta). FDC networks are in cyan (anti-CD21/35) (n = 6 LNs; 3 experiments). b) Image analysis to quantify antigen distribution within each B cell follicle. c) Confocal image of an FDC network (cyan) after immunization with two subsequent ICs in PBS analyzed 7 days after the first immunization (IC-PE; magenta) and 24 hours after the second (IC-488; yellow). Single-color images of IC-488 (left) and IC-PE (right) are shown below. Cyan line demarcates FDC network boundary based on anti-CD21/35 staining. Right, quantification of the distribution of both antigens on the FDC network. (n = 8 LNs; 2 experiments). d) Confocal image of an FDC network (cyan) after immunization with two subsequent ICs as in C for 14 and 7 days. Single-color images of IC-488 (left) and IC-PE (right) are shown below. Cyan line demarcates the FDC network boundary based on anti-CD21/35 staining. Right, quantification of the distribution of both antigens on the FDC network (n = 12 LNs; 2 experiments). e) Image of an LN FDC network (cyan) 56 days after immunization with IC-PE (magenta). Right, single-color image of IC-PE (gray) with cyan line demarcating the FDC network boundary based on anti-CD21/35 staining (n = 4 LNs; 1 experiment). f) Image of a draining LN 21 days after immunization with IC-PE (red). Naïve B cells are shown in gray (anti-IgD). (n = 4 LNs; 2 experiments). g) Image of a draining LN 7 days after immunization with AF555-labeled mi3-Spycatcher nanoparticles coated with YU-gp120-Spytag HIV envelope protein (magenta). FDC networks are shown in cyan (anti-CD21/35). White square indicates the region magnified. Cyan line demarcating the FDC network boundary based on anti-CD21/35 staining (n = 3 LNS; 2 experiments). h) Flow cytometry gating strategy to analyze FDCs. Quantitative data show the mean ± SD analysis by two-tailed t-test or one-way ANOVA with multiple comparisons.

Source data

Extended Data Fig. 2 B cell activation is required for FDC expansion.

a) Quantification of the FDC network volume per LN based on anti-CD21/35 staining in clarified LNs from non-immunized (n = 8 mice) and 13 days post-immunized (n = 9) mice with IC-PE. b) Representative immunofluorescence images of LN B cell GCs from mice immunized for 14 days with IC-PE. Upper row shows the merged image of GL7 (yellow), IC-PE (magenta), and anti-CD21/35 (cyan) and the corresponding single-color images (gray). Yellow line demarcates GL7 staining and magenta line IC-PE localization (n = 4 mice). Lower row shows the merged image of anti-PD1 (yellow), IC-PE (magenta) and anti-CD21/35 (cyan) and the corresponding single-color images (gray). Yellow line demarcates PD1 staining and magenta line IC-PE localization (n = 4 mice). c) FDC numbers in non-transgenic C57BL/6 (WT; n = 5) and BCR-transgenic B1-8f (B1-8flox Igκ−/−; n = 6) and MD4 mice (n = 2) 24 hours after immunization with IC-PE. d) Representative confocal images of LNs from non-tg (WT), B1-8f and MD4 mice 24 hours after immunization with IC-PE (magenta). FDC networks are shown in cyan (anti-CD21/35). The white line delimits the edges of the organs. e) Percentage of GC B cells in non-immunized (light gray; n = 4 mice) and IC-immunized mice treated with anti-CD40L (orange; n = 8 mice) or isotype control antibody (black; n = 8 mice) as described in Fig. 2b. Quantitative data show means ± SD and analysis by two-tailed one-way ANOVA with multiple comparisons.

Source data

Extended Data Fig. 3 IC-binding receptor expression on FDCs.

a) CR2, FCGR2B and FCER2A membrane expression on IC+ and IC− FDCs 7 days after immunization (n = 7 mice; 2 experiments). b) Histogram showing Myosin heavy chain 11 (MYH11) expression in IC-PE+ and IC-PE− FDCs 7 days after immunization. Quantitative data show means ± SD and analysis by two-tailed paired t-test.

Source data

Extended Data Fig. 4 ScRNAseq of follicular stromal cells.

a) Experimental workflow for scRNAseq of Cxcl13-TdTomato+ LN cells. Cxcl13-TdTomato mice were immunized consecutively with two ICs separated by 7 days or only with one IC. 24 h after the last immunization, draining LNs were dissociated into a single-cell suspension, stained and live cells were flow-sorted based on PDPN and TdTomato positivity. Single-sorted cells were used for 10x RNA sequencing. b) Feature plots showing expression of markers for hematopoietic cells (H2-Aa) and cytokines important for LN organization, Cxcl12 and Cxcl13. c) Violin plots showing the expression of Myh11 and Fcer2a on the three FDC clusters from Fig. 4c (LZ 1 in green, LZ 2 in red, and DZ in blue). One-tail adjusted P for multiple comparisons. d) Confocal image of a LN from a mouse after 7 days postimmunization with IC-PE (yellow). CR2 staining is shown in cyan and CD16/32 in magenta (n = 4 LNs; 2 experiments).

Extended Data Fig. 5 Antigen degradation by FDCs.

a) Quantification of Atto488 intensity after treating the control sensor lacking the BHQ-1 quencher or antigen-degradation sensor (1:9 antigen:quencher molar ratio) with protease for 30 min at 37 °C. b) Naïve B cells were incubated with beads coated with anti-IgM and the antigen-degradation sensor (green) or control sensor (orange) at indicated times at 37 °C. Plots illustrate Atto488 and AF647 intensity on B cells containing degradation sensor beads or control sensor beads. Graphs show the percentage of B cells containing quenched antigen (% of Atto488-low) and the levels of antigen degradation on B cells containing beads (measured as Atto488/AF647 intensity ratio) (2 experiments). c) Contour plots show Atto488 and AF647 levels on B cells (blue) and FDCs (gray) containing IC-antigen-degradation sensor at different time points post-immunization. d) Quantification of the antigen degradation levels in antigen+ B cells and FDCs at different time points post-immunization. (n = 7 mice; 2 experiments)). All quantitative data show means ± SD analyzed by two-tailed unpaired t-test.

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Extended Data Fig. 6 In vivo IC deposition on FDCs requires CR2 expression.

a) CR2, FCER2A and FCGR2B membrane expression on FDCs from mice described in Fig. 2c. b) Experimental workflow. Lethally irradiated CD45.2 WT (n = 6) and Cr2-KO (n = 4) mice reconstituted with bone marrow cells from WT CD45.1/CD45.2 mice and immunized with IC-PE and IC-488 6 days later. c) Gating strategy based on FCGR2B and VCAM1 expression to analyze FDCs in CD45.2 WT and Cr2-KO mice reconstituted with WT CD45.1/CD45.2 BM. In gray, FCGR2B and VCAM1 expression on WT FDCs (PDPN+ CD31− Madcam1+ CD21/35hi); in red, on PDPN+ CD31− stromal cells. d) CR2 expression on WT and Cr2-KO FDCs from mice as described in (B). e) Percentage of FDCs loaded with IC−PE and IC-488 from mice described in (B). f) Quantity of IC loaded in the FDC network from mice described in (B). g) Surface CR2 density on IC+ (red) or IC− (gray) FDCs 7 days after immunization. Plots show the median (line), the 25th and 75th percentiles (box) and 1.5x the interquartile range (whiskers) of the number of CR2 molecules/μm2 (n = 8 mice; 2 experiments). h) Schematic of the binding of a C3d-coated IC to the surface of an FDC illustrating the mathematical modelling parameters. i) Percentage of FDCs loaded with IC-PE and amount of IC-PE 24 h after IC-PE immunization and injection of anti-CD21/35 blocking antibody (n = 2 mice). j) Immunization workflow to analyze the effect of blocking CR2-C3d binding using an anti-CD21/35 antibody (7G6). k) Percentage of FDCs within the stromal PDPN+ cells in LNs from mice untreated (black) or treated (blue) for 7 days with anti-CD21/35 (7G6) blocking antibody as shown in J (n = 4 mice). l) Number of FDCs per LN and expression of FCGR2B and VCAM1 in WT (black; n = 5), Cr2-HET (dark blue; n = 5) and Cr2-KO mice (light blue; n = 4) bone-marrow reconstituted with WT CD45.1 cells. m) Representative images of a FDC network from mice described in L. Cyan line demarcates FDC network based on FDCM1 stain (n = 5 mice; 2 experiments). Quantitative data show means ± SD and analysis by two-tailed unpaired t-test or One-way ANOVA with multiple comparisons.

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Extended Data Fig. 7 Central FDCs can get partially saturated.

a) Gating strategy to analyze IgG1+ cells within GC CD45.1+ donor B cells in WT, Cr2-HET and Cr2-KO mice bone-marrow reconstituted with CD45.1+ WT cells and immunized for 21 days with IC-NP. Non-immunized mice shown as control. b) Gating strategy to analyze NP-specific B cells within GC CD45.1+ donor B cells from mice described in A. c) Gating strategy to analyze plasmablasts (CD138 + NP+ within CD45.1+/−) from mice described in A. d) Gating strategy to analyze memory B cells (CD38hi PDL-2+ within the NP-specific CD45.1+ donor cells) from mice described in A. e) Representative confocal images of FDC networks from LNs of WT, Cr2-HET and Cr2-KO mice bone-marrow reconstituted with WT CD45.1 cells and immunized for 21 days with IC NP-PE (n = 3 mice). Upper panel shows FDCM1 staining. Lower panel shows NP-PE. f) Gating strategy to analyze FDCs containing different combinations of ICs from consecutive immunizations with three (lower panel) or four (upper panel) different fluorescent antigen-ICs as indicated in Fig. 7a, b. g) Workflow to analyze antigen displacement by subsequent immunizations. Mice were immunized with IC-405 alone or followed by four subsequent immunizations with different ICs. Graphs show the percentage of FDCs loaded with the first antigen and the quantity (MFI) of the loaded antigen in the two groups of mice (n = 7 mice). h) Immunization workflow to analyze the antigen-specific antibody response generated to NP under non-saturating (2-IC) or saturating conditions (4-IC). i) Titers of high-affinity (NP(7)-BSA) and total (NP(25)-BSA) NP-specific IgG1 in sera of mice 21 and 56 days after immunization with either two antigen-ICs (No saturation condition; black; n = 8 mice) or four antigen-ICs (Saturation condition; green; n = 7 mice) as in D. j) Ratio of binding to NP(7) and NP(25), as measured by ELISA in mice immunized as in I. Quantitative data show means ± SD and analysis by two-tailed unpaired t-test with multiple comparisons.

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Martínez-Riaño, A., Wang, S., Boeing, S. et al. Long-term retention of antigens in germinal centers is controlled by the spatial organization of the follicular dendritic cell network. Nat Immunol 24, 1281–1294 (2023). https://doi.org/10.1038/s41590-023-01559-1

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