Toll-like receptor 9 antagonizes antibody affinity maturation

Article metrics

Abstract

Key events in T cell–dependent antibody responses, including affinity maturation, are dependent on the B cell’s presentation of antigen to helper T cells at critical checkpoints in germinal-center formation in secondary lymphoid organs. Here we found that signaling via Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture, process and present antigen and to activate antigen-specific helper T cells in vitro. In a mouse model in vivo and in a human clinical trial, the TLR9 agonist CpG enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling might enhance antibody titers at the expense of the ability of B cells to engage in germinal-center events that are highly dependent on B cells’ capture and presentation of antigen.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: The effect of TLR9 signaling on the outcome of B cell responses to antigen.
Fig. 2: TLR9 signaling antagonizes the trafficking of BCR-bound antigen into late endosomal compartments.
Fig. 3: The effect of TLR9 signaling on B cell responses to membrane bound antigen.
Fig. 4: The effect of TLR9 and BCR signaling on B cell transcription and the expression of B cell surface proteins.
Fig. 5: TLR9 signaling decreases the ability of antigen-specific B cells to interact with and activate antigen-specific helper T cells in response to soluble antigen.
Fig. 6: TLR9 signaling decreases the ability of antigen-specific B cells to activate antigen-specific helper T cells in response to membrane bound antigen.
Fig. 7: B cell–intrinsic expression of MyD88 affects the outcome of T cell dependent antibody response in vivo.
Fig. 8: CpG does not induce high-affinity antibodies in humans.

References

  1. 1.

    De Silva, N. S. & Klein, U. Dynamics of B cells in germinal centres. Nat. Rev. Immunol. 15, 137–148 (2015).

  2. 2.

    Kurosaki, T., Kometani, K. & Ise, W. Memory B cells. Nat. Rev. Immunol. 15, 149–159 (2015).

  3. 3.

    Mesin, L., Ersching, J. & Victora, G. D. Germinal center B cell dynamics. Immunity 45, 471–482 (2016).

  4. 4.

    Bannard, O. & Cyster, J. G. Germinal centers: programmed for affinity maturation and antibody diversification. Curr. Opin. Immunol. 45, 21–30 (2017).

  5. 5.

    DeFranco, A. L., Rookhuizen, D. C. & Hou, B. Contribution of Toll-like receptor signaling to germinal center antibody responses. Immunol. Rev. 247, 64–72 (2012).

  6. 6.

    Rawlings, D. J., Schwartz, M. A., Jackson, S. W. & Meyer-Bahlburg, A. Integration of B cell responses through Toll-like receptors and antigen receptors. Nat. Rev. Immunol. 12, 282–294 (2012).

  7. 7.

    Akkaya, M. et al. B cells produce type 1 IFNs in response to the TLR9 agonist CpG-A conjugated to cationic lipids. J. Immunol. 199, 931–940 (2017).

  8. 8.

    Roche, P. A. & Furuta, K. The ins and outs of MHC class II-mediated antigen processing and presentation. Nat. Rev. Immunol. 15, 203–216 (2015).

  9. 9.

    Rookhuizen, D. C. & DeFranco, A. L. Toll-like receptor 9 signaling acts on multiple elements of the germinal center to enhance antibody responses. Proc. Natl Acad. Sci. USA 111, E3224–E3233 (2014).

  10. 10.

    Akkaya, M. et al. T cell-dependent antigen adjuvanted with DOTAP-CpG-B but not DOTAP-CpG-A induces robust germinal center responses and high affinity antibodies in mice. Eur. J. Immunol. 47, 1890–1899 (2017).

  11. 11.

    Gavin, A. L. et al. Adjuvant-enhanced antibody responses in the absence of toll-like receptor signaling. Science 314, 1936–1938 (2006).

  12. 12.

    Coffman, R. L., Sher, A. & Seder, R. A. Vaccine adjuvants: putting innate immunity to work. Immunity 33, 492–503 (2010).

  13. 13.

    Francica, J. R. et al. Analysis of immunoglobulin transcripts and hypermutation following SHIV(AD8) infection and protein-plus-adjuvant immunization. Nat. Commun. 6, 6565 (2015).

  14. 14.

    Basso, K. & Dalla-Favera, R. Roles of BCL6 in normal and transformed germinal center B cells. Immunol. Rev. 247, 172–183 (2012).

  15. 15.

    Fleire, S. J. et al. B cell ligand discrimination through a spreading and contraction response. Science 312, 738–741 (2006).

  16. 16.

    Weber, M. et al. Phospholipase C-γ2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen. J. Exp. Med. 205, 853–868 (2008).

  17. 17.

    Tolar, P., Sohn, H. W., Liu, W. & Pierce, S. K. The molecular assembly and organization of signaling active B-cell receptor oligomers. Immunol. Rev. 232, 34–41 (2009).

  18. 18.

    Liu, W., Meckel, T., Tolar, P., Sohn, H. W. & Pierce, S. K. Intrinsic properties of immunoglobulin IgG1 isotype-switched B cell receptors promote microclustering and the initiation of signaling. Immunity 32, 778–789 (2010).

  19. 19.

    Akkaya, B. et al. A simple, versatile antibody-based barcoding method for flow cytometry. J. Immunol. 197, 2027–2038 (2016).

  20. 20.

    Crompton, P. D. et al. The TLR9 ligand CpG promotes the acquisition of Plasmodium falciparum-specific memory B cells in malaria-naive individuals. J. Immunol. 182, 3318–3326 (2009).

  21. 21.

    Lanzavecchia, A. Antigen presentation by B lymphocytes: a critical step in T-B collaboration. Curr. Top. Microbiol. Immunol. 130, 65–78 (1986).

  22. 22.

    Eckl-Dorna, J. & Batista, F. D. BCR-mediated uptake of antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation. Blood 113, 3969–3977 (2009).

  23. 23.

    Genestier, L. et al. TLR agonists selectively promote terminal plasma cell differentiation of B cell subsets specialized in thymus-independent responses. J. Immunol. 178, 7779–7786 (2007).

  24. 24.

    Hoogeboom, R. & Tolar, P. Molecular mechanisms of B cell antigen gathering and endocytosis. Curr. Top. Microbiol. Immunol. 393, 45–63 (2016).

  25. 25.

    Mason, D. Y., Jones, M. & Goodnow, C. C. Development and follicular localization of tolerant B lymphocytes in lysozyme/anti-lysozyme IgM/IgD transgenic mice. Int. Immunol. 4, 163–175 (1992).

  26. 26.

    Ho, W. Y., Cooke, M. P., Goodnow, C. C. & Davis, M. M. Resting and anergic B cells are defective in CD28-dependent costimulation of naive CD4+ T cells. J. Exp. Med. 179, 1539–1549 (1994).

  27. 27.

    Akkaya, M., Kwong, L. S., Akkaya, E., Hatherley, D. & Barclay, A. N. Rabbit CD200R binds host CD200 but not CD200-like proteins from poxviruses. Virology 488, 1–8 (2016).

  28. 28.

    Dadaglio, G., Nelson, C. A., Deck, M. B., Petzold, S. J. & Unanue, E. R. Characterization and quantitation of peptide-MHC complexes produced from hen egg lysozyme using a monoclonal antibody. Immunity 6, 727–738 (1997).

  29. 29.

    Cauerhff, A., Goldbaum, F. A. & Braden, B. C. Structural mechanism for affinity maturation of an anti-lysozyme antibody. Proc. Natl. Acad. Sci. USA 101, 3539–3544 (2004).

  30. 30.

    Traba, J., Miozzo, P., Akkaya, B., Pierce, S. K. & Akkaya, M. An optimized protocol to analyze glycolysis and mitochondrial respiration in lymphocytes. J. Vis. Exp. 117, e54918 (2016).

  31. 31.

    Akkaya, B. et al. Ex-vivo iTreg differentiation revisited: Convenient alternatives to existing strategies. J. Immunol. Meth. 441, 67–71 (2017).

  32. 32.

    Mullen, G. E. et al. Phase 1 trial of AMA1-C1/Alhydrogel plus CPG 7909: an asexual blood-stage vaccine for Plasmodium falciparum malaria. PLoS ONE 3, e2940 (2008).

  33. 33.

    Tomlinson, D. C., L’Hôte, C. G., Kennedy, W., Pitt, E. & Knowles, M. A. Alternative splicing of fibroblast growth factor receptor 3 produces a secreted isoform that inhibits fibroblast growth factor-induced proliferation and is repressed in urothelial carcinoma cell lines. Cancer. Res. 65, 10441–10449 (2005).

  34. 34.

    Akkaya, M., Aknin, M. L., Akkaya, B. & Barclay, A. N. Dissection of agonistic and blocking effects of CD200 receptor antibodies. PLoS ONE 8, e63325 (2013).

  35. 35.

    Sohn, H. W., Tolar, P., Brzostowski, J. & Pierce, S. K. A method for analyzing protein-protein interactions in the plasma membrane of live B cells by fluorescence resonance energy transfer imaging as acquired by total internal reflection fluorescence microscopy. Methods. Mol. Biol. 591, 159–183 (2010).

  36. 36.

    Natkanski, E. et al. B cells use mechanical energy to discriminate antigen affinities. Science 340, 1587–1590 (2013).

  37. 37.

    Liu, W., Meckel, T., Tolar, P., Sohn, H. W. & Pierce, S. K. Antigen affinity discrimination is an intrinsic function of the B cell receptor. J. Exp. Med. 207, 1095–1111 (2010).

  38. 38.

    Davey, A. M. & Pierce, S. K. Intrinsic differences in the initiation of B cell receptor signaling favor responses of human IgG+ memory B cells over IgM+ naive B cells. J. Immunol. 188, 3332–3341 (2012).

  39. 39.

    Wang, J., Sohn, H., Sun, G., Milner, J. D. & Pierce, S. K. The autoinhibitory C-terminal SH2 domain of phospholipase C-γ2 stabilizes B cell receptor signalosome assembly. Sci. Signal. 7, ra89 (2014).

  40. 40.

    Offerdahl, D. K., Dorward, D. W., Hansen, B. T. & Bloom, M. E. A three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines. PLoS ONE 7, e47912 (2012).

  41. 41.

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

  42. 42.

    Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

  43. 43.

    Gordon, E. B. et al. Targeting glutamine metabolism rescues mice from late-stage cerebral malaria. Proc. Natl Acad. Sci. USA 112, 13075–13080 (2015).

  44. 44.

    Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).

  45. 45.

    Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).

Download references

Acknowledgements

We thank S. Akira (Osaka University) for TLR9-deficient mice; I. Gery (US National Institutes of Health) for 3A9 mice; N. Barclay (University of Oxford) for mouse mAb OX68; P. Roche (US National Institutes of Health) for the hybridoma secreting mouse mAb AW3.18; L. Lantz (US National Institutes of Health) for producing purified mouse mAb AW3.18; J. Charles-Guery and D. Hudrisier (University of Toulouse) for mouse mAb F10.6; O. Voss (US National Institutes of Health) for the NIH3T3 mouse fibroblast cell line; P. Tolar (The Francis Crick Institute) for Matlab code; and J. Brzostowski and S. Bolland for suggestions. Supported by the Intramural Research Program of the US National Institutes of Health, National Institute of Allergy and Infectious Diseases.

Author information

M.A. and S.K.P. conceived of the project, designed the experiments and wrote the manuscript; M.A., B.A., A.S.K., P.M., H.S., M.P., A.S.R., B.P.T., T.H. and J.L. carried out the experiments; M.A., B.A., A.S.K., P.M., H.S., J.K., J.L., D.W.D., E.D., J.S., L.H.M. and S.K.P. analyzed the data; and S.K.P. secured funding.

Correspondence to Susan K. Pierce.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 The effects of BCR and or TLR9 signaling in B cell functions.

(a) Representative flow cytometry plots showing the differential phosphorylation of kinases, described in Fig. 1a–d. (b) The effects of Anti-IgM concentration on the phosphorylation of downstream kinases are shown. B cells were stimulated with 1 or 10 µg/ml Anti-IgM either individually or in combination with 1µM CpG. Phosphorylation of kinases was measured using phosphoflow as described in Fig. 1. Data are representative of two independent experiments performed in duplicates(a) or triplicates (b). (c) Changes in the phosphorylation of kinases in B cells obtained from TLR-9 KO mice in comparison with WT mice upon stimulation with CpG are shown. Data represent two independent experiments each carried out with duplicates (d) Calcium response of MD4 HEL transgenic B cells upon stimulation with 1 µg/ml HEL and/or 1 µM CpG are shown using a strategy described in Fig. 1e. Data are representative of three independent experiments. (e) Representative flow cytometry plots indicating the proliferation levels for B cells incubated with Anti-IgM (1 µg/ml) or CpG alone (0.04 µM) or with both from the experiment outlined in Fig. 1h. (f)The proliferative response of B cells in response to a range of Anti-IgM (Fab’2) antibody concentrations are shown. Data are representatives of two independent experiments each carried out with triplicates. (g) Percentage of IgG positive B cells in the live B cell population upon negative magnetic separation (left) or negative magnetic separation with additional IgG depletion (right) are shown. Data are representatives of two independent experiments.

Supplementary Figure 2 TLR9 signaling does not affect the internalization dynamics of BCR bound soluble antigen.

The internalization of BCR bound soluble antigen was quantified in splenic B cells purified from WT or MD4 mice. (a) The percent internalization of Alexa-647-labeled Anti-IgM by WT B cells in the presence or absence of CpG was measured with time at 37 °C as detailed in Methods. (b) The internalization of HEL by MD4 HEL-specific B cells was determined by measuring the decrease in surface BCR over time at 37 °C as described in the Methods section. Data are representative of three independent experiments each of which were performed in duplicates. (c-g) To visualize the cytoplasmic compartments containing internalized antigen, purified B cells were incubated with Miltenyi metal particles coated with Anti-IgM in the presence or absence of 1µM CpG. Cells were fixed at 60 min and analyzed later using tomographic TEM. Representative reconstructed images are shown (c). White arrows indicate vesicles containing internalized particles. N marks the nucleus. Sections taken from 12 different cells per group were used to quantify the number of bead-containing vesicles in each section (d), the number of beads per each vesicle (e), volume of the bead-containing vesicles (f) and number of beads per vesicular volume (g). Lines indicate mean values. Statistical significance was measured by two sided unpaired t-test.

Supplementary Figure 3 Compartmentalization of the internalized antigen.

(a) Flow cytometry plots showing the histogram overlays of select time points for the pHrodo HEL stimulated samples described in Fig. 2a. (b-c) 10 min post-stimulation, colocalization of internalized HEL with LAMP-1 (b) and with H2M (c) are shown for the samples described in Fig. 2. Left panels show the representative confocal microscopy images and the right panels show the 3D colocalization indices of 25 cells per group. Lines indicate mean values. Statistical significance was measured using a two sided Mann-Whitney U test.

Supplementary Figure 4 Cell surface expression dynamics of B and T cell markers.

(a-d) Mouse splenic B cells were purified from either WT or TLR9 KO mice. Cells were cultured in vitro in the presence or absence of CpG (1 µM) and/or Anti-IgM (5 µg/ml). Cell surface expression levels of CD86 (a), CD80 (b), MHC-II (c) and CD69 (d) at 24 and 48h post stimulation are shown as histogram overlays (left) and MFI graphs (right). Data represents more than three independent experiments, each performed with triplicates. (e-h) Time course of the change in the Fold MFI for CD86 (e), MHC-II (f) and CD69 (g). Data are representative of two independent experiments performed in triplicates. (h) MFI graph for T cell CD44 expression for the experiment outlined in Fig. 5d. Data represents three independent experiements each carried out in triplicates. Statistical significance was measured using two sided unpaired t-test (n.s.= P>0.05; *=0.01<P≤0.05; **= 0.001< P ≤0.01).

Supplementary Figure 5 Gating strategy for spleen cell analyses of chimeric mice and quantitation of TFH cells.

(a) Chimeric mice were generated as schematized. (b) After at least 8 weeks, the levels of reconstitution were evaluated by flow cytometry. Shown are flow cytometry plots of the distribution of CD45.1+ and CD45.2 + cells in the B cell gate. (c) Spleens from chimeric mice were analyzed 14 days post immunization with NP-CGG-alum plus CpG and immune cell subpopulations were quantified. Gating strategy to determine the total number of GC B cells, isotype switched B cells, cells of the PC lineage, TFH cells and the percent antigen-specific GC B cells and isotype switched B cells are schematized. (d,e) Shown are quantification of the number of TFH cells per spleen for µMT-MyD88 KO (red circles) or µMT-WT chimeras (blue triangles)(d) and representative flow cytometry plots for TFH cells (e). Each symbol in (d) represents an individual mouse. Data were pooled from two independent experiments. Dotted lines indicate mean values. Statistical significance was calculated using two sided Welch’s t test.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1-5 and Supplementary Table 1

Life Sciences Reporting Summary

Videos

Supplementary Video 1a

Time lapse TIRF images for 5 min showing DyLight 649 Anti-IgM Fab-labeled untreated MD4 mouse splenic B cells (No CpG) spreading and contracting over the PLB containing HEL antigen

Supplementary Video 1b

Time lapse TIRF images for 5 min showing DyLight 649 Anti-IgM Fab-labeled 20 min CpG pretreated MD4 mouse splenic B cells (20min CpG) spreading and contracting over the PLB containing HEL antigen

Supplementary Video 1c

Time lapse TIRF images for 5 min showing DyLight 649 Anti-IgM Fab-labeled 60 min CpG pretreated MD4 mouse splenic B cells (60 min CpG) spreading and contracting over the PLB containing HEL antigen

Supplementary Video 2a

3D reconstructed surface images using Imaris program from confocal Z stack images for CpG-untreated MD4 mouse splenic B cells labeled with DyLight 649 Anti-IgM Fab in response to the CM-DiI-labeled PMS containing Alexa Fluor 488-HEL. Shown is the movie for the 3D surface views followed by XZ, XY and YZ sliced views of the three channel surface images for BCR (Cyan), HEL (Magenta) and membrane (Yellow)

Supplementary Video 2b

3D reconstructed surface images using Imaris program from confocal Z stack images for CpG-treated MD4 mouse splenic B cells labeled with DyLight 649 Anti-IgM Fab in response to the CM-DiI-labeled PMS containing Alexa Fluor 488-HEL. Shown is the movie for the 3D surface views followed by XZ, XY and YZ sliced views of the three channel surface images for BCR (Cyan), HEL (Magenta) and membrane (Yellow)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Akkaya, M., Akkaya, B., Kim, A.S. et al. Toll-like receptor 9 antagonizes antibody affinity maturation. Nat Immunol 19, 255–266 (2018) doi:10.1038/s41590-018-0052-z

Download citation

Further reading