Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Molecular basis for potent B cell responses to antigen displayed on particles of viral size

Nature Immunology volume 24, pages 1762–1777 (2023)Cite this article

Subjects

Abstract

Multivalent viral epitopes induce rapid, robust and T cell-independent humoral immune responses, but the biochemical basis for such potency remains incompletely understood. We take advantage of a set of liposomes of viral size engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme. Particulate Ag induces potent ‘all-or-none’ B cell responses that are density dependent but affinity independent. Unlike soluble Ag, particulate Ag induces signal amplification downstream of the B cell receptor by selectively evading LYN-dependent inhibitory pathways and maximally activates NF-κB in a manner that mimics T cell help. Such signaling induces MYC expression and enables even low doses of particulate Ag to trigger robust B cell proliferation in vivo in the absence of adjuvant. We uncover a molecular basis for highly sensitive B cell responses to viral Ag display that is independent of encapsulated nucleic acids and is not merely accounted for by avidity and B cell receptor cross-linking.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: SVLS induce highly potent B cell responses that depend on ED but not affinity.
Fig. 2: pHEL triggers robust signaling downstream of the BCR that is insensitive to epitope affinity.
Fig. 3: Particulate Ag triggers bimodal signaling that is sensitive to dose and ED but not epitope affinity.
Fig. 4: pHEL signaling is independent of MYD88/IRAK1/IRAK4 but requires the canonical BCR pathway.
Fig. 5: pHEL triggers robust signal amplification downstream of the BCR.
Fig. 6: Soluble but not particulate Ag is restrained by LYN-dependent inhibitory signaling.
Fig. 7: pHEL triggers highly robust NF-κB signaling that mimics co-stimulation.
Fig. 8: pHEL triggers robust proliferation with a lower NUR77–GFP threshold than soluble stimuli.

Similar content being viewed by others

Data availability

No large data sets were generated for this manuscript. All data generated or analyzed during this study are included in the published article or are available upon request. Source data depicting unprocessed western blots and all statistical analyses are provided with this paper.

References

  1. Bachmann, M. F. & Zinkernagel, R. M. Neutralizing antiviral B cell responses. Annu. Rev. Immunol. 15, 235–270 (1997).

    CAS  PubMed  Google Scholar 

  2. Feldmann, M. & Easten, A. The relationship between antigenic structure and the requirement for thymus-derived cells in the immune response. J. Exp. Med. 134, 103–119 (1971).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Abbott, R. K. et al. Precursor frequency and affinity determine B cell competitive fitness in germinal centers, tested with germline-targeting HIV vaccine immunogens. Immunity 48, 133–146 (2018).

    CAS  PubMed  Google Scholar 

  4. Kato, Y. et al. Multifaceted effects of antigen valency on B cell response composition and differentiation in vivo. Immunity 53, 548–563 (2020).

  5. Dosenovic, P. et al. Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity. Proc. Natl Acad. Sci. USA 115, 4743–4748 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Desaymard, C. & Howard, J. G. Role of epitope density in the induction of immunity and tolerance with thymus-independent antigens. II. Studies with 2,4-dinitrophenyl conjugates in vivo. Eur. J. Immunol. 5, 541–545 (1975).

    CAS  PubMed  Google Scholar 

  7. Dintzis, H. M., Dintzis, R. Z. & Vogelstein, B. Molecular determinants of immunogenicity: the immunon model of immune response. Proc. Natl Acad. Sci. USA 73, 3671–3675 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Bachmann, M. F. et al. The influence of antigen organization on B cell responsiveness. Science 262, 1448–1451 (1993).

    CAS  PubMed  Google Scholar 

  9. Ingale, J. et al. High-density array of well-ordered HIV-1 spikes on synthetic liposomal nanoparticles efficiently activate B cells. Cell Rep. 15, 1986–1999 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Tolar, P. & Pierce, S. K. Unveiling the B cell receptor structure. Science 377, 819–820 (2022).

    CAS  PubMed  Google Scholar 

  11. Veneziano, R. et al. Role of nanoscale antigen organization on B-cell activation probed using DNA origami. Nat. Nanotechnol. 15, 716–723 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Pierce, S. K. & Liu, W. The tipping points in the initiation of B cell signalling: how small changes make big differences. Nat. Rev. Immunol. 10, 767–777 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Mattila, P. K., Batista, F. D. & Treanor, B. Dynamics of the actin cytoskeleton mediates receptor cross talk: an emerging concept in tuning receptor signaling. J. Cell Biol. 212, 267–280 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Gold, M. R. & Reth, M. G. Antigen receptor function in the context of the nanoscale organization of the B cell membrane. Annu. Rev. Immunol. 37, 97–123 (2019).

    CAS  PubMed  Google Scholar 

  15. Kwak, K., Akkaya, M. & Pierce, S. K. B cell signaling in context. Nat. Immunol. 20, 963–969 (2019).

    CAS  PubMed  Google Scholar 

  16. Tolar, P., Hanna, J., Krueger, P. D. & Pierce, S. K. The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens. Immunity 30, 44–55 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Batista, F. D., Iber, D. & Neuberger, M. S. B cells acquire antigen from target cells after synapse formation. Nature 411, 489–494 (2001).

    CAS  PubMed  Google Scholar 

  18. Batista, F. D. & Neuberger, M. S. B cells extract and present immobilized antigen: implications for affinity discrimination. EMBO J. 19, 513–520 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Batista, F. D. & Neuberger, M. S. Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. Immunity 8, 751–759 (1998).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  21. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Ambegaonkar, A. A. et al. Expression of inhibitory receptors by B cells in chronic human infectious diseases restricts responses to membrane-associated antigens. Sci. Adv. 6, eaba6493 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Sohn, H. W., Pierce, S. K. & Tzeng, S. J. Live cell imaging reveals that the inhibitory FcγRIIB destabilizes B cell receptor membrane–lipid interactions and blocks immune synapse formation. J. Immunol. 180, 793–799 (2008).

    CAS  PubMed  Google Scholar 

  24. Carrasco, Y. R. & Batista, F. D. B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27, 160–171 (2007).

    CAS  PubMed  Google Scholar 

  25. Pape, K. A., Catron, D. M., Itano, A. A. & Jenkins, M. K. The humoral immune response is initiated in lymph nodes by B cells that acquire soluble antigen directly in the follicles. Immunity 26, 491–502 (2007).

    CAS  PubMed  Google Scholar 

  26. Aung, A. et al. Low protease activity in B cell follicles promotes retention of intact antigens after immunization. Science 379, eabn8934 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Junt, T. et al. Subcapsular sinus macrophages in lymph nodes clear lymph-borne viruses and present them to antiviral B cells. Nature 450, 110–114 (2007).

    CAS  PubMed  Google Scholar 

  28. Gonzalez, S. F. et al. Trafficking of B cell antigen in lymph nodes. Annu. Rev. Immunol. 29, 215–233 (2011).

    CAS  PubMed  Google Scholar 

  29. Phan, T. G., Grigorova, I., Okada, T. & Cyster, J. G. Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nat. Immunol. 8, 992–1000 (2007).

    CAS  PubMed  Google Scholar 

  30. Cyster, J. G. B cell follicles and antigen encounters of the third kind. Nat. Immunol. 11, 989–996 (2010).

    CAS  PubMed  Google Scholar 

  31. Wholey, W. Y. et al. Synthetic liposomal mimics of biological viruses for the study of immune responses to infection and vaccination. Bioconjug. Chem. 31, 685–697 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Cheng, W. The density code for the development of a vaccine. J. Pharm. Sci. 105, 3223–3232 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Paus, D. et al. Antigen recognition strength regulates the choice between extrafollicular plasma cell and germinal center B cell differentiation. J. Exp. Med. 203, 1081–1091 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Zikherman, J., Parameswaran, R. & Weiss, A. Endogenous antigen tunes the responsiveness of naive B cells but not T cells. Nature 489, 160–164 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Goodnow, C. C. et al. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334, 676–682 (1988).

    CAS  PubMed  Google Scholar 

  36. Tan, C. et al. NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting. Nat. Immunol. 21, 1267–1279 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Wholey, W.-Y. et al. Initiation of neutralizing antibody response probed using synthetic virus-like structures. Preprint at bioRxiv https://doi.org/10.1101/2023.02.20.529089 (2023).

  38. Cyster, J. G. & Goodnow, C. C. Antigen-induced exclusion from follicles and anergy are separate and complementary processes that influence peripheral B cell fate. Immunity 3, 691–701 (1995).

    CAS  PubMed  Google Scholar 

  39. Chou, M. Y., Liu, D., An, J., Xu, Y. & Cyster, J. G. B cell peripheral tolerance is promoted by cathepsin B protease. Proc. Natl Acad. Sci. USA 120, e2300099120 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Martin, F. & Kearney, J. F. Marginal-zone B cells. Nat. Rev. Immunol. 2, 323–335 (2002).

    CAS  PubMed  Google Scholar 

  41. Dal Porto, J. M. et al. B cell antigen receptor signaling 101. Mol. Immunol. 41, 599–613 (2004).

    CAS  PubMed  Google Scholar 

  42. Lowe, J., Joseph, R. E. & Andreotti, A. H. Conformational switches that control the TEC kinase–PLCγ signaling axis. J. Struct. Biol. X 6, 100061 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Dickson, E. J. & Hille, B. Understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids. Biochem. J. 476, 1–23 (2019).

    CAS  PubMed  Google Scholar 

  44. Getahun, A. Role of inhibitory signaling in peripheral B cell tolerance. Immunol. Rev. 307, 27–42 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Stokoe, D. et al. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science 277, 567–570 (1997).

    CAS  PubMed  Google Scholar 

  46. Alessi, D. R. et al. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα. Curr. Biol. 7, 261–269 (1997).

    CAS  PubMed  Google Scholar 

  47. Chung, J. K. et al. Switch-like activation of Bruton’s tyrosine kinase by membrane-mediated dimerization. Proc. Natl Acad. Sci. USA 116, 10798–10803 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Depoil, D. et al. CD19 is essential for B cell activation by promoting B cell receptor–antigen microcluster formation in response to membrane-bound ligand. Nat. Immunol. 9, 63–72 (2008).

    CAS  PubMed  Google Scholar 

  49. Mattila, P. K. et al. The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling. Immunity 38, 461–474 (2013).

    CAS  PubMed  Google Scholar 

  50. Fearon, D. T. & Carroll, M. C. Regulation of B lymphocyte responses to foreign and self-antigens by the CD19/CD21 complex. Annu. Rev. Immunol. 18, 393–422 (2000).

    CAS  PubMed  Google Scholar 

  51. Chan, V. W., Meng, F., Soriano, P., DeFranco, A. L. & Lowell, C. A. Characterization of the B lymphocyte populations in Lyn-deficient mice and the role of Lyn in signal initiation and down-regulation. Immunity 7, 69–81 (1997).

    CAS  PubMed  Google Scholar 

  52. Xu, Y., Harder, K. W., Huntington, N. D., Hibbs, M. L. & Tarlinton, D. M. Lyn tyrosine kinase: accentuating the positive and the negative. Immunity 22, 9–18 (2005).

    PubMed  Google Scholar 

  53. Shinohara, H. & Kurosaki, T. Comprehending the complex connection between PKCβ, TAK1, and IKK in BCR signaling. Immunol. Rev. 232, 300–318 (2009).

    CAS  PubMed  Google Scholar 

  54. Finkin, S., Hartweger, H., Oliveira, T. Y., Kara, E. E. & Nussenzweig, M. C. Protein amounts of the MYC transcription factor determine germinal center B cell division capacity. Immunity 51, 324–336 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Au-Yeung, B. B. et al. A sharp T-cell antigen receptor signaling threshold for T-cell proliferation. Proc. Natl Acad. Sci. USA 111, E3679–E3688 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Heinzel, S. et al. A Myc-dependent division timer complements a cell-death timer to regulate T cell and B cell responses. Nat. Immunol. 18, 96–103 (2017).

    CAS  PubMed  Google Scholar 

  57. Tan, C., Noviski, M., Huizar, J. & Zikherman, J. Self-reactivity on a spectrum: a sliding scale of peripheral B cell tolerance. Immunol. Rev. 292, 37–60 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Dolmetsch, R. E., Lewis, R. S., Goodnow, C. C. & Healy, J. I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386, 855–858 (1997).

    CAS  PubMed  Google Scholar 

  59. Healy, J. I. et al. Different nuclear signals are activated by the B cell receptor during positive versus negative signaling. Immunity 6, 419–428 (1997).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Kalinke, U. et al. The role of somatic mutation in the generation of the protective humoral immune response against vesicular stomatitis virus. Immunity 5, 639–652 (1996).

    CAS  PubMed  Google Scholar 

  62. Nowosad, C. R., Spillane, K. M. & Tolar, P. Germinal center B cells recognize antigen through a specialized immune synapse architecture. Nat. Immunol. 17, 870–877 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Macauley, M. S. et al. Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis. J. Clin. Invest. 123, 3074–3083 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Scanlan, C. N., Offer, J., Zitzmann, N. & Dwek, R. A. Exploiting the defensive sugars of HIV-1 for drug and vaccine design. Nature 446, 1038–1045 (2007).

    CAS  PubMed  Google Scholar 

  65. de La Vega, M. A., Wong, G., Kobinger, G. P. & Qiu, X. The multiple roles of sGP in Ebola pathogenesis. Viral Immunol. 28, 3–9 (2015).

    PubMed  Google Scholar 

  66. Maity, P. C. et al. B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation. Sci. Signal 8, ra93 (2015).

    PubMed  Google Scholar 

  67. Ubelhart, R. et al. Responsiveness of B cells is regulated by the hinge region of IgD. Nat. Immunol. 16, 534–543 (2015).

    PubMed  Google Scholar 

  68. Gasparrini, F. et al. Nanoscale organization and dynamics of the siglec CD22 cooperate with the cytoskeleton in restraining BCR signalling. EMBO J. 35, 258–280 (2016).

    CAS  PubMed  Google Scholar 

  69. Shinohara, H. et al. Positive feedback within a kinase signaling complex functions as a switch mechanism for NF-κB activation. Science 344, 760–764 (2014).

    CAS  PubMed  Google Scholar 

  70. Berry, C. T., May, M. J. & Freedman, B. D. STIM- and Orai-mediated calcium entry controls NF-κB activity and function in lymphocytes. Cell Calcium 74, 131–143 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Berry, C. T. et al. BCR-induced Ca2+ signals dynamically tune survival, metabolic reprogramming, and proliferation of naive B cells. Cell Rep. 31, 107474 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Forsdyke, D. R. Two signal half-century: from negative selection of self-reactivity to positive selection of near-self-reactivity. Scand. J. Immunol. 89, e12746 (2019).

    PubMed  Google Scholar 

  73. Chackerian, B., Durfee, M. R. & Schiller, J. T. Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model. J. Immunol. 180, 5816–5825 (2008).

    CAS  PubMed  Google Scholar 

  74. Chackerian, B., Lowy, D. R. & Schiller, J. T. Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies. J. Clin. Invest. 108, 415–423 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Chen, Z. et al. Self-antigens displayed on liposomal nanoparticles above a threshold of epitope density elicit class-switched autoreactive antibodies independent of T cell help. J. Immunol. 204, 335–347 (2020).

    CAS  PubMed  Google Scholar 

  76. Hou, B., Reizis, B. & DeFranco, A. L. Toll-like receptors activate innate and adaptive immunity by using dendritic cell-intrinsic and -extrinsic mechanisms. Immunity 29, 272–282 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Rickert, R. C., Roes, J. & Rajewsky, K. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25, 1317–1318 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Wholey, W. Y., Yoda, S. T. & Cheng, W. Site-specific and stable conjugation of the SARS-CoV-2 receptor-binding domain to liposomes in the absence of any other adjuvants elicits potent neutralizing antibodies in BALB/c mice. Bioconjug. Chem. 32, 2497–2506 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. DeSantis, M. C., Kim, J. H., Song, H., Klasse, P. J. & Cheng, W. Quantitative correlation between infectivity and Gp120 density on HIV-1 virions revealed by optical trapping virometry. J. Biol. Chem. 291, 13088–13097 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Pang, Y., Song, H., Kim, J. H., Hou, X. & Cheng, W. Optical trapping of individual human immunodeficiency viruses in culture fluid reveals heterogeneity with single-molecule resolution. Nat. Nanotechnol. 9, 624–630 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Gallagher, M. P., Conley, J. M. & Berg, L. J. Peptide antigen concentration modulates digital NFAT1 activation in primary mouse naive CD8+ T cells as measured by flow cytometry of isolated cell nuclei. Immunohorizons 2, 208–215 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Lu, W. et al. The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P2 replenishment in response to TCR signaling during T cell development and survival. Nat. Immunol. 24, 136–147 (2023).

    CAS  PubMed  Google Scholar 

  83. Prado, D. S. et al. Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice. J. Biol. Chem. 297, 101330 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the following funders: R01AI155653 (W.C. and J.Z.), R01AI148487 (J.Z.) and R01AR069520 (J.Z.), an AAI Postdoctoral Fellowship (J.F.B. and J.Z.), NIH Training Grants T32 AI007334 (J.R. and H.V.N.) and T32 AR079068 (H.V.N.) and Rheumatology Research Foundation Scientist Development Award A138750 (H.V.N.). We thank A. Weiss for helpful discussions and critical reading of the manuscript. We thank A. Roque for help with mouse husbandry.

Author information

Author notes

  1. These authors contributed equally: Jeremy F. Brooks, Julianne Riggs.

Authors and Affiliations

  1. Division of Rheumatology, Rosalind Russell and Ephraim P. Engleman Rheumatology Research Center, Department of Medicine, University of California, San Francisco, CA, USA

    Jeremy F. Brooks, Julianne Riggs, James L. Mueller, Raisa Mathenge, Vivasvan S. Vykunta, Hailyn V. Nielsen & Julie Zikherman

  2. Biomedical Sciences Graduate Program, University of California, San Francisco, CA, USA

    Julianne Riggs

  3. Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI, USA

    Wei-Yun Wholey, Alexander R. Meyer, Sekou-Tidiane Yoda & Wei Cheng

  4. Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA

    Wei Cheng

Authors
  1. Jeremy F. Brooks
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julianne Riggs
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. James L. Mueller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raisa Mathenge
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei-Yun Wholey
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alexander R. Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sekou-Tidiane Yoda
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Vivasvan S. Vykunta
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hailyn V. Nielsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Wei Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Julie Zikherman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.F.B., J.R., J.L.M. and J.Z. designed the study, performed experiments and analyzed data. W.C. helped design the study and interpret data. R.M. performed experiments and analyzed data. W.-Y.W., A.R.M., S.-T.Y. and W.C. designed, generated and characterized all SVLSs and recombinant HEL protein used in this study. V.S.V. performed proliferation assays with titration of soluble protein affinity and dose. H.V.N. prepared cell lysates and performed western blots for Fig. 7. J.F.B., W.C. and J.Z. wrote the manuscript. J.R. helped write and edit the manuscript. J.Z. and W.C. conceptualized and funded the study. All authors read and approved the manuscript.

Corresponding authors

Correspondence to Wei Cheng or Julie Zikherman.

Ethics declarations

Competing interests

J.Z. serves as a scientific consultant for Walking Fish Therapeutics. W.C. has a patent pending on SVLSs. All other authors have no competing interests.

Peer review

Peer review information

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

Additional information

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

Extended data

Extended Data Fig. 1 pHEL SVLS library and in vitro potency.

a. Hen egg lysozyme (HEL) engineered with c-terminal cysteine residue and point mutations (zoomed box) described to reduce binding affinity to Hy10 BCR38. b. Schematic of SVLS library constructed with varying densities of HELD or HELT conjugated to liposomes yielding pHELD/T particles (3–500 epitope density/particle, calculated as [HEL]/[lip] in M). Each bar represents a single pHEL preparation to illustrate range. c. Congenically-marked NUR77-GFP reporter MD4 (Hy10) mixed with wild-type splenocytes and stimulated with pHELD or naked control liposome (pLIP02) for 24 hr. GFP was analysed in B220+ cells of each gt as in Main Fig. 1c. d. NUR77-GFP expression was analysed in MD4 B cells at 24 hr post-stimulation with the same batch of pHELD at various times post-synthesis, normalized to the pLIP02 condition in each experiment. e. As in main Fig. 1e but with pHELT at varying ED. Data is from a single experiment and representative of 3 independent experiments. f. As in E, but stimulated with high or ultra-low density pHELD/T. Data is from a single experiment. g. As in E, but depicting CD69 after 24 hr with pHELD/T. Data are from a single experiment and representative of 3 independent experiments. Curves were modelled with three-parameter nonlinear regression (C, E, F, G). h, i. SVLS with HELD conjugated at ED 124 and encapsulated AF594, herein termed pHELD(AF594), mixed at serial dilutions with MD4 cells for 24 hr and analyzed by flow cytometry. H. Quantification of AF594 fluorescence in B220+ lymphocytes. I. Representative histograms (left) depict AF594 fluorescence in B220+ cells. Inset values: AF594 MFI. Average particles/B cell in culture based on concentrations of each. Right-hand histograms depict 0.156pM-stimulated B cells sub-gated according to AF594 fluorescence to represent capture of particles. Data are from a single experiment and representative of 3 independent experiments.

Extended Data Fig. 2 pHEL signaling is sensitive to ED but not affinity and is potent in both follicular and MZ B cells.

a-c. MD4 splenocytes and lymphocytes were stimulated as in Main Fig. 2A,B but assessed for intra-cellular pS6 over a time course. Graphs (left) and histograms (middle) depict pS6 in B220+ cells from a single time course and is representative of 2 independent experiments. Graphs (right) depict B cell pS6 MFI after 20 min. Data are pooled from n = 5 (HELD), n = 6 (HELT) experiments. C. Graph depicts pHELT and pHELD data from A, B graphed together for comparison. A-C were compared by two-tailed paired parametric T-test, with mean depicted. d. As in 2 A, B but comparing pErk in stimulated B220+ splenocytes and lymph node cells separately at 20 mins. Histograms are representative of 2 independent experiments. e-g. As in 2 A, but splenocytes were stained with B220, CD21, and CD23 to identify pErk expression in subsets. Representative histograms show pErk in gated subsets following sHEL-WT 1 μg/ml, 1pM pHELT-HD (ED 256), or PMA stimulation. F. Quantification and mean of %pErk+ cells from three independent experiments (G). Groups were compared by one-way ANOVA. Data in E-G are representative of 8 independent experiments. h, i. Pooled MD4 splenocytes and lymphocytes were loaded with Indo-1 calcium indicator dye, stained with surface markers to detect B220+ subsets (H) as in S2E, and stimulated with anti-IgM (10 μg/ml), sHELD (1 μg/ml), or pHELD-HD (1pM). Calcium entry was assessed by flow cytometry for >= 3 minutes. MFI of bound/unbound Indo-1 fluorescence over time is plotted (I). Data are representative of 4 independent experiments. J. As in Main Fig. 3a,b except CD23+ B cells stimulated with 10, 1, or 0.1pM doses of either pHELT-HD (ED 256) or pHELT (ED 3). Data are representative of 4 independent experiments.

Extended Data Fig. 3 CD19 is dispensable in vitro but promotes B cell expansion in response to particulate Ag in vivo.

a. 20’ Phosflow assay to detect pErk in Cd19+/– or Cd19–/– MD4 splenocytes after stimulation with either control pLIP02 1pM, 10 μg/ml anti-IgM, 1 mg/ml sHELD, or 1pM pHELD-HD. Samples were co-stained with B220, CD23, and CD21 to detect B cell subsets. Histograms depict CD23+ B cells and are representative of 4 independent experiments. b-g. As in Main Fig. 1i, lymphocytes from MD4 mice were loaded with CTV and those from MD4.CD19-/- mice were loaded with CTY. Cells were mixed at a 1:1 ratio prior to adoptive transfer i.v. of 5 × 106 cells into CD45.1 hosts. Time course, immunization, and d3 harvest as in Fig. 1i. MD4 data correspond to those displayed in Main Fig. 1i–k. b. Experimental schematic. c. Representative gating scheme to identify donor IgM[a]+ B cells with either CTV or CTY fluorescence; L/D, LIVE/DEAD stain. d. Representative histograms depict live IgM[a]+ donor lymphocytes in unimmunized (top) or immunized hosts. e. Graph depicts mean + SEM for % distribution of IgM[a]+ donor B cells of each genotype across cell divisions. Statistical analysis by Two-way ANOVA in supplement. f. Graphs depict division and proliferation indices. g. Ratio of CTV/CTY for donor T cells and IgM[a]+ donor B220+ B cells are plotted for each host according to immunization condition. Data in F and G depict mean of 4 biological replicates and were compared by a two-tailed paired parametric T-test.

Extended Data Fig. 4 pHEL evades LYN-dependent inhibition.

a, b. As in Main Fig. 6b,c but with broader titration of pHELT-HD (ED 256) dose from 0.1–10 pM. Histograms depict pErk in CD23+ B220+ cells. MD4 histograms in A correspond to those depicted in Extended Data Fig. 2j and are representative of 3 independent experiments. c. As in Main Fig. 6b, but with sHELT and pHELT (HD/LD 256, 64) stimuli to compare varied epitope density. Data are representative of 4 independent experiments. d. As in Main Fig. 6b,c, except pHELD ultra-low ED 6 and pHELT ultra-low ED 3 are used at 10 pM and 1 pM doses. Data from unstimulated and anti-IgM conditions are re-plotted from panel B for ease of comparison. Graphs in B and D each depict data from 3 independent experiments with mean. Pre-specified groups were compared by two-tailed unpaired parametric T-tests. e-h. corresponding to Main Fig. 6e, lymphocytes from MD4 mice were loaded with CTV and those from MD4.Lyn-/- mice were loaded with CTY and mixed at a 1:3 ratio for adoptive transfer of 5 × 106 cells into CD45.1 hosts prior to immunization one day later with pHELT-HD ~ 0.1 μg. e. Representative gating scheme to identify live donor IgM[a]+ B220+ cells with either CTV or CTY fluorescence; L/D, LIVE/DEAD stain. f. Ratio of CTV/CTY in donor T cells and IgM[a]+ donor B220+ cells are plotted for each host according to immunization condition with mean depicted. g. Graph depicts mean + SEM for % distribution of IgM[a]+ donor B cells of each genotype across cell divisions and data was compared by two-way ANOVA with Holm-Sidak correction for multiple comparisons. h. Graphs depict division and proliferation indices. Lines connecting samples indicate donor cells of each genotype from an individual host. Groups in H were compared by a two-tailed parametric paired T-test. Data in E-H represents 5 biological replicates.

Extended Data Fig. 5 Soluble stimuli dominantly suppress pHEL signaling.

a. As in Main Fig. 6f, but with inclusion of anti-IgM 10 μg/ml as second stimulus following pHELD pre-treatment. Data are representative of 4 independent experiments. b. Data correspond to Main Fig. 6g but depict same samples with single cell resolution. c. As in Main Fig. 6g but with sHEL 1 μg/ml added at peak calcium response following pHEL stimulation. Data are representative of 3 independent experiments.

Extended Data Fig. 6 pHEL robustly trigger NF-κB independently of MYD88.

a. As in Main Fig. 7b-d except histograms showing IκBα degradation at 20 minutes post-stimulation for soluble stimuli sHELD/T and pHELT/D with high or low ED (HD vs. LD). Inset triangles represent decreasing concentration of pHEL (10, 1, 0.1 pM). Data are representative of >= 4 independent experiments. b. As in Main Fig. 6b, except splenocytes from Lyn+/+ and Lyn–/– MD4 mice were stained to detect intracellular IκB in CD23+ B cells at 20 minutes. Data are representative of 3 independent experiments. c. Graph corresponds to data in Main Fig. 7g,h except nuclear p65 MFI rather than % p65 positive nuclei are quantified. Graph depicts data with mean from 3 independent experiments. Groups were compared by paired two-tailed parametric T-tests. d. Nuclear p65 translocation is detected as in Fig. 7f except stimuli are applied on ice for 15 min each, first 10pM pHELT-HD +/- superimposed soluble stimuli (anti-IgM 10 μg/ml or sHEL-WT 1 μg/ml), followed by 20 min 37 C incubation as in Main Fig. 6f. e. As in Main Fig. 4c,d except stained to detect intracellular IκB in CD23 + B cells at 20 minutes. Data are representative of 3 independent experiments. In panels A, B and E, line in offset histograms references internal negative control.

Extended Data Fig. 7 pHEL reduces NUR77-GFP threshold for B cell division.

a. MD4.NUR77-GFP lymphocytes were cultured for 24 hr with sHELD 1 μg/ml or 1pM pHEL. Ratio of NUR77-GFP and intra-cellular MYC among B220+ cells from a single experiment, representative of 3 independent experiments. b. MD4 lymphocytes incubated at 4 C with 1st stimulus (1pM pHELT-HD) for 15 min, followed by 2nd stimulus (or media) for 15 min. Soluble stimuli: 1 μg/ml anti-IgM or 0.1 μg/ml sHEL-WT. After 48 hr culture at 37 C without BAFF, viable frequencies of B220+ cells are plotted from 4 independent experiments, compared by a two-tailed unpaired parametric T-test. c. As in Main Fig. 8e except histograms depict B220 + FSC-A, representative of 4 independent experiments. d. CTV-loaded NUR77-GFP splenocytes (+/-MD4 Tg) cultured for 72 hr with 20 ng/ml BAFF + either 6.4 μg/ml anti-IgM or 30 ng/ml sHEL, and ibrutinib titration (0–50 nM). Plots depict CTV and GFP expression in B220+ cells, representative of 3 independent experiments. e. NUR77-GFP splenocytes cultured with 20 ng/ml BAFF as in D except with anti-IgM +/-10ng/ml IL-4. Plots depict CTV and GFP expression in B220+ cells, representative of 6 independent experiments. f. As in Main Fig. 8f,g MD4.NUR77-GFP lymphocytes were cultured with stimuli +20 ng/ml BAFF for 72 hrs: 1 μg/ml sHELD/T, 1pM pHELD/T-HD/LD. These data correspond to Fig. 8f,g but include plots and quantification for pHELD/T-LD for comparison. Shaded bars reference NUR77-GFP levels in dividing B cells stimulated with sHELD/sHELT. (Bottom) Quantification of NUR77-GFP levels in the first division. Data are representative of 5 independent experiments. g. As in Main Fig. 8h, but comparing intra-cellular MYC among CTV-loaded B220+ cells after 72 hr culture: pHELT (1, 0.1, 0.01pM) and sHELT (500, 50, 5 ng/ml). Inset values: MFI of division 1 cells. Data are representative of 3 independent experiments. h. As in E, but cultured with anti-IgM(6.4 μg/ml) and titration of ibrutinib, comparing MYC among CTV-loaded B220+ cells after 72 hr culture. Data are representative of 6 independent experiments.

Extended Data Fig. 8 pHEL partially activates anergic B cells.

a, b. As in Main Figs. 2e,f and 3c except cells from MD4 or MD4.ML5 mice were stimulated with indicated reagents: anti-IgM 10 μg/ml, sHELD 1 μg/ml, or pHELD-HD (ED 286) 1 pM. Indo-1 ratio is shown on a linear scale. Gated in B are corresponding time gates to capture fraction of responding or non-responding B cells. Data are representative of 3 independent experiments. c, d. As in 8 A, B except pooled splenocytes and lymphocytes from MD4 (naïve) or MD4.ML5 (anergic) mice were incubated with indicated stimuli for 24 hours (sHEL-WT 1 μg/ml or pHELT-HD 1pM). Histograms and graphs depict MFI of CD86, CD69, or intra-cellular MYC in live B220 B cells. Graphs depict data and mean from and histograms are representative of 3 (MD4/ML5) or 4 (MD4) independent experiments. MD4 data in graphs corresponds to a subset of data plotted in Fig. 8b. Data were compared by two-tailed unpaired parametric t-test with Holm-Sidak correction. e-g. Viable frequencies of CTV-loaded MD4 (naïve) or MD4.ML5 (anergic) lymph node B cells in culture at 72-hours post-stimulation with sHELD/T and pHELD/T in the presence or absence of 20 ng/ml BAFF assessed via live/dead indicator dye. E. Representative plots depict gating to identify live B cells from total B220+ cells at 72 hr with corresponding histograms below depicting vital dye dilution of live B220+ cells at the same time point. Data are representative of 4 independent experiments. F. Absolute live B cell counts at sequential time points assessed using counting beads from a single experiment. G. Graphs depict %live B220 + B cells from total lymphocyte gate. Each datapoint is a sample from 4 independent experiments (except N = 3 for MD4 with no BAFF) independent experiments; lines connect samples from the same experiment. Groups were compared by a two-tailed paired parametric T-test.

Extended Data Fig. 9 Model.

a. Signalosome assembly in naïve B cells upon BCR stimulation by soluble Ag. BCR signal transduction requires sequential action of Src family kinases (SFKs) and SYK kinase. CD19 engagement amplifies PI3K activation and production of PI(3,4,5)P3. While multiple SFKs can mediate ITAM signaling downstream of the BCR, the SFK LYN plays a non-redundant role in phosphorylating ITIM-containing inhibitory coreceptors which in turn recruit PTPases SHP1 and SHIP1 that suppress PIP3. Dynamic regulation of PIP3 at the plasma membrane controls amplitude of signaling by recruiting downstream mediators including AKT, BTK, and PLCγ2 to orchestrate transcriptional programs mediated by NFAT, NF-κB and other factors. b. SVLS with appropriately spaced epitopes robustly engage pre-existing BCR nanoclusters but evade co-inhibitory receptors and results in downstream signal amplification. SVLS do not rely upon CD19 engagement for signal amplification in vitro. In the absence of inhibitory PTPase engagement via ITIM -containing inhibitory receptors, PIP3 accumulates at the plasma membrane leading to enhanced and prolonged signalosome assembly and activity downstream of SVLS stimulation. Robust NFAT and NF-κB accumulation in the nucleus and AKT-dependent signals mimic co-stimulation and promote MYC expression, resulting in T-independent cell growth, survival, and proliferation.

Supplementary information

Source data

Source Data Fig. 5

Unprocessed western blots for Fig. 5.

Source Data Fig. 6

Unprocessed western blots for Fig. 6a.

Source Data Fig. 7

Unprocessed western blots for Fig. 7a.

Source Data

Statistical source data for all main and Extended Data figures.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Brooks, J.F., Riggs, J., Mueller, J.L. et al. Molecular basis for potent B cell responses to antigen displayed on particles of viral size. Nat Immunol 24, 1762–1777 (2023). https://doi.org/10.1038/s41590-023-01597-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41590-023-01597-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing