Abstract

Immunoglobulin G3 (IgG3) has an uncertain role in the response to infection with and vaccination against human immunodeficiency virus (HIV). Here we describe a regulatory role for IgG3 in dampening the immune system–activating effects of chronic HIV viremia on B cells. Secreted IgG3 was bound to IgM-expressing B cells in vivo in HIV-infected chronically viremic individuals but not in early-viremic or aviremic individuals. Tissue-like memory (TLM) B cells, a population expanded by persistent HIV viremia, bound large amounts of IgG3. IgG3 induced clustering of B cell antigen receptors (BCRs) on the IgM+ B cells, which was mediated by direct interactions between soluble IgG3 and membrane IgM of the BCR (IgM-BCR). The inhibitory IgG receptor CD32b (FcγRIIb), complement component C1q and inflammatory biomarker CRP contributed to the binding of secreted IgG3 onto IgM-expressing B cells of HIV-infected individuals. Notably, IgG3-bound TLM B cells were refractory to IgM-BCR stimulation, thus demonstrating that IgG3 can regulate B cells during chronic activation of the immune system.

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References

  1. 1.

    Lane, H. C. et al. Abnormalities of B-cell activation and immunoregulation in patients with the acquired immunodeficiency syndrome. N. Engl. J. Med. 309, 453–458 (1983).

  2. 2.

    Moir, S. & Fauci, A. S. B-cell responses to HIV infection. Immunol. Rev. 275, 33–48 (2017).

  3. 3.

    Moir, S. et al. B cells in early and chronic HIV infection: evidence for preservation of immune function associated with early initiation of antiretroviral therapy. Blood 116, 5571–5579 (2010).

  4. 4.

    Moir, S. et al. Evidence for HIV-associated B cell exhaustion in a dysfunctional memory B cell compartment in HIV-infected viremic individuals. J. Exp. Med. 205, 1797–1805 (2008).

  5. 5.

    Ho, J. et al. Two overrepresented B cell populations in HIV-infected individuals undergo apoptosis by different mechanisms. Proc. Natl. Acad. Sci. USA 103, 19436–19441 (2006).

  6. 6.

    Meffre, E. et al. Maturational characteristics of HIV-specific antibodies in viremic individuals. JCI Insight 1, e84610 (2016).

  7. 7.

    Karnell, J. L. et al. Role of CD11c+ T-bet+ B cells in human health and disease. Cell. Immunol. 321, 40–45 (2017).

  8. 8.

    Naradikian, M. S., Hao, Y. & Cancro, M. P. Age-associated B cells: key mediators of both protective and autoreactive humoral responses. Immunol. Rev. 269, 118–129 (2016).

  9. 9.

    Portugal, S., Obeng-Adjei, N., Moir, S., Crompton, P. D. & Pierce, S. K. Atypical memory B cells in human chronic infectious diseases: An interim report. Cell. Immunol. 321, 18–25 (2017).

  10. 10.

    Pupovac, A. & Good-Jacobson, K. L. An antigen to remember: regulation of B cell memory in health and disease. Curr. Opin. Immunol. 45, 89–96 (2017).

  11. 11.

    Knox, J. J., Kaplan, D. E. & Betts, M. R. T-bet-expressing B cells during HIV and HCV infections. Cell. Immunol. 321, 26–34 (2017).

  12. 12.

    Barnett, B. E. et al. Cutting edge: B cell-intrinsic T-bet expression is required to control chronic viral infection. J. Immunol. 197, 1017–1022 (2016).

  13. 13.

    Rubtsov, A. V. et al. Toll-like receptor 7 (TLR7)-driven accumulation of a novel CD11c+ B-cell population is important for the development of autoimmunity. Blood 118, 1305–1315 (2011).

  14. 14.

    Rubtsova, K. et al. B cells expressing the transcription factor T-bet drive lupus-like autoimmunity. J. Clin. Invest. 127, 1392–1404 (2017).

  15. 15.

    Peng, S. L., Szabo, S. J. & Glimcher, L. H. T-bet regulates IgG class switching and pathogenic autoantibody production. Proc. Natl. Acad. Sci. USA 99, 5545–5550 (2002).

  16. 16.

    Pekkarinen, P. T. et al. Dysregulation of adaptive immune responses in complement C3-deficient patients. Eur. J. Immunol. 45, 915–921 (2015).

  17. 17.

    Mortensen, R. et al. Adaptive immunity against Streptococcus pyogenes in adults involves increased IFN-γ and IgG3 responses compared with children. J. Immunol. 195, 1657–1664 (2015).

  18. 18.

    Knox, J. J. et al. T-bet+ B cells are induced by human viral infections and dominate the HIV gp140 response. JCI Insight 2, e92943 (2017).

  19. 19.

    Buckner, C. M. et al. Characterization of plasmablasts in the blood of HIV-infected viremic individuals: evidence for nonspecific immune activation. J. Virol. 87, 5800–5811 (2013).

  20. 20.

    Roux, K. H., Strelets, L. & Michaelsen, T. E. Flexibility of human IgG subclasses. J. Immunol. 159, 3372–3382 (1997).

  21. 21.

    Lefranc, M. P. & Lefranc, G. Human Gm, Km, and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism. Methods Mol. Biol. 882, 635–680 (2012).

  22. 22.

    Michaelsen, T. E., Sandlie, I., Bratlie, D. B., Sandin, R. H. & Ihle, O. Structural difference in the complement activation site of human IgG1 and IgG3. Scand. J. Immunol. 70, 553–564 (2009).

  23. 23.

    Morell, A., Terry, W. D. & Waldmann, T. A. Metabolic properties of IgG subclasses in man. J. Clin. Invest. 49, 673–680 (1970).

  24. 24.

    Dugast, A. S. et al. Independent evolution of Fc- and Fab-mediated HIV-1-specific antiviral antibody activity following acute infection. Eur. J. Immunol. 44, 2925–2937 (2014).

  25. 25.

    Chung, A. W. et al. Dissecting polyclonal vaccine-induced humoral immunity against HIV using systems serology. Cell 163, 988–998 (2015).

  26. 26.

    Scharf, O. et al. Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1. J. Virol. 75, 6558–6565 (2001).

  27. 27.

    Yates, N. L. et al. Vaccine-induced Env V1-V2 IgG3 correlates with lower HIV-1 infection risk and declines soon after vaccination. Sci. Transl. Med. 6, 228ra39 (2014).

  28. 28.

    Yates, N. L. et al. Multiple HIV-1-specific IgG3 responses decline during acute HIV-1: implications for detection of incident HIV infection. AIDS 25, 2089–2097 (2011).

  29. 29.

    Wesemann, D. R. et al. Immature B cells preferentially switch to IgE with increased direct Sμ to Sε recombination. J. Exp. Med. 208, 2733–2746 (2011).

  30. 30.

    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).

  31. 31.

    Lu, J. et al. Structural recognition and functional activation of FcγR by innate pentraxins. Nature 456, 989–992 (2008).

  32. 32.

    Bang, R. et al. Analysis of binding sites in human C-reactive protein for FcγRI, FcγRIIA, and C1q by site-directed mutagenesis. J. Biol. Chem. 280, 25095–25102 (2005).

  33. 33.

    Bharadwaj, D., Stein, M. P., Volzer, M., Mold, C. & Du Clos, T. W. The major receptor for C-reactive protein on leukocytes is Fcgγ receptor II. J. Exp. Med. 190, 585–590 (1999).

  34. 34.

    Bruhns, P. et al. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. Blood 113, 3716–3725 (2009).

  35. 35.

    Seeling, M., Brückner, C. & Nimmerjahn, F. Differential antibody glycosylation in autoimmunity: sweet biomarker or modulator of disease activity? Nat. Rev. Rheumatol. 13, 621–630 (2017).

  36. 36.

    Sjögren, J. et al. EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein. Biochem. J. 455, 107–118 (2013).

  37. 37.

    Noto, A. & Pantaleo, G. B-cell abnormalities and impact on antibody response in HIV infection. Curr. Opin. HIV AIDS 12, 203–208 (2017).

  38. 38.

    Pallikkuth, S., de Armas, L., Rinaldi, S. & Pahwa, S. T follicular helper Cells and B cell dysfunction in aging and HIV-1 infection. Front. Immunol. 8, 1380 (2017).

  39. 39.

    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).

  40. 40.

    Liu, W., Won Sohn, H., Tolar, P., Meckel, T. & Pierce, S. K. Antigen-induced oligomerization of the B cell receptor is an early target of FcγRIIB inhibition. J. Immunol. 184, 1977–1989 (2010).

  41. 41.

    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).

  42. 42.

    Sörman, A., Zhang, L., Ding, Z. & Heyman, B. How antibodies use complement to regulate antibody responses. Mol. Immunol. 61, 79–88 (2014).

  43. 43.

    Ugurlar, D. et al. Structures of C1-IgG1 provide insights into how danger pattern recognition activates complement. Science 359, 794–797 (2018).

  44. 44.

    Hogarth, P. M. & Pietersz, G. A. Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond. Nat. Rev. Drug Discov. 11, 311–331 (2012).

  45. 45.

    Xu, L. et al. Impairment on the lateral mobility induced by structural changes underlies the functional deficiency of the lupus-associated polymorphism FcγRIIB-T232. J. Exp. Med. 213, 2707–2727 (2016).

  46. 46.

    Clatworthy, M. R. et al. Systemic lupus erythematosus-associated defects in the inhibitory receptor FcγRIIb reduce susceptibility to malaria. Proc. Natl. Acad. Sci. USA 104, 7169–7174 (2007).

  47. 47.

    Willcocks, L. C. et al. A defunctioning polymorphism in FCGR2B is associated with protection against malaria but susceptibility to systemic lupus erythematosus. Proc. Natl. Acad. Sci. USA 107, 7881–7885 (2010).

  48. 48.

    Son, M., Diamond, B. & Santiago-Schwarz, F. Fundamental role of C1q in autoimmunity and inflammation. Immunol. Res. 63, 101–106 (2015).

  49. 49.

    Nezlin, R., & Ghetie, V. Interactions of immunoglobulins outside the antigen-combining site. Adv. Immunol. 82, 155–215 (2004).

  50. 50.

    Molinos-Albert, L. M., Clotet, B., Blanco, J. & Carrillo, J. Immunologic insights on the membrane proximal external region: a major human immunodeficiency virus type-1 vaccine target. Front. Immunol. 8, 1154 (2017).

  51. 51.

    Sohn, H. W., Krueger, P. D., Davis, R. S. & Pierce, S. K. FcRL4 acts as an adaptive to innate molecular switch dampening BCR signaling and enhancing TLR signaling. Blood 118, 6332–6341 (2011).

  52. 52.

    Cerutti, A. et al. CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM+IgD+ B cell line. J. Immunol. 160, 2145–2157 (1998).

  53. 53.

    Brunner, M. & Sigal, L. H. Immune complexes from serum of patients with Lyme disease contain Borrelia burgdorferi antigen and antigen-specific antibodies: potential use for improved testing. J. Infect. Dis. 182, 534–539 (2000).

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Acknowledgements

We thank the study volunteers for their participation in this study; C. Rehm and S. Jones for specimen procurement; A.S. Fauci for discussions and suggestions. This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

Author information

Author notes

  1. These authors contributed equally to this work: Lela Kardava, Haewon Sohn.

Affiliations

  1. Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    • Lela Kardava
    • , Christine Youn
    • , James W. Austin
    • , Wei Wang
    • , Clarisa M. Buckner
    • , J. Shawn Justement
    • , Valerie A. Melson
    • , Marissa A. Hand
    • , Michael C. Sneller
    • , Tae-Wook Chun
    •  & Susan Moir
  2. Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    • Haewon Sohn
    • , Gwynne E. Roth
    • , Peter D. Sun
    •  & Susan K. Pierce
  3. Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD, USA

    • Kathleen R. Gittens
    •  & Richard W. Kwan
  4. Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD, USA

    • Yuxing Li
  5. Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA

    • Yuxing Li

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Contributions

L.K., H.S. and J.W.A. designed and performed experiments, analyzed data and helped write the manuscript; C.Y., W.W., C.M.B., J.S.J., V.A.M, G.E.R and T.-W.C. designed and performed experiments and analyzed data; M.A.H., K.R.G. and T.-W.C. recruited study subjects and coordinated procedures and sample collection; R.W.K. and M.C.S. oversaw clinical aspects of study-subject eligibility, participation and care; Y.L. provided critical resources and scientific input; P.D.S and S.K.P. provided scientific input and helped design experiments; S.K.P. helped write the manuscript; and S.M. supervised the study, oversaw protocol logistics and wrote the manuscript.

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The authors declare no competing interests.

Corresponding author

Correspondence to Susan Moir.

Integrated supplementary information

  1. Supplementary Figure 1 Additional cell-surface analyses.

    (a) Flow cytometry of CD20-gated B cells isolated from the peripheral blood of HIV-negative, HIV-aviremic and chronically infected HIV-viremic individuals, and stained for IgG1 and total IgG (tIgG) with pan anti-IgG mAb. (b) Flow cytometry of CD20-gated B cells isolated from the peripheral blood of a chronically infected HIV-viremic individual, and stained for IgG3 and tIgG with pan anti-IgG mAb clones G18-145, M1310G05 and ICO-97. (c) Gating strategy to evaluate mean fluorescence intensities (MFI) of B cell-bound IgG3. Staining for IgG3 and tIgG (clone G18-145) was used to identify cell-bound IgG3 by excluding tIgG/IgG3 double positive CD20-gated B cells with a clear diagonal pattern. IgG3 MFI was then determined for all B cells, as well as tissue-like memory (TLM) and naïve (N) B cells defined by expression of CD27 and CD21, as indicated in the representative plot and histograms. Data are representative of 10 (HIV-negative), 14 (HIV-aviremic) and 49 (HIV-viremic) individual experiments (a) or one experiment representative of eight independent experiments (b) or one representative of 106 individual experiments (c).

  2. Supplementary Figure 2 Factors that influence IgG3+IgM+ B cells.

    (a) Longitudinal flow cytometry for IgG3 and IgM of CD20-gated B cells isolated from an HIV-infected individual during early viremia and one year later in chronic stage of viremia. Similarly, B cells from a chronically infected HIV-viremic individual were stained for IgG3 and IgM before and 10 years after sustained suppression of viremia by ART. (b) Comparison by stage of HIV infection and status of viremia of frequencies of IgG3+IgM+ and TLM B cells measured by flow cytometry, as well as of HIV viral load and CD4+ T-cell counts. Dotted line represents limit of detection. (c) Comparison by stage of HIV infection and status of viremia of mean fluorescence intensities (MFI) for IgG3 measured by flow cytometry of CD20-gated TLM and naïve B cells where IgG3-expressing B cells were excluded, as described in Methods and Supplementary Fig. 1c. (d) Comparison of IgG3 MFI on TLM versus naïve B cells of 92 HIV-viremic individuals, all stages of disease combined. Red horizontal bars represent medians; each symbol (b,c) represents an individual in early viremia (n = 16), chronic viremia (n = 73) or aviremia (n = 17); * P< 0.05, ** P< 0.01, *** P 0.001 and **** P< 0.0001; n.s., not significant (two-tailed Mann-Whitney test after obtaining significance by Kruskal-Wallis ANOVA test on full set (b,c) or two-tailed Wilcoxon matched-pairs signed rank test (d)). Data are representative of five (early to chronic viremic) and seven (chronic viremic to aviremic) individual experiments (a).

  3. Supplementary Figure 3 PEG-IgG3 isolated from serum of HIV-viremic individuals with high-intensity IgG3 on IgM+ B cells binds to B cells of HIV-negative individuals.

    (a) Flow cytometry of CD20-gated B cells of an HIV-negative individual incubated without (no treatment) or with PEG-IgG3 isolated from sera of HIV-viremic individuals with low- or high-intensity IgG3 on IgM+ B cells. Bottom: cumulative data from paired sera of HIV-viremic individuals with high (yellow; n = 12) or low (light green; n = 12) intensity IgG3 on IgM+ B cells. Heat map depicts % of IgM-expressing B cells from HIV-negative individuals with bound IgG3 following incubation with serum-derived PEG precipitate or bead-enriched IgG3. PEG-IgG3 isolated with serum from individuals with high-intensity IgG3 on IgM+ B cells bound B cells of HIV-negative individuals with a median 39%, compared to 0% from those with low-intensity IgG3 on IgM+ B cells (P = 0.005). For bead-enriched IgG3, the medians were 8.2% for high versus 1.8% for low-intensity IgG3 on IgM+ B cells (P = 0.009). P values, two-tailed Wilcoxon matched-pairs signed rank test. (b) Flow cytometry of CD20-gated B cells of an HIV-negative individual incubated without (no treatment) or with PEG-IgG3 isolated from the serum of HIV-viremic individuals with low- or high-intensity IgG3 on IgM+ B cells. The plots show IgG3 and IgM staining for TLM and naïve B cells, defined by the expression of CD21 and CD27. (c) Comparison of calcium uptake by HIV-negative IgD+ (D) TLM and naïve (N) B-cell IgG3+/– (G3) populations treated with PEG-IgG3 isolated from serum of chronically infected HIV-viremic individuals with moderate to high-intensity IgG3 on IgM+ B cells. Each color coded symbol represents an individual (n = 6); black horizontal bars represent medians; * P< 0.05; n.s., not significant (two-tailed Wilcoxon matched-pairs signed rank test after obtaining significance by Friedman ANOVA test on full set). Data are representative of 12 individual experiments (b).

  4. Supplementary Figure 4 IgG3 serum levels and binding to HIV-negative B cells.

    (a) Comparison of levels and ratios of IgG subclasses in serum of HIV-viremic individuals with high (n = 12) or low (n = 12) intensity IgG3 on IgM+ B cells (same serum used in Supplementary Fig. 3). Red horizontal bars represent medians; *** P< 0.001 and **** P< 0.0001; n.s., not significant (two-tailed Mann-Whitney test). (b) Flow plots of CD20-gated B cells of an HIV-negative individual incubated without (no treatment) or with heat-aggregated IgG3 fractionated into SEC-1 or SEC-2. (c) Flow plots of CD20-gated B cells of an HIV-negative individual incubated without (no treatment) or with PEG precipitate or with SEC-1 or SEC-2 fractions isolated from PEG-precipitated serum of an HIV-viremic individual with high-intensity IgG3 on IgM+ B cells. Right, cumulative data from sera of HIV-viremic individuals with moderate- to high-intensity IgG3 on IgM+ B cells. Each color coded symbol represents an individual (n = 6); black horizontal bars represent medians; * P< 0.05 (two-tailed Wilcoxon matched-pairs signed rank test after obtaining significance by Friedman ANOVA test on full set). Data are representative of three (b) or six (c) independent experiments.

  5. Supplementary Figure 5 Role of CD32b, C1q, CRP and glycosylation in non-aggregated IgG3 binding to B cells of HIV-negative individuals.

    (a) Immunoblot analysis of C1q, IgG3 and CRP content in PEG precipitate (PEG), SEC-1, SEC-2 or SEC-2 depleted of C1q (C1q-d SEC-2) using serum of an HIV-viremic individual with moderate-intensity IgG3 on IgM+ B cells. The gels are shown in the order the membrane was sequentially stained and stripped. Lanes marked IgG3 and C1q indicate respective protein controls. (b) Flow cytometry of CD20-gated B cells of an HIV-negative individual incubated without PEG-IgG3 (no treatment), with PEG-IgG3 from serum of an HIV-viremic individual with moderate-intensity IgG3 on IgM+ B cells treated with control rabbit IgG or rabbit anti-C1q or rabbit anti-CRP antibody. Right, cumulative pattern from sera of chronically infected HIV-viremic individuals with moderate- to high-intensity IgG3 on IgM+ B cells. (c) Flow cytometry of CD20-gated B cells of an HIV-negative individual stained for IgG3 and IgM. The B cells were untreated or treated with antibody to CD32b or control goat IgG prior to addition of PEG-IgG3 from an HIV-viremic individual with high-intensity IgG3 on IgM+ B cells. Graph: cumulative effect of pre-treating B cells with anti-CD32b prior to addition of PEG-IgG3 from HIV-viremic individuals with moderate- to high-intensity IgG3 on IgM+ B cells (n = 6). (d) Effect of treating PEG-IgG3 prepared from individuals with moderate- to high-intensity IgG3 on IgM+ B cells with PNGase F or EndoS2 on binding to B cells of HIV-negative individuals. Each color coded symbol (b,d) represents an individual (b, n = 7; d, n = 6); black horizontal bars represent medians; * P< 0.05; n.s., not significant (two-tailed Wilcoxon matched-pairs signed rank test, after (b,d) obtaining significance by Friedman ANOVA test on full set). Data are representative of eight independent experiments (a).

Supplementary information

  1. Supplementary Figures

    Supplementary Figures 1–5, Supplementary Table 1

  2. Reporting Summary

  3. List of antibodies and related reagents used in this study

    Supplementary Table 2

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https://doi.org/10.1038/s41590-018-0180-5

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