Circulating autoreactive proteinase 3+ B cells and tolerance checkpoints in ANCA-associated vasculitis

Alvise Berti, Sophie Hillion, Amber M Hummel, Young Min Son, Nedra Chriti, Tobias Peikert, Eva M Carmona, Wayel H Abdulahad, Peter Heeringa, Kristina M Harris, E William St Clair, Paul Brunetta, Fernando C Fervenza, Carol A Langford, Cees Gm Kallenberg, Peter A Merkel, Paul A Monach, Philip Seo, Robert F Spiera, John H Stone, Guido Grandi, Jie Sun, Jacques-Olivier Pers, Ulrich Specks, Divi Cornec, RAVE-ITN Research Group, Alvise Berti, Sophie Hillion, Amber M Hummel, Young Min Son, Nedra Chriti, Tobias Peikert, Eva M Carmona, Wayel H Abdulahad, Peter Heeringa, Kristina M Harris, E William St Clair, Paul Brunetta, Fernando C Fervenza, Carol A Langford, Cees Gm Kallenberg, Peter A Merkel, Paul A Monach, Philip Seo, Robert F Spiera, John H Stone, Guido Grandi, Jie Sun, Jacques-Olivier Pers, Ulrich Specks, Divi Cornec, RAVE-ITN Research Group

Abstract

BACKGROUNDLittle is known about the autoreactive B cells in antineutrophil cytoplasmic antibody-associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3-reactive (PR3+) B cells.METHODSMulticolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets).RESULTSThe frequency of PR3+ B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%-6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%-5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%-2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3+ B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001).CONCLUSIONThis study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells.Trial registrationClinicalTrials.gov NCT00104299.FundingThe Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.

Keywords: Autoimmune diseases; Autoimmunity; Immunology; Vasculitis.

Conflict of interest statement

Conflict of interest: PB was an employee of Genentech during the conduct of the RAVE trial and previously received salary and Genentech stock. JS’s laboratory received funding from Evive Biotech. JS and RFS participated in the GiACTA trial (sponsored by Roche) blocking the IL-6 receptor with tocilizumab in giant cell arteritis and reported grant support and personal fees from Roche. US, PAM, FCF, JHS, and RFS have received research grants from Roche/Genentech. PAM and RFS also report consulting fees from Roche/Genentech. Genentech and Biogen Idec provided partial funding for the study and donated the study medication but had no determinative role in the study design, the analyses, or the preparation of the manuscript. ANCA ELISA kits were donated by EUROIMMUN.

Figures

Figure 1. Circulating B cells in patients…
Figure 1. Circulating B cells in patients with PR3-AAV and MPO-AAV and HCs.
Gating strategy used to define B cell subsets (A). CD19+ cells were first categorized based on CD24 and CD38 expression, in transitional B cells (transi, CD24hiCD38hi), plasmablasts (PB, CD24–CD38hi), and mature B cells (CD24+CD38+). Mature B cells were further classified into 4 populations: naive (CD27–IgD+), unswitched memory (UnSW; CD27+IgD+), switched memory (SW; CD27+IgD–), and double negative (DN, CD27–IgD–). B cell frequency and subset distribution were overall similar in patients with PR3-AAV (n = 105) and MPO-AAV (n = 49) but different compared with HCs (n = 27) (B and C). Principal component analysis of the 200 B cell clusters obtained with spanning-tree progression analysis of density-normalized events (SPADE) representing HCs and PR3-AAV trial participants (D) and participants with MPO-AAV and PR3-AAV (E). Data represent median (25%–75% IQR). Multiple comparisons among more than 2 groups were performed with Kruskal-Wallis test. *P < 0.05, **P < 0.01, ***P < 0.001 after correction for FDR with Benjamini and Hochberg test.
Figure 2. Circulating PR3 + B cells…
Figure 2. Circulating PR3+ B cells and PR3-ANCA production in patients with PR3-AAV and MPO-AAV and HCs.
Representative examples of the gating of PR3+ B cells among total CD19+ cells in a patient with PR3-AAV, a patient with MPO-AAV, and a HC (A). PR3+ B cell frequency and count were increased in patients with PR3-AAV (n = 105) compared with patients with MPO-AAV (n = 49) and HCs (n = 27) (B and C). PBMCs were cultured to promote differentiation into antibody-secreting cells, after which PR3-ANCA secretion was analyzed by means of a Phadia ImmunoCAP 250 analyzer (D). Only patients with PR3-AAV can produce PR3-ANCA IgG in vitro. Correlation of circulating (in vivo) PR3-ANCA IgG with secreted (in vitro) PR3-ANCA IgG in patients with PR3-AAV (E). Data represent median (25%–75% IQR). Multiple comparisons among more than 2 groups were performed with Kruskal-Wallis test. *P < 0.05, ***P < 0.001 after correction for FDR with Benjamini and Hochberg test.
Figure 3. Frequency of PR3 + B…
Figure 3. Frequency of PR3+ B cells within each B cell subset.
Scatter plots depicting the frequency of PR3+ B cells within each B cell subset in HCs (n = 27), patients with MPO-AAV (n = 49), and patients with PR3-AAV (n = 105) (A). B cell subset distribution within PR3+ pool in patients with PR3-AAV and MPO-AAV and HCs (B). Data represent median (25%–75% IQR). Multiple comparisons among more than 2 groups were performed with Kruskal-Wallis test. **P < 0.01, ***P < 0.001 after correction for FDR with Benjamini and Hochberg test.
Figure 4. Selected PR3-reactive B cell clusters…
Figure 4. Selected PR3-reactive B cell clusters are significantly more represented or activated in patients with PR3-AAV.
Six of 200 clusters showed a stable expression of PR3 on the membrane of the B cells (A). A processed SPADE explanatory image of 1 patients with PR3-AAV, showing the most relevant clusters grouped by conventional subsets (B). In red, the six clusters of B cells with a stable increased reactivity for PR3 across all the samples, and their frequencies (C). The MFI of 4 of these 6 clusters is reduced in AAV compared with HCs (D). Additional 5 clusters with significantly varied MFI between HCs and patients with AAV: the MFIs of clusters within the SW memory and PB compartments are increased in patients with AAV compared with HCs (E), and 2 clusters within the DN showed a relative MFI increase in patients with PR3-AAV compared with patients with MPO-AAV and HCs (F). A more conservative gating approach (PR3hi) (G, left), showing the increase of PR3hi B cells in patients with PR3-AAV compared with those with MPO-AAV and HCs (G, middle). Among B cell subsets, PR3hi B cells were significantly increased only in the DN subset in participants with PR3-AAV compared with participants with MPO-AAV and HCs (G, right). Each point represents the frequency in an individual; horizontal lines show the median with 25%–75% IQR; each histogram represents mean ± SD. Multiple comparisons among more than 2 groups were performed with 1-way ANOVA or Kruskal-Wallis test, where appropriate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 after correction for FDR with Benjamini and Hochberg test. MFI, mean fluorescence intensity.
Figure 5. Maturation of PR3 + B…
Figure 5. Maturation of PR3+ B cells among different participant groups.
Scatter plots depicting the different frequency of PR3+ B cells within each B cell subset through the maturation process in HCs (n = 27), participants with MPO-AAV (n = 49), and participants with PR3-AAV (n = 105). The multiple comparisons on B cell maturation were analyzed by using mixed-effects modeling. (A). Paired comparisons between PR3+ naive and SW mature PR3+ B cells, showing the enrichment of memory B cells in participants with PR3-AAV, but not in participants with MPO-AAV and HCs (B). Ratio between the frequency of PR3+ B cells among IgD– switched memory and the frequency of PR3+ B cells among naive B cells (C). Each point represents the frequency in an individual; horizontal lines show the median with 25%–75% IQR. Multiple comparisons among more than 2 groups were performed with Kruskal-Wallis test. Selected cytokine (BAFF, CXCL13, IL-6) levels by SW memory/naive PR3+ B cell ratio in patients with PR3-AAV. P values were determined by 2-tailed Mann-Whitney test (D). *P < 0.05, **P < 0.01, ***P < 0.001 after correction for FDR with Benjamini and Hochberg test.
Figure 6. SW memory/naive PR3 + B…
Figure 6. SW memory/naive PR3+ B cell ratio and clinical manifestations in patients with PR3-AAV.
In participants with PR3-AAV (n = 105), the SW memory/naive PR3+ B cell ratio did not correlate with age (A), sex (B), or renal manifestations (C), but higher levels of CRP and ESR were associated with a ratio of more than 1 (D). The ratio did not correlate with disease activity as assessed by BVAS/WG (E). Associations with complete remission definitions (F), future relapse and severe relapse (G), and time-to-complete remission (H) are represented. When evaluating associations with remission, the patients that underwent crossover (n = 7) or experienced early treatment failure (n = 6) during the trial time were excluded from the analysis. Data represent median (25%–75% IQR), while histograms represent proportions. P values were determined by 2-tailed Mann-Whitney test, or Fisher’s test, where appropriate. Spearman’s test and the Kaplan-Meier method with the log-rank test were used to test correlations and time to event, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

References

    1. Kitching AR, et al. ANCA-associated vasculitis. Nat Rev Dis Prim. 2020;6(1):71. doi: 10.1038/s41572-020-0204-y.
    1. Lionaki S, et al. Classification of antineutrophil cytoplasmic autoantibody vasculitides: the role of antineutrophil cytoplasmic autoantibody specificity for myeloperoxidase or proteinase 3 in disease recognition and prognosis. Arthritis Rheum. 2012;64(10):3452–3462. doi: 10.1002/art.34562.
    1. Cornec D, et al. ANCA-associated vasculitis — clinical utility of using ANCA specificity to classify patients. Nat Rev Rheumatol. 2016;12(10):570–579. doi: 10.1038/nrrheum.2016.123.
    1. Wardemann H, et al. Predominant autoantibody production by early human B cell precursors. Science. 2003;301(5638):1374–1377. doi: 10.1126/science.1086907.
    1. Yurasov S, et al. B-cell tolerance checkpoints in healthy humans and patients with systemic lupus erythematosus. Ann N Y Acad Sci. 2005;1062:165–172. doi: 10.1196/annals.1358.019.
    1. Yurasov S. et al. Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med. 2005;201(5):703–711. doi: 10.1084/jem.20042251.
    1. Nemazee D. Mechanisms of central tolerance for B cells. Nat Rev Immunol. 2017;17(5):281–294. doi: 10.1038/nri.2017.19.
    1. Voswinkel J, et al. Is PR3-ANCA formation initiated in Wegener’s granulomatosis lesions? Granulomas as potential lymphoid tissue maintaining autoantibody production. Ann N Y Acad Sci. 2005;1051:12–19. doi: 10.1196/annals.1361.042.
    1. de la Varga-Martínez R, et al. Autoreactive B-lymphocytes in SLE and RA patients: isolation and characterisation using extractable nuclear and citrullinated antigens bound to immunobeads. Eur J Immunol. 2019;49(7):1107–1116. doi: 10.1002/eji.201848065.
    1. Sweet RA, et al. A new site-directed transgenic rheumatoid factor mouse model demonstrates extrafollicular class switch and plasmablast formation. Autoimmunity. 2010;43(8):607–618. doi: 10.3109/08916930903567500.
    1. Erikson J, et al. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature. 1999;349(6307):331–334.
    1. Sibilia J, et al. Structural analysis of human antibodies to proteinase 3 from patients with Wegener granulomatosis. J Immunol. 1997;159(2):712–719.
    1. Weppner G, et al. In situ detection of PR3-ANCA+ B cells and alterations in the variable region of immunoglobulin genes support a role of inflamed tissue in the emergence of auto-reactivity in granulomatosis with polyangiitis. J Autoimmun. 2018;93:89–103. doi: 10.1016/j.jaut.2018.07.004.
    1. Xiao H, et al. Alternative complement pathway in the pathogenesis of disease mediated by anti-neutrophil cytoplasmic autoantibodies. Am J Pathol. 2007;170(1):52–64. doi: 10.2353/ajpath.2007.060573.
    1. Schreiber A, et al. C5a receptor mediates neutrophil activation and ANCA-induced glomerulonephritis. J Am Soc Nephrol. 2009;20(2):289–298. doi: 10.1681/ASN.2008050497.
    1. Xiao H, et al. Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest. 2002;110(7):955–963. doi: 10.1172/JCI0215918.
    1. Shochet L, et al. Animal models of ANCA associated vasculitis. Front Immunol. 2020;11:525. doi: 10.3389/fimmu.2020.00525.
    1. Cornec D, et al. Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls. J Autoimmun. 2017;84:122–131. doi: 10.1016/j.jaut.2017.08.006.
    1. Lepse N, et al. Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission. Rheumatology (Oxford) 2014;53(9):1683–1692. doi: 10.1093/rheumatology/keu149.
    1. Md Yusof MY, et al. Repeat cycles of rituximab on clinical relapse in ANCA-associated vasculitis: identifying B cell biomarkers for relapse to guide retreatment decisions. Ann Rheum Dis. 2015;74(9):1734–1738. doi: 10.1136/annrheumdis-2014-206496.
    1. Aybar LT, et al. Reduced CD5(+) CD24(hi) CD38(hi) and interleukin-10(+) regulatory B cells in active anti-neutrophil cytoplasmic autoantibody-associated vasculitis permit increased circulating autoantibodies. Clin Exp Immunol. 2015;180(2):178–188. doi: 10.1111/cei.12483.
    1. Unizony S, et al. Peripheral CD5+ B cells in antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Rheumatol. 2015;67(2):535–544. doi: 10.1002/art.38916.
    1. Todd SK, et al. Regulatory B cells are numerically but not functionally deficient in anti-neutrophil cytoplasm antibody-associated vasculitis. Rheumatology (Oxford) 2014;53(9):1693–1703. doi: 10.1093/rheumatology/keu136.
    1. Havenar-Daughton C, et al. CXCL13 is a plasma biomarker of germinal center activity. Proc Natl Acad Sci U S A. 2016;113(10):2702–2707. doi: 10.1073/pnas.1520112113.
    1. Zhao Y, et al. Circulating T follicular helper cell and regulatory T cell frequencies are influenced by B cell depletion in patients with granulomatosis with polyangiitis. Rheumatology (Oxford) 2014;53(4):621–630. doi: 10.1093/rheumatology/ket406.
    1. Bynoe MS, et al. Characterization of anti-DNA B cells that escape negative selection. Eur J Immunol. 1999;29(4):1304–1313. doi: 10.1002/(SICI)1521-4141(199904)29:04<1304::AID-IMMU1304>;2-6.
    1. Mandik-Nayak L, et al. Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: Localization to the T-B interface of the splenic follicle. J Exp Med. 1997;186(8):1257–1267. doi: 10.1084/jem.186.8.1257.
    1. Li H, et al. Editors and editing of anti-DNA receptors. Immunity. 2001;15(6):947–957. doi: 10.1016/S1074-7613(01)00251-5.
    1. Pewzner-Jung Y, et al. B cell deletion, anergy, and receptor editing in “knock in” mice targeted with a germline-encoded or somatically mutated anti-DNA heavy chain. J Immunol. 1998;161(9):4634–4645.
    1. Ohlsson S, et al. Increased circulating levels of proteinase 3 in patients with anti-neutrophilic cytoplasmic autoantibodies-associated systemic vasculitis in remission. Clin Exp Immunol. 2003;131(3):528–535. doi: 10.1046/j.1365-2249.2003.02083.x.
    1. Lyons PA, et al. Genetically distinct subsets within ANCA-associated vasculitis. N Engl J Med. 2012;367(3):214–223. doi: 10.1056/NEJMoa1108735.
    1. Jenks SA, et al. Distinct effector B cells induced by unregulated Toll-like receptor 7 contribute to pathogenic responses in systemic lupus erythematosus. Immunity. 2018;49(4):725–739. doi: 10.1016/j.immuni.2018.08.015.
    1. MacLennan ICM. Germinal centers. Annu Rev Immunol. 2016;34:335–368. doi: 10.1146/annurev-immunol-041015-055605.
    1. Chan TD, et al. Elimination of germinal-center-derived self-reactive B cells is governed by the location and concentration of self-antigen. Immunity. 2012;37(5):893–904. doi: 10.1016/j.immuni.2012.07.017.
    1. Reed JH, et al. Clonal redemption of autoantibodies by somatic hypermutation away from self-reactivity during human immunization. J Exp Med. 2016;213(7):1255–1265. doi: 10.1084/jem.20151978.
    1. MacLennan I, Toellner KM. Extrafollicular antibody responses. Immunol Rev. 2003;194:8–18. doi: 10.1034/j.1600-065X.2003.00058.x.
    1. Monach PA, et al. Serum proteins reflecting inflammation, injury and repair as biomarkers of disease activity in ANCA-associated vasculitis. Ann Rheum Dis. 2013;72(8):1342–1350. doi: 10.1136/annrheumdis-2012-201981.
    1. Stone JH, et al. Rituximab versus cyclophosphamide for ANCA-associated vasculitis. N Engl J Med. 2010;363(3):221–232. doi: 10.1056/NEJMoa0909905.
    1. Stone JH, et al. A disease-specific activity index for Wegener’s granulomatosis: modification of the Birmingham Vasculitis Activity score. International network for the study of the systemic vasculitides (INSSYS) Arthritis Rheum. 2001;44(4):912–920. doi: 10.1002/1529-0131(200104)44:4<912::AID-ANR148>;2-5.
    1. Sun J, et al. A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide. Clin Exp Immunol. 1998;114(2):320–326. doi: 10.1046/j.1365-2249.1998.00730.x.
    1. Capizzi SA, et al. Effects of carboxy-terminal modifications of proteinase 3 (PR3) on the recognition by PR3-ANCA. Kidney Int. 2003;63(2):756–760. doi: 10.1046/j.1523-1755.2003.00765.x.
    1. Rasmussen SM, et al. Stable phenotype of B-cell subsets following cryopreservation and thawing of normal human lymphocytes stored in a tissue biobank. Cytometry B Clin Cytom. 2015;88(1):40–49. doi: 10.1002/cytob.21192.
    1. Sun J, et al. Capture-ELISA based on recombinant PR3 is sensitive for PR3-ANCA testing and allows detection of PR3 and PR3-ANCA/PR3 immunecomplexes. J Immunol Methods. 1998;211(1–2):111–123.
    1. Kimball AK, et al. A beginner’s guide to analyzing and visualizing mass cytometry data. J Immunol. 2018;200(1):3–22. doi: 10.4049/jimmunol.1701494.
    1. Lepse N, et al. Toll-like receptor 9 activation enhances B cell activating factor and interleukin-21 induced anti-proteinase 3 autoantibody production in vitro. Rheumatology (Oxford) 2016;55(1):162–172. doi: 10.1093/rheumatology/kev293.
    1. Li A, Barber RF. Multiple testing with the structure-adaptive Benjamini–Hochberg algorithm. J R Stat Soc Ser B Stat Methodol. 2019;81(1):45–74. doi: 10.1111/rssb.12298.

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