Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection

Katy A. Lloyd; Johanna Steen; Khaled Amara; Philip J. Titcombe; Lena Israelsson; Susanna L. Lundström; Diana Zhou; Roman A. Zubarev; Evan Reed; Luca Piccoli; Cem Gabay; Antonio Lanzavecchia; Dominique Baeten; Karin Lundberg; Daniel L Mueller; Lars Klareskog; Vivianne Malmström; Caroline Grönwall

doi:10.1002/eji.201747446

Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection

Katy A. Lloyd, Johanna Steen, Khaled Amara, Philip J. Titcombe, Lena Israelsson, Susanna L. Lundström, Diana Zhou, Roman A. Zubarev, Evan Reed, Luca Piccoli, Cem Gabay, Antonio Lanzavecchia, Dominique Baeten, Karin Lundberg, Daniel L Mueller, Lars Klareskog, Vivianne Malmström, Caroline Grönwall

Medicine - Rheumatic and Autoimmune

Research output: Contribution to journal › Article › peer-review

37 Scopus citations

Abstract

Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico, and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decrease in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA⁺ B cells, possibly through non-antigen driven mechanisms.

Original language	English (US)
Pages (from-to)	1030-1045
Number of pages	16
Journal	European Journal of Immunology
Volume	48
Issue number	6
DOIs	https://doi.org/10.1002/eji.201747446
State	Published - Jun 2018

Bibliographical note

Funding Information:
We would like to thank Dr Fredrik Wermeling (Karolinska Institutet) for reagents and valuable discussions, Dr Yan Wang (Karolinska Institutet) for sequence analysis advice, and Ragnhild Stålesen and Dr Monika Hansson (Karolinska Institutet) for support in antibody production, validation and characterization. We thank Dr Christian Busse (German Cancer Research Center) for kindly providing the paired V-region sequences from B cells isolated from malaria patients. We also thank UCB Pharma for support with technology and antibody production as well as financial support for Katy Lloyd's postdoctoral training within the IMI BTCure project. This work was supported by the Swedish Research Council, the Swedish Rheumatism Association, King Gustaf V's 80-year Foundation, and the IMI EU funded project BeTheCure 115142.

Funding Information:
Acknowledgements: We would like to thank Dr Fredrik Wermel-ing (Karolinska Institutet) for reagents and valuable discussions, Dr Yan Wang (Karolinska Institutet) for sequence analysis advice, and Ragnhild Stålesen and Dr Monika Hansson (Karolinska Insti-tutet) for support in antibody production, validation and characterization. We thank Dr Christian Busse (German Cancer Research Center) for kindly providing the paired V-region sequences from B cells isolated from malaria patients. We also thank UCB Pharma for support with technology and antibody production as well as financial support for Katy Lloyd’s postdoctoral training within the IMI BTCure project. This work was supported by the Swedish Research Council, the Swedish Rheumatism Association, King Gustaf V’s 80-year Foundation, and the IMI EU funded project BeTheCure 115142.

Publisher Copyright:
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Keywords

Anti-CCP
Anti-citrullinated protein autoantibodies
Autoreactive B cells
Fab glycosylation
N-linked glycosylation

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Lloyd, K. A., Steen, J., Amara, K., Titcombe, P. J., Israelsson, L., Lundström, S. L., Zhou, D., Zubarev, R. A., Reed, E., Piccoli, L., Gabay, C., Lanzavecchia, A., Baeten, D., Lundberg, K., Mueller, D. L., Klareskog, L., Malmström, V., & Grönwall, C. (2018). Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection. European Journal of Immunology, 48(6), 1030-1045. https://doi.org/10.1002/eji.201747446

Lloyd, KA, Steen, J, Amara, K, Titcombe, PJ, Israelsson, L, Lundström, SL, Zhou, D, Zubarev, RA, Reed, E, Piccoli, L, Gabay, C, Lanzavecchia, A, Baeten, D, Lundberg, K, Mueller, DL, Klareskog, L, Malmström, V & Grönwall, C 2018, 'Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection', European Journal of Immunology, vol. 48, no. 6, pp. 1030-1045. https://doi.org/10.1002/eji.201747446

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abstract = "Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico, and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decrease in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly through non-antigen driven mechanisms.",

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note = "Funding Information: We would like to thank Dr Fredrik Wermeling (Karolinska Institutet) for reagents and valuable discussions, Dr Yan Wang (Karolinska Institutet) for sequence analysis advice, and Ragnhild St{\aa}lesen and Dr Monika Hansson (Karolinska Institutet) for support in antibody production, validation and characterization. We thank Dr Christian Busse (German Cancer Research Center) for kindly providing the paired V-region sequences from B cells isolated from malaria patients. We also thank UCB Pharma for support with technology and antibody production as well as financial support for Katy Lloyd's postdoctoral training within the IMI BTCure project. This work was supported by the Swedish Research Council, the Swedish Rheumatism Association, King Gustaf V's 80-year Foundation, and the IMI EU funded project BeTheCure 115142. Funding Information: Acknowledgements: We would like to thank Dr Fredrik Wermel-ing (Karolinska Institutet) for reagents and valuable discussions, Dr Yan Wang (Karolinska Institutet) for sequence analysis advice, and Ragnhild St{\aa}lesen and Dr Monika Hansson (Karolinska Insti-tutet) for support in antibody production, validation and characterization. We thank Dr Christian Busse (German Cancer Research Center) for kindly providing the paired V-region sequences from B cells isolated from malaria patients. We also thank UCB Pharma for support with technology and antibody production as well as financial support for Katy Lloyd{\textquoteright}s postdoctoral training within the IMI BTCure project. This work was supported by the Swedish Research Council, the Swedish Rheumatism Association, King Gustaf V{\textquoteright}s 80-year Foundation, and the IMI EU funded project BeTheCure 115142. Publisher Copyright: {\textcopyright} 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim",

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AU - Lloyd, Katy A.

AU - Steen, Johanna

AU - Amara, Khaled

AU - Titcombe, Philip J.

AU - Israelsson, Lena

AU - Lundström, Susanna L.

AU - Zhou, Diana

AU - Zubarev, Roman A.

AU - Reed, Evan

AU - Piccoli, Luca

AU - Gabay, Cem

AU - Lanzavecchia, Antonio

AU - Baeten, Dominique

AU - Lundberg, Karin

AU - Mueller, Daniel L

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AU - Malmström, Vivianne

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N2 - Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico, and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decrease in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly through non-antigen driven mechanisms.

AB - Autoreactive B cells have a central role in the pathogenesis of rheumatoid arthritis (RA), and recent findings have proposed that anti-citrullinated protein autoantibodies (ACPA) may be directly pathogenic. Herein, we demonstrate the frequency of variable-region glycosylation in single-cell cloned mAbs. A total of 14 ACPA mAbs were evaluated for predicted N-linked glycosylation motifs in silico, and compared to 452 highly-mutated mAbs from RA patients and controls. Variable region N-linked motifs (N-X-S/T) were strikingly prevalent within ACPA (100%) compared to somatically hypermutated (SHM) RA bone marrow plasma cells (21%), and synovial plasma cells from seropositive (39%) and seronegative RA (7%). When normalized for SHM, ACPA still had significantly higher frequency of N-linked motifs compared to all studied mAbs including highly mutated HIV broadly-neutralizing and malaria-associated mAbs. The Fab glycans of ACPA-mAbs were highly sialylated, contributed to altered charge, but did not influence antigen binding. The analysis revealed evidence of unusual B-cell selection pressure and SHM-mediated decrease in surface charge and isoelectric point in ACPA. It is still unknown how these distinct features of anti-citrulline immunity may have an impact on pathogenesis. However, it is evident that they offer selective advantages for ACPA+ B cells, possibly through non-antigen driven mechanisms.

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KW - Anti-citrullinated protein autoantibodies

KW - Autoreactive B cells

KW - Fab glycosylation

KW - N-linked glycosylation

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Variable domain N-linked glycosylation and negative surface charge are key features of monoclonal ACPA: Implications for B-cell selection

Abstract

Bibliographical note

Keywords

UN SDGs

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