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Detection of experimental and clinical immune complexes by measuring SHIP-1 recruitment to the inhibitory FcγRIIB

Detection of experimental and clinical immune complexes by measuring SHIP-1 recruitment to the inhibitory FcγRIIB
Detection of experimental and clinical immune complexes by measuring SHIP-1 recruitment to the inhibitory FcγRIIB
Fc gamma receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of monoclonal antibody (mAb) therapeutics and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, immunoglobulin G (IgG) immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signalling pathway of the inhibitory FcγRIIB (CD32B), utilised NanoLuc® Binary Interaction Technology (NanoBiT™) to measure recruitment of the Src homology 2 (SH2) domain-containing inositol phosphatase 1 (SHIP-1) to the immunoreceptor tyrosine-based inhibitory motif (ITIM) of this receptor. Such recruitment required prior crosslinking of an immunoreceptor tyrosine-based activation motif (ITAM)-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signalling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, Rituximab:anti-idiotype complexes and anti-trinitrophenol (TNP)-TNP complexes in a sensitive manner (≤1μg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.
Stopforth, R.J.
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Oldham, R.J.
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Tutt, A.L.
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Duriez, P.
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Chan, H.T.C.
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Binkowski, B.
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Zimprich, C.
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Li, D.
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Hargreaves, P.
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Cong, M.
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Reddy, V.
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Leandro, M.
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Cambridge, G.
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Lux, A.
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Nimmerjahn, F.
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Cragg, M.S.
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Stopforth, R.J.
2d3e18ff-5563-4247-9150-f0f337fb585f
Oldham, R.J.
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Tutt, A.L.
46ce577b-aea1-412d-84ea-fc4dab794469
Duriez, P.
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Chan, H.T.C.
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Binkowski, B.
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Zimprich, C.
4db401a0-262b-4c1e-b6db-376d3c197d8b
Li, D.
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Hargreaves, P.
019c11f6-4787-4c4d-9998-02fbf22cf391
Cong, M.
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Reddy, V.
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Leandro, M.
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Cambridge, G.
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Lux, A.
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Nimmerjahn, F.
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Cragg, M.S.
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Stopforth, R.J., Oldham, R.J., Tutt, A.L., Duriez, P., Chan, H.T.C., Binkowski, B., Zimprich, C., Li, D., Hargreaves, P., Cong, M., Reddy, V., Leandro, M., Cambridge, G., Lux, A., Nimmerjahn, F. and Cragg, M.S. (2018) Detection of experimental and clinical immune complexes by measuring SHIP-1 recruitment to the inhibitory FcγRIIB. The Journal of Immunology. (doi:10.4049/jimmunol.1700832).

Record type: Article

Abstract

Fc gamma receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of monoclonal antibody (mAb) therapeutics and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, immunoglobulin G (IgG) immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signalling pathway of the inhibitory FcγRIIB (CD32B), utilised NanoLuc® Binary Interaction Technology (NanoBiT™) to measure recruitment of the Src homology 2 (SH2) domain-containing inositol phosphatase 1 (SHIP-1) to the immunoreceptor tyrosine-based inhibitory motif (ITIM) of this receptor. Such recruitment required prior crosslinking of an immunoreceptor tyrosine-based activation motif (ITAM)-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signalling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, Rituximab:anti-idiotype complexes and anti-trinitrophenol (TNP)-TNP complexes in a sensitive manner (≤1μg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

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Accepted/In Press date: 20 December 2017
e-pub ahead of print date: 19 January 2018

Identifiers

Local EPrints ID: 417550
URI: https://eprints.soton.ac.uk/id/eprint/417550
PURE UUID: 6da40d56-fa9d-46cc-be4b-1e0196b3051a
ORCID for R.J. Oldham: ORCID iD orcid.org/0000-0002-8007-1145
ORCID for M.S. Cragg: ORCID iD orcid.org/0000-0003-2077-089X

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Date deposited: 02 Feb 2018 17:30
Last modified: 14 Mar 2019 01:49

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Contributors

Author: R.J. Stopforth
Author: R.J. Oldham ORCID iD
Author: A.L. Tutt
Author: P. Duriez
Author: H.T.C. Chan
Author: B. Binkowski
Author: C. Zimprich
Author: D. Li
Author: P. Hargreaves
Author: M. Cong
Author: V. Reddy
Author: M. Leandro
Author: G. Cambridge
Author: A. Lux
Author: F. Nimmerjahn
Author: M.S. Cragg ORCID iD

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