Abstract
The presence of autoantibodies is a defining feature of many autoimmune diseases. The number of unique autoantibody clones is conceivably limited by immune tolerance mechanisms, but unknown due to limitations of the currently applied technologies. Here, we introduce an autoantigen-specific liquid chromatography-mass spectrometry-based IgG1 Fab profiling approach using the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA) as an example. We show that each patient harbors a unique and diverse ACPA IgG1 repertoire dominated by only a few antibody clones. In contrast to the total plasma IgG1 antibody repertoire, the ACPA IgG1 sub-repertoire is characterised by an expansion of antibodies that harbor one, two or even more Fab glycans, and different glycovariants of the same clone can be detected. Together, our data indicate that the autoantibody response in a prominent human autoimmune disease is complex, unique to each patient and dominated by a relatively low number of clones.
Introduction
Antibodies play a central role in protecting the host from pathogens. To provide protection in a specific and tunable manner, each antibody harbors a highly variable region in its antigen-binding fragments (Fab). This variable domain is initially generated by recombination of variable (V), diversity (D) and joining (J) gene segments during which nucleotides are deleted, and palindromic and non-templated nucleotides are inserted at the junctions of the assembled gene segments1,2,3. Together with somatic hypermutation of the variable domain upon antigen encounter4,5, the various processes may eventually give rise to billions of unique antibodies, including various clones capable of binding the same antigen6. This high diversity and flexibility of the antibody repertoire ultimately allows antigen-specific immune responses even against newly arising or continuously evolving pathogens.
To prevent the formation of antibodies that target the body itself, known as autoantibodies, various tolerance mechanisms are in place. These mechanisms identify autoreactive B cell clones and exclude them from the repertoire by, for instance, clonal deletion or editing of the variable domain7. Failure of tolerance mechanisms can ultimately lead to autoimmune diseases, currently estimated to affect about 1 in 10 individuals8. Notably, many autoimmune diseases are responsive to B cell-targeting therapies9 and are accompanied by disease-specific autoantibodies10.
Serological studies have provided insights into the association of autoantibodies with disease development, progression or treatment10. Consequently, autoantibodies are widely used as biomarkers for diagnosis and prognosis, for disease classification and for guiding treatment choices10,11,12. Yet, insights into the extent of autoantibody repertoires, i.e., the extent of tolerance failure, are lacking due to the limitations of the currently applied technologies.
We recently introduced a method that enables to study plasma antibody repertoires at the protein level with molecular resolution13. This liquid chromatography-mass spectrometry (LC-MS)-based Fab profiling approach selectively generates IgG1 Fab fragments from affinity enriched plasma IgG and analyzes these Fab molecules by LC-MS, thereby resolving the diversity of polyclonal antibody mixtures and repertoires based on the unique mass and retention time of each Fab molecule. The application of this approach revealed that plasma as well as virus-specific IgG1 repertoires are unique and polyclonal, with a few clones showing particularly high abundances13,14. This diversity of repertoires against infectious agents may, however, differ from that of autoreactive antibody repertoires as a result of the exclusion of autoreactive antibody clones by tolerance mechanisms as well as the different nature of and context in which autoantigens may be recognized. Here, we therefore introduce in-depth autoantigen-specific Fab profiling and resolve an autoreactive plasma antibody sub-repertoire at the molecular level, using the prominent autoantibody response of anti-citrullinated protein antibodies (ACPA) as an example.
ACPA target proteins containing citrulline, a post-translational modification of the amino acid arginine, and are highly specific to rheumatoid arthritis (RA), a systemic and highly prevalent autoimmune disease8,15,16. ACPA are detected in 50 to 75% of RA patients and associate with severe bone erosions, disease progression and poor treatment responses17,18. Moreover, ACPA can be present years before RA diagnosis19 and frequently harbor N-linked glycans in their variable domains – also referred to as Fab glycans due to their localization in the Fab20,21. Intriguingly, the level of ACPA IgG Fab glycosylation in ACPA-positive healthy subjects correlates with transition to RA22. ACPA are thus not only a pivotal diagnostic and prognostic biomarker for RA, but also a promising candidate to predict RA development.
By adapting the total plasma IgG1 Fab profiling approach to the ACPA response, we here resolve the ACPA antibody sub-repertoire at molecular detail. We show that the ACPA repertoire in plasma is polyclonal, albeit dominated by only a few clones. We confirm that ACPA IgG1 are, relatively to total plasma IgG1, extensively Fab-glycosylated and, moreover, reveal that their repertoire is unique to each patient. Remarkably, the ACPA IgG1 repertoire is characterized by an expansion of antibodies harboring two or more Fab glycans, while at the same time a substantial fraction of ACPA IgG1 do not harbor any. Together, this study provides a means to characterize human autoantibody repertoires in-depth and unveils the complexity and unique features of a prominent human disease-specific autoantibody repertoire.
Results
ACPA-specific Fab repertoire profiling by LC-MS
To enable insights into the extent and molecular composition of autoreactive antibody repertoires, we introduce an in-depth autoantigen-specific Fab profiling approach using the prototypic autoantibody response in RA, the ACPA response, as an example. The procedure of ACPA IgG1 Fab profiling consists of three parts (Fig. 1a). First, ACPA are affinity purified from patient plasma using the cyclic citrullinated peptide 2 (CCP2), a “golden standard” antigen used in clinical practice for diagnostic and prognostic purposes, and its non-modified, arginine-containing version CArgP2; the specificity of the ACPA purification is monitored by enzyme-linked immunosorbent assays (ELISAs). Second, IgG is affinity captured from purified ACPA spiked with known concentrations of two monoclonal IgG1 antibodies; and ACPA IgG1 Fab fragments are generated by subsequent on-bead enzymatic digestion using the IgG1-specific protease immunoglobulin degrading enzyme (IgdE), which cleaves IgG1 just above the hinge region. Finally, the collected ACPA IgG1 Fab fragments are profiled using the recently developed intact mass LC-MS-based method described in Bondt, et al.13; and the quantity and plasma concentrations of each Fab molecule detected are determined based on the monoclonal antibodies spiked.
