Anti-centromere antibodies target centromere-kinetochore macrocomplex: a comprehensive autoantigen profiling.

OBJECTIVES: Anti-centromere antibodies (ACAs) are detected in patients with various autoimmune diseases such as Sjögren's syndrome (SS), systemic sclerosis (SSc) and primary biliary cholangitis (PBC). However, the targeted antigens of ACAs are not fully elucidated despite the accumulating understanding of the molecular structure of the centromere. The aim of this study was to comprehensively reveal the autoantigenicity of centromere proteins. METHODS: A centromere antigen library including 16 principal subcomplexes composed of 41 centromere proteins was constructed. Centromere protein/complex binding beads were used to detect serum ACAs in patients with SS, SSc and PBC. ACA-secreting cells in salivary glands obtained from patients with SS were detected with green fluorescent protein-fusion centromere antigens and semiquantified with confocal microscopy. RESULTS: A total of 241 individuals with SS, SSc or PBC and healthy controls were recruited for serum ACA profiling. A broad spectrum of serum autoantibodies was observed, and some of them had comparative frequency as anti-CENP-B antibody, which is the known major ACA. The prevalence of each antibody was shared across the three diseases. Immunostaining of SS salivary glands showed the accumulation of antibody-secreting cells (ASCs) specific for kinetochore, which is a part of the centromere, whereas little reactivity against CENP-B was seen. CONCLUSIONS: We demonstrated that serum autoantibodies target the centromere-kinetochore macrocomplex in patients with SS, SSc and PBC. The specificity of ASCs in SS salivary glands suggests kinetochore complex-driven autoantibody selection, providing insight into the underlying mechanism of ACA acquisition.

Antigen-driven selection of antibodies against SSA, SSB and the centromere 'complex', including a novel antigen, MIS12 complex, in human salivary glands.

OBJECTIVES: Recent evidences have revealed that anti-SSA/SSB antibodies, the major autoantibodies in Sjögren's syndrome (SS), are produced in salivary glands. This study aims to clarify overall of autoantibody production at lesion site, including anti-centromere antibody (ACA)-positive SS. METHODS: Antibodies of antibody-secreting cells in human salivary glands were produced as recombinant antibodies. The reactivity of these antibodies and their revertants were investigated by ELISA and newly developed antigen-binding beads assay, which can detect conformational epitopes. The target of uncharacterised antibodies was identified by immunoprecipitation and mass spectrometry. Autoantibody-secreting cells in salivary gland tissue were identified by immunohistochemistry using green fluorescent protein-autoantigen fusion proteins. RESULTS: A total of 256 lesion antibodies were generated, and 69 autoantibodies including 24 ACAs were identified among them. Beads assay could detect more autoantibodies than ELISA, suggesting autoantibodies target to antigens with native conformation. After somatic hypermutations were reverted, autoantibodies drastically decreased antigen reactivity. We showed that MIS12 complex, a novel target of ACA, and CENP-C are major targets of ACA produced in salivary glands by examining cloned antibodies and immunohistochemistry, whereas few anti-CENP-B antibodies were detected. The target profiling of serum ACA from 269 patients with SS, systemic sclerosis (SSc), primary biliary cirrhosis (PBC) and healthy controls revealed that ACA-positive patients have antibodies against various sites of centromere complex regardless of disease. CONCLUSION: We showed direct evidences of antigen-driven maturation of anti-SSA/SSB antibody and ACA in SS lesion. ACA recognises centromere 'complex' rather than individual protein, and this feature is common among patients with SS, SSc and PBC.