Sequencing and functional analyses of antibody repertoires in autoimmune diseases [electronic resource]

While autoantibodies have long been known to play critical roles in autoimmune disease pathogenesis, many questions remain regarding their origin, evolution, structure, and function. Recent advances in next-generation sequencing are now enabling the large-scale analysis of patient antibody repertoires and thus furthering our ability to study autoantibodies directly derived from patients. Using a dual-barcoded single-cell sequencing method designed to capture antibody repertoires from patient plasmablasts, we have studied autoantibodies present in two different autoimmune diseases, rheumatoid arthritis and neonatal lupus-associated congenital heart block. Anti-citrullinated protein antibodies (ACPA) are one of the hallmarks of rheumatoid arthritis (RA). We developed citrullinated-peptide tetramers to use as a sorting reagent to identify ACPA-producing B cells by flow cytometry, and then we sorted and sequenced antibody repertoires from 6 CCP+ RA patients. After recombinant expression and functional testing by synovial planar array of sequenced antibodies, we identified three clonal families which exhibited differential citrullinated antigen reactivities between clonal family members. Furthermore, expression of ancestral antibodies from these clonal families also showed differential binding. This provided evidence that affinity maturation of ACPA during clonal expansion results in epitope-spreading against citrullinated antigens. In an attempt to artificially replicate somatic hypermutation leading to epitope-spreading and to identify the regions most critical for antigen-binding, we performed molecular modeling and mutation studies using pairs of clonally-related antibodies. We were successful in altering the antigen-specifities of some of the mutated antibodies. Additionally, we identified sequences important for antigen binding, including residues located within a framework region. Neonatal lupus-associated congenital heart block is typically associated with maternal antibodies against Ro and La antigens; however, in sequencing the antibody repertoires of mothers whose children had congenital heart block, we discovered a novel autoantibody reactivity. Three unrelated monoclonal antibodies derived from one patient were found to bind in an ANA assay to both mitotic spindle and to mitochondria. Dual-staining experiments have confirmed that the antibodies do not bind to either of the known mitotic spindle autoantigens, NuMA or Eg5. Western blot studies indicate that the antigen is approximately 55 kDa. In rheumatoid arthritis, we describe a novel mechanism by which epitope-spreading could be occurring in patients. In congenital heart block, we have discovered a novel autoantibody reactivity against both mitotic spindle and mitochondria. Together, these findings represent advancements in the field of autoantibody research made possible by the use of next-generation sequencing and antibody repertoire capture methods.

Advisor: Robinson William (William Hewitt) primary advisor. Thesis advisor
Engleman Edgar G. advisor. Thesis advisor
Fathman C. Garrison
Utz Paul advisor. Thesis advisor