Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY.
Objective. Hepatitis C virus (HCV) is the most frequent cause of mixed cryoglobulinemia (MC), which is characterized by endothelial deposition of rheumatoid factor (RF)-containing immune complexes and end-organ vasculitis. MC is a lymphoproliferative disorder of B cells expressing RF-like immunoglobulin (Ig), yet its precise antigenic stimulus is unknown. We have proposed that IgG/HCV immune complexes stimulate B cell expansion and somatic hypermutation (SHM)-induced affinity maturation in part via engagement of an RF-like B cell receptor. Here, we test the hypothesis that SHM augments RF activity. Methods. RFs cloned from single B cells of four HCV MC patients were expressed as IgM, IgG, or IgG Fab. Selected Igs were reverted to germline. RF activity of the somatically mutated and germline reverted Igs was determined by ELISA. Results. Igs containing SHM had RF activity, with a preference for IgG1 >IgG2 ~IgG4 >IgG3 . In contrast, reverted germline Igs had markedly diminished activities for IgG. Competition with 1 μg/ml Protein A abrogated RF activity, suggesting specificity for IgG Fc. Swapping of mutated heavy and light chain pairs also abrogated RF activity, suggesting that context-specific pairing of appropriate IgH and Igκ, in addition to SHM, is necessary for RF activity. Conclusion. SHM significantly contributes to RF activity in HCV MC patients, suggesting that autoreactivity in these patients arises through antigen-dependent SHM, as opposed to non-deletion of autoreactive germline Igs.