Somatic hypermutations confer rheumatoid factor activity in hepatitis C virus-associated mixed cryoglobulinemia.

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 in which B cells express RF-like 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. This study was undertaken to test the hypothesis that SHM augments RF activity. METHODS: RFs cloned from single B cells from 4 patients with HCV-associated MC (HCV-MC) were expressed as IgM, IgG, or IgG Fab. Selected Ig were reverted to germline. RF activity of somatically mutated Ig and germline-reverted Ig was determined by enzyme-linked immunosorbent assay. RESULTS: Ig with SHM had RF activity, with the preference for binding being highest for IgG1, followed by IgG2 and IgG4, and lowest for IgG3, where there was no detectable binding. In contrast, reverted germline IgG exhibited markedly diminished RF activity. Competition with 1 µg/ml of protein A abrogated RF activity, suggesting specificity for IgG Fc. Swapping of mutated heavy-chain pairs 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 nondeletion of autoreactive germline Ig.

A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.

Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment-containing B cells. The cloned variable regions are expressed as IgM and produced during culture at concentrations between 5 and 20 µg/ml. Using this system, we show that clonal Igs from patients with HCV-related mixed cryoglobulinemia, when expressed as IgM, have RF activity. We anticipate that this system will be useful for the cloning and expression of mAbs partially encoded by VH1-69 and for determination of the reactivity patterns of polyspecific, low-affinity IgMs of human pathogenic importance.