197. Somatic Hypermutations Confer Rheumatoid Factor Activity in Hepatitis C Virus-Associated Mixed Cryoglobulinemia
Session: Abstracts: Oral Abstract Session: Virology
Friday, October 22, 2010: 11:15 AM
109-110
Background: Hepatitis C virus (HCV) is the most frequent cause of type II mixed cryoglobulinemia (MC), which is characterized by endothelial deposition of rheumatoid factor (RF)-containing immune complexes (ICs), causing end-organ vasculitis. MC is a B cell lymphoproliferative disorder, yet its precise antigenic stimulus is unknown. We have reported that HCV+MC+ patients have clonal expansions of mutated IgMκ RF-bearing memory B cells. We have proposed that IgG/HCV ICs simulate B cell expansion and somatic hypermutation (SHM)-induced affinity maturation via engagement of the B cell receptor and toll-like receptor 7. We hypothesize that SHM induced by these ICs is essential for RF activity. We have created an IgM expression system to measure these clonal Igs’ RF activities and test whether these activities depend upon SHM.

Methods: Ig heavy (H) and light chain variable (V) region RT-PCR was performed on an HCV+MC+ patient’s singly-sorted RF+ B cells. Overlap PCR was used to create framework region 1-3 germline counterparts for each Ig. After ligation into IgμC- and IgκC-containing expression vectors, Ig was transfected into J chain-expressing 293T cells and secreted pentameric IgM was purified. RF activity was determined by ELISA.

Results: 8 mutated and 4 germline Igs were cloned and expressed. All VH utilized VH1-69/D3-22/JH4 gene segments, with two clonal (6/8 and 2/8 of total mutated IgVH) complementarity determining region 3 (CDR3)-expressing populations. All Vκ utilized Vκ3-20/Jκ2 gene segments and contained one clonal IgVκ CDR3. Nonsynonymous mutations ranged from 0-6 for VH and 0-4 for Vκ. The mutated Igs had RF activity, with a preference for IgG1>IgG2~IgG4>IgG3. Competition with 0.05 μg/ml Protein A abrogated RF activities, suggesting specificity for IgG Fc. In contrast, reverted germline Igs had markedly diminished avidities for IgG1, IgG2, IgG3 and IgG4.

Conclusion: SHM significantly contributes to RF activity in HCV+MC+ patients, suggesting that autoreactivity in these patients arises from antigen-dependent SHM, as opposed to non-deletion of autoreactive germline Igs.


Subject Category: V. Virology including clinical and basic studies of viral infections, including hepatitis

Speakers:
Edgar D. Charles, MD , Laboratory of Virology and Infectious Diseases, Rockefeller University, New York, NY
Michael I.M. Orloff , Laboratory of Virology and Infectious Diseases, Rockefeller University, New York, NY
Charles M. Rice, PhD , Laboratory of Virology and Infectious Diseases, Rockefeller University, New York, NY
Lynn B. Dustin, PhD , Laboratory of Virology and Infectious Diseases, Rockefeller University, New York, NY

Disclosures:

E. D. Charles, None

M. I. M. Orloff, None

C. M. Rice, None

L. B. Dustin, None

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