Clonal expansion of immunoglobulin M+CD27+ B cells in HCV-associated mixed cryoglobulinemia

Abstract

Hepatitis C virus (HCV) is associated with B-cell lymphoproliferative disorders such as mixed cryoglobulinemia (MC) and B-cell non-Hodgkin lymphoma (B-NHL). The pathogenesis of these disorders remains unclear, and it has been proposed that HCV drives the pro-liferation of B cells. Here we demonstrate that certain HCV(+)MC(+) subjects have clonal expansions of immunoglobulin M (IgM)(+)kappa(+)IgD(low/-)CD21(low)CD27(+) B cells. Using RT-PCR to amplify Ig from these singly sorted cells, we show that these predominantly rheumatoid factor-encoding V(H)1-69/J(H)4 and V(kappa)3-20 gene segment-restricted cells have low to moderate levels of somatic hypermutations. Ig sequence analysis suggests that antigen selection drives the generation of mutated clones. These findings lend further support to the notion that specific antigenic stimulation leads to B-cell proliferation in HCV MC and that chronic B-cell stimulation may set the stage for malignant transformation and the development of B-NHL. The finding that these hypermutated, marginal zone-like IgM(+)CD27(+) B cells are clonally expanded in certain subjects with MC offers insight into mechanisms of HCV-associated MC and B-cell malignancy. This study was registered at www.clinicaltrials.gov as NCT00219999.

Figure 1

Ig CDR3 PCR. DNA was isolated from PBMCs, amplified following an established protocol (see “CDR3 PCR”) and run on a 12% polyacrylamide gel. Polyclonal CDR3s are indicated by the presence of DNA smears between 80 and 120 bp, the expected size range of CDR3. Monoclonal and oliglonal CDR3s are reflected by the presence of solitary, or 2 to 3 bands, respectively, in the expected size range (A). Densitometric analysis of gel bands (B). *Patients with MC.

Figure 2

Phylogenetic analysis of V_H (left) and V_κ (right) from subject 1432's IgM⁺κ⁺ CD27⁺CD21^low B cells. Trees were constructed using neighbor-joining analysis of Tamura-Nei distances calculated from pairwise comparisons of Ig DNA sequences. Percentages of bootstrap support (> 50%) from 1000 replicate samples (analyzed by the neighbor joining method) are indicated to the left of the support node. V_H and V_κ from B cell A5 of patient 1403 serve as reference sequences.