The V_H1-69–expressing marginal zone B cells expanded in HCV-associated mixed cryoglobulinemia display proliferative anergy irrespective of CD21^low phenotype

To the editor:

We read with great interest the article by Charles et al¹  describing anergy of V_H1-69⁺ CD21^low B cells from patients with hepatitis C virus (HCV)–associated mixed cryoglobulinemia (MC). Anergy of these cells was made evident by decreased calcium mobilization and low production of IgM. These authors reported that the V_H1-69⁺ B cells with a CD21^high phenotype were not anergic, but data from our laboratory challenge this conclusion. It is worth noting that in Charles et al's study, calcium mobilization in different subsets of B cells was very variable, and that the analyses could be biased by contaminating V_H1-69⁻ CD21^high B cells because G6⁺ cells were not gated in those experiments. Furthermore, IgM production does not appear to be a reliable marker of anergy in CD21^low B cells, because the CD21^low B cells of patients with common variable immunodeficiency or with HIV infection have defects of proliferative responses although they produce significant amounts of IgM.^2,3  Unfortunately, proliferation studies were not reported in Charles et al's article.

We investigated the functional properties of V_H1-69⁺ B cells from several patients with HCV-associated MC. As with Charles's study, we exploited the G6 antibody⁴  (provided by R. Jefferis, University of Birmingham, Birmingham, United Kingdom) to identify these cells. Phenotypic characteristics suggest that the V_H1-69⁺ CD21^high (IgM⁺CD27⁺) marginal zone (MZ)–like B cells are the precursors of their CD21^low (CD11c⁺) counterparts, considering that some of them express low-level CD11c (Figure 1A,C). The proliferative responses of V_H1-69⁺ B cells were analyzed by the CFSE dilution flow cytometric method⁵ ; the strategy of analysis is illustrated in Figure 1B and D. We found that both the CD21^low (Figure 1B) and the CD21^high (Figure 1D) V_H1-69⁺ B cells failed to proliferate in response to the stimulation of Toll-like receptor (TLR) 9 with CpG; similar findings were obtained with the TLR7 ligand R848 (not shown). Furthermore, V_H1-69⁺ B cells failed to differentiate to CD20^low/neg plasmablasts (Figure 1B,D). The data shown in Figure 1D are representative of the findings in 3 HCV⁺ MC patients with highly enriched (> 75% of total B cells) CD21^high V_H1-69⁺ B cells.

Clonal B cells of HCV⁺ MC patients provide a unique model for investigating the pathophysiology of human MZ B cells. Murine MZ B cells expand massively on stimulation by blood-borne microbial antigens⁶  and, similarly, the antigenic pressure of HCV appears to result in the robust clonal expansion of V_H1-69⁺ MZ B cells in MC.⁷  We provide evidence that these cells undergo premature proliferative anergy, which takes place when the V_H1-69⁺ B cells still retain a CD21^high MZ-like phenotype and have not yet shifted to a fully “exhausted”³  CD21^low phenotype. Interestingly, microarray gene expression profiling studies⁸  showed that murine MZ B cells stimulated in vivo by Streptococcus pneumoniae rapidly up-regulate Stra13, a negative regulator of B-cell proliferation. Thus, early proliferative anergy may serve to constrain the excessive expansion of MZ B cells activated by microbial antigens, and to attenuate the risk of autoimmune disease and of malignant transformation.