Response: proliferative versus functional anergy

We appreciate the interest shown in our work. In their comment, Visentini et al used cell proliferation studies to conclude that V_H1-69-positive CD21^high B cells in HCV patients with mixed cryoglobulinemia are anergic.¹  However, B-cell anergy was initially operationally defined as the inability to differentiate into antibody-producing cells on stimulation.²  By this definition, our data (Figure 7 in Charles et al³ ) demonstrate that CD21^low, V_H1-69-positive B cells are anergic, as these cells fail to produce high levels of antibody after stimulation with CD40L, IL-2, and IL-10.³  In contrast, we find that the CD21^high subset does produce antibody after stimulation in vitro. We speculate that this population may be a source of the excessive IgM rheumatoid factor that is typically seen in HCV patients with mixed cryoglobulinemia.

Visentini et al also question whether our calcium mobilization assays were affected by contaminating V_H1-69–negative CD21^high B cells. Positive selection or gating of V_H1-69–expressing B cells for these assays would entail the use of the anti-idiotype antibody, G6, which could stimulate the B-cell antigen receptor (BCR) and impact subsequent analyses. Rather, we used negative selection and gating methods to avoid stimulating the V_H1-69–expressing B cells. B cells for calcium mobilization were gated on the IgG-negative population, which in these subjects consisted mostly of V_H1-69⁺ IgM⁺ cells. For transcriptome analysis, we extracted RNA from negatively isolated IgM⁺κ⁺CD27⁺ B cells. We maintain that our experimental design represents a reasonable trade-off between cell population purity and absence of unintended BCR stimulation. We disagree with the statement that calcium mobilization in different B-cell subsets was very variable. In fact, in 6 of 8 patients, the CD27⁺CD21^low B-cell fraction had decreased calcium mobilization compared with the CD27⁺CD21^high B-cell fraction. Similarly, in 7 of 8 patients, the CD27⁻CD21^low B-cell fraction had lower calcium mobilization compared with the CD27⁻CD21^high B-cell fraction. We welcome continued research into the induction of attenuation mechanisms in these autoreactive B cells.