key: cord-0696316-v18u9lzp
authors: Charles, Edgar D.; Dustin, Lynn B.
title: Response: proliferative versus functional anergy
date: 2011-09-22
journal: Blood
DOI: 10.1182/blood-2011-07-368092
sha: 016c55930d5ae94bbc4e100f193f75112b07b14c
doc_id: 696316
cord_uid: v18u9lzp

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We appreciate the interest shown in our work. In their comment, Visentini et al used cell proliferation studies to conclude that V H 1-69-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 al 3 ) demonstrate that CD21 low , V H 1-69-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 H 1-69-negative CD21 high B cells. Positive selection or gating of V H 1-69-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 H 1-69-expressing B cells. B cells for calcium mobilization were gated on the IgG-negative population, which in these subjects consisted mostly of V H 1-69 ϩ IgM ϩ cells. For transcriptome analysis, we extracted RNA from negatively isolated IgM ϩ ϩ CD27 ϩ 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 ϩ CD21 low B-cell fraction had decreased calcium mobilization compared with the CD27 ϩ CD21 high B-cell fraction. Similarly, in 7 of 8 patients, the CD27 Ϫ CD21 low B-cell fraction had lower calcium mobilization compared with the CD27 Ϫ CD21 high B-cell fraction. We welcome continued research into the induction of attenuation mechanisms in these autoreactive B cells. To the editor: Figure 1A-B) . FISH analysis using a BCL2 break-apart probe (LSI BCL2 BAP, Vysis) confirmed a BCL2 breakpoint in both areas suggestive of the t(14;18)(q32;21) translocation ( Figure 1B; insert) . IGH PCR clonality analysis performed in microdissected tissue showed identical monoclonal peaks in both lesions ( Figure 1C-D) . Sequence analysis of the BCL2 breakpoint region confirmed the clonal identity of both lesions ( Figure 1E ). To explain the discordant BCL2 reactivity, exon 1 of the BCL2 gene, where the epitope of the BCL2 antibody resides, was amplified and sequenced. A point mutation resulting in amino acid substitution (c.144 C Ͼ G; p.I48M ATC-ATG) was found in the mFL, whereas the FLIS showed a wild type sequence ( Figure 1F -G). Array CGH (244K platform, Agilent Technologies)

The VH1-69-expressing marginal zone B cells expanded in HCV-associated mixed cryoglobulinemia display proliferative anergy irrespective of CD21low phenotype

Clonal anergy: persistence in tolerant mice of antigenbinding B lymphocytes incapable of responding to antigen or mitogen