Response: common variable immunodeficiency patients with increased CD21^−/lo B cells suffer from altered receptor editing and defective central B-cell tolerance

The letter by Rakhmanov et al¹  suggested that normal κ/λ ratios in CD21⁺ naive B cells and increased Igλ-chain usage by CD21^−/lo B cells in common variable immunodeficiency disease (CVID) patients with expanded CD21^−/lo B-cell populations (CVID group-Ia² ) were evidence of normal or increased receptor editing during central selection. Although increased Igλ-chain usage could correlate with extensive secondary recombination because Igλ genes normally rearrange after Igκ genes, the best evidence of receptor editing and central tolerance defects is found in proximal B-cell populations, specifically immature B cells in marrow or new emigrant/transitional B cells in peripheral blood. These subpopulations, similar in antibody repertoire and reactivity, offer an accurate historical record of central events uninfluenced by proliferation.^3,4  Accordingly, the analysis by Warnatz and colleagues¹  of Igλ-chain usage in CD21⁺ naive B cells and CD21^−/lo B cells from 4 CVID group-Ia patients does not inform central events but implies peripheral selection in 2 B-cell subpopulations with well-documented histories of homeostatic expansion.^4,5  Therefore, we assessed if receptor editing is functional in CVID group-Ia patients by analyzing the antibody repertoire and reactivity of new emigrant/transitional B cells from these patients.

We report here the Igκ repertoire from 1226 single CD19⁺CD10⁺⁺IgM^hiCD27⁻ new emigrant/transitional B cells from 49 individuals including 21 healthy controls, 11 CVID group-Ia patients and 17 CVID non group-Ia patients with CD21^−/lo B cells < 20% of total B cells.²  Secondary recombination mediating receptor editing replaces previous Igκ gene rearrangements with variable (Vκ)-joining (Jκ) gene segment joints resulting in increased Vκ genes located upstream of the locus combined to downstream Jκ genes (Figure 1A).⁶  We found that the Igκ repertoires of new emigrant/transitional B cells from CVID group-Ia patients were biased toward Jκ1, the most upstream Jκ gene segment, suggesting decreased secondary recombination (Figure 1B). CVID group-Ia Igκ repertoires were also characterized by decreased usage of upstream Vκ segments, genes overrepresented in diseases marked by extensive secondary recombination (Figure 1C).^7,8  Moreover, bivariate analysis of Jκ1 and upstream Vκ usage further demonstrated CVID group-Ia new emigrant/transitional B cells occupy a distinct Igκ repertoire niche reflecting a dearth of secondary recombination (Figure 1D). The molecular basis of secondary recombination defects in these patients remains unknown and its correlation with the presence of peripheral CD21^−/lo B cells is not understood. One clue about the regulation of secondary recombination came from BTK-deficient patients who display impaired B-cell receptor (BCR) signaling correlating with extensive secondary recombination events potentially through a failure to down-regulate RAG gene expression.⁷  Increased BCR signaling might therefore induce the premature RAG gene down-regulation resulting in the diminished secondary recombination activity observed in CVID group-Ia patients. This hypothesis, however, is not supported by BCR-induced calcium flux data demonstrating normal signal strength in CD21⁺ mature naive B cells from such patients.^5,9  Alternatively, decreased recombination events in CVID group-Ia Igκ repertoires may suggest defective V(D)J recombination and/or DNA repair. The presence of such defects may account for radiosensitivity and malignancy susceptibility reported in some CVID patients.¹⁰ 

Finally, using an RT-PCR method to clone and express in vitro antibodies express by single B cells,³  we found that CVID group-Ia patients displayed increased frequencies of polyreactive clones in their new emigrant/transitional B-cell compartment, demonstrating a defective central B-cell tolerance checkpoint in these individuals (Figure 1E). Hence, the altered regulation of secondary recombination/receptor editing in developing B cells from CVID group-Ia patients correlates with the impaired counterselection of autoreactive clones in their bone marrow.