Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients
Francesca Pala, Henner Morbach, Maria Carmina Castiello, Jean-Nicolas Schickel, Samantha Scaramuzza, Nicolas Chamberlain, Barbara Cassani, Salome Glauzy, Neil Romberg, Fabio Candotti, Alessandro Aiuti, Marita Bosticardo, Anna Villa, Eric Meffre
Francesca Pala, Henner Morbach, Maria Carmina Castiello, Jean-Nicolas Schickel, Samantha Scaramuzza, Nicolas Chamberlain, Barbara Cassani, Salome Glauzy, Neil Romberg, Fabio Candotti, Alessandro Aiuti, Marita Bosticardo, Anna Villa, Eric Meffre
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Research Article Immunology

Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients

Abstract

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients.

Authors

Francesca Pala, Henner Morbach, Maria Carmina Castiello, Jean-Nicolas Schickel, Samantha Scaramuzza, Nicolas Chamberlain, Barbara Cassani, Salome Glauzy, Neil Romberg, Fabio Candotti, Alessandro Aiuti, Marita Bosticardo, Anna Villa, Eric Meffre

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Figure 6

GT treatment restores peripheral B cell tolerance checkpoint in WAS patients.

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GT treatment restores peripheral B cell tolerance checkpoint in WAS pati...
(A) Recombinant antibodies expressed by mature naive (CD19+CD10–CD21+IgM+CD27–) B cells from 4 WAS GT-treated patients were tested by ELISA for reactivity against HEp-2 cell lysate. Dotted lines show ED38+ control. Horizontal lines show cutoff OD405 for positive reactivity. The frequencies of HEp-2–reactive and non–HEp-2–reactive clones are summarized in the pie charts, with the number of antibodies tested shown in the center. The frequencies of HEp-2–reactive (B), polyreactive (C), and antinuclear (E) mature naive B cells in HDs and WAS patients before and after GT are represented (left panels). The evolution of the frequencies of HEp-2–reactive (B), polyreactive (C), and antinuclear (E) B cell clones between new emigrant and mature naive B cells in HDs (open diamonds) and post-GT WAS patients (filled diamonds) is shown in the right panels. Each symbol represents an individual, and horizontal bars display means. (D) Autoreactive antibodies expressed by mature naive B cells in post-GT WAS patients mostly recognize cytoplasmic structures, besides mnWAS3p-k77 with ANA reactivity. Original magnification, ×40. (B, C, and E) Differences between patients and HDs were analyzed for statistical significance using the Mann-Whitney test. Differences between samples before and after GT were analyzed using paired t tests. **P < 0.01.