N-WASP is required for B-cell–mediated autoimmunity in Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by eczema, thrombocytopenia, immunodeficiency, and autoimmunity.^1,2  By generating a mouse lacking expression of the WAS protein (WASP) selectively in B lymphocytes (B/WcKO), we and others have revealed a nonredundant B-cell-intrinsic role of WASP in immune homeostasis and prevention of autoimmunity, as well as in marginal zone (MZ) development and regulation of the germinal center (GC) reaction.^3-5  Neural WASP (N-WASP, encoded by the Wasl gene) is another member of the WASP family of proteins; it is ubiquitously expressed and shares 50% homology with WASP.⁶  Similar to WASP, N-WASP undergoes a conformational change upon activation that enables initiation of actin polymerization,^7,8  thereby linking cellular activation to cytoskeletal modifications.⁹  Selective deletion of N-WASP in B lymphocytes of Was knockout (WKO) mice resulted in the aggravation of B-cell abnormalities, including a strong decrease of intracellular calcium flux and Bruton’s tyrosine kinase (Btk) and Src homology 2-containing inositol 5′ phosphatase phosphorylation upon B-cell receptor (BCR) stimulation,¹⁰  further worsening of MZ B-cell depletion,¹¹  and defective somatic hypermutation.¹²  However, lack of WASP expression in multiple hematopoietic cells may have indirectly contributed to B-cell abnormalities in these models.

To investigate the B-cell intrinsic role played by WASP and N-WASP in immune homeostasis and regulation more specifically, we have developed a double conditional mouse model (B/DcKO) in which deletion of both Was and Wasl floxed alleles in B lymphocytes is driven by the Cre recombinase expressed under the B-cell-specific promoter mb1. Here we show that deletion of N-WASP in B cells impairs B-cell activation and T-cell-dependent antibody responses and reduces manifestations of immune dysregulation seen in B/WcKO mice.

Detailed methods are presented in supplemental Data (available on the Blood Web site). B/DcKO mice were generated by breeding B/WcKO³  with Wasl^fl/fl 13  mice. Lymphocyte subsets were analyzed by fluorescence-activated cell sorting (FACS) and immunofluorescence staining of spleen sections. FACS-sorted spleen follicular (Fo) and MZ B cells were analyzed for proliferation by assessing carboxyfluorescein succinimidyl ester dilution at day 4 after stimulation with anti-immunoglobulin M (anti-IgM) and CpG 1826. Intraperitoneal immunization with 2,4,6-trinitrophenyl hapten-Keyhole limpet hemocyanin (TNP-KLH) was performed as described.¹⁴  Ig serum levels were analyzed by enzyme-linked immunosorbent assay.¹⁴  Levels of serum autoantibodies were assessed by enzyme-linked immunosorbent assay or by using a protein array (University of Texas Southwestern Medical Center).^3,15  Pathological scoring of periodic acid–Schiff-stained kidney sections from 7- to 20-month-old mice was assessed blindly by a trained nephrologist as previously described.³ 

FACS analysis of the B-cell compartment yielded similar results in B/DcKO and B/WcKO mice. In particular, B-lymphocyte progenitors were normally represented in the bone marrow of B/DcKO mice (supplemental Figure 1A-B); however, the proportion of B220^hiIgM⁺ bone marrow mature recirculating B cells was markedly reduced (Figure 1A). Furthermore, B/DcKO mice had a normal frequency and absolute count of transitional and mature Fo B cells in the spleen (supplemental Figure 1C-D) but a marked reduction of MZ B cells (Figure 1B). Analysis of serum Ig levels showed that B/DcKO mice lacked the increase of IgM and IgE serum levels observed in WKO and B/WcKO mice³  and had lower IgG levels (supplemental Figure 2). The distribution and count of CD4⁺ and CD8⁺ splenic T cells were unaffected in B/DcKO mice (data not shown).

We have previously shown that spontaneous GC formation is a prominent feature of immune dysregulation in B/WcKO mice.³  By contrast, B/DcKO mice did not present spontaneous GC formation, as shown by the low proportion of PNA⁺GL7⁺ GC B cells and lack of peanut agglutinin staining in the spleen follicles of naïve mice (Figure 1C). These results suggest that concurrent deletion of N-WASP in the B-cell lineage of B/WcKO mice restrains spontaneous GC formation.

To test the hypothesis that the combined N-WASP and WASP deletion may affect B-cell activation, we stimulated sorted spleen Fo and MZ B cells from B/DcKO, B/WcKO, and wild-type mice with anti-IgM antibody and CpG. Upon in vitro stimulation, proliferation and viability of B/DcKO Fo B lymphocytes, but not MZ B cells, were markedly impaired (Figure 1D). These data are consistent with data recently reported by others.¹⁰  To determine whether these functional abnormalities of B/DcKO Fo B cells may have important implications in vivo, we immunized mice with the T-cell-dependent antigen TNP-KLH. Upon immunization, robust GC formation (as indicated by peanut agglutinin staining) and an increased proportion of CD19^lowCD138⁺ plasma cells were observed in the spleens of B/WcKO but not B/DcKO mice (Figure 1E). Furthermore, both low- and high-affinity IgG1 anti-TNP antibody responses were reduced in B/DcKO compared with B/WcKO mice (Figure 1F). Altogether, these data indicate that activation of Fo B cells and in vivo response to T-cell-dependent antigens are impaired in B/DcKO mice.

Autoimmunity is a prominent feature in B/WcKO mice, with increased production of IgM and IgG autoantibodies (Figure 2A-B and supplemental Figure 3A).⁶  By contrast, B/DcKO mice lacked IgG autoantibodies to double-stranded DNA and single-stranded DNA (Figure 2A) and to a broad range of self-antigens, as tested by a protein array (Figure 2B). However, they showed increased levels of IgM autoantibodies, which were also observed in WKO and B/WcKO mice (supplemental Figure 3A). Moreover, unimmunized B/DcKO mutant mice had higher levels of IgM anti-TNP polyreactive antibodies (supplemental Figure 3B), as previously shown for B/WcKO mice.³  Finally, whereas older B/WcKO and especially WKO mice developed kidney immunopathology, as previously reported,⁶  none of the B/DcKO mice studied (up to age 14 months) showed signs of kidney disease (Figure 2C-D). All three mutant strains, but not wild-type mice, had increased glomerular deposits of IgG. No differences in the amount of IgM, IgG, or C3 deposits were observed among WKO, B/WcKO, and B/DcKO mice (supplemental Figure 4). Overall, in comparison with B/WcKO mice, B/DcKO mice show reduced levels of IgG autoantibodies and lack of tissue immunopathology in spite of the presence of IgM autoantibodies.

Recent data have suggested a direct role of WASP in shaping the immune repertoire through negative selection of autoreactive progenitors¹⁶  and skewing of the BCR repertoire.¹⁷  The strength of BCR signaling controls central mechanisms of B-cell tolerance, including deletion, receptor editing, and anergy.^18-20  We have shown that simultaneous deletion of WASP and N-WASP affects proliferation of Fo B cells but not MZ B cells in response to BCR and Toll-like receptor 9 (TLR9) signaling. Our results support a model in which deletion of both WASP and N-WASP causes a defect in the BCR signaling responsible for the accumulation of IgM autoantibody-secreting plasma cells, whereas GC responses and generation of class-switched autoreactive B cells are impaired.^10,12  Defective migration of B cells devoid of both WASP and N-WASP to the GC¹²  and impaired class switching in response to CD40 or TLR signaling may both contribute to this defect. Finally, although both WKO and B/WcKO mice had similar levels of IgM autoantibodies and higher levels of IgG autoantibodies were observed in B/WcKO mice, renal damage was more severe in WKO mice. No tissue immunopathology was observed in B/DcKO mice in spite of IgM autoantibodies. These apparent discrepancies may be reconciled with the observation that other blood lineages, in particular regulatory T cells and plasmacytoid dendritic cells, play important, WASP-dependent roles in immune homeostasis,^21-24  independently of autoantibody production.²⁵  In conclusion, our data broaden the understanding of the molecular mechanisms underlying immune dysregulation in WAS and suggest that N-WASP may be an attractive novel target for pharmacological control of autoimmunity in patients with this disease.

This work was supported by grant 5P01HL059561 from the National Heart, Lung, and Blood Institute, National Institutes of Health (L.D.N.) and grant CHUV-UNIL CGRB 29583 from the University Hospital of Lausanne and the Suisse National Science Foundation (F.C.). A.J.T. is supported by the Wellcome Trust.

Contribution: S.V. performed and analyzed most experiments and wrote the manuscript; E.S., K.A., C.I.M.D., M.R., K.C., E.C., D.R., and D.M. performed experiments; M.M. performed experiments and contributed to the writing of the paper; G.C.T. and L.S.W. supervised some experiments; S.S. and A.J.T. contributed vital experimental tools; F.C. supervised some experiments and contributed to the writing of the paper; and L.D.N. designed, analyzed, and supervised all experiments and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Luigi D. Notarangelo, Division of Immunology, Boston Children’s Hospital, Karp Research Building, Room 10217, 1 Blackfan Circle, Boston, MA 02115; e-mail: luigi.notarangelo@childrens.harvard.edu.