Systemic autoimmunity and defective Fas ligand secretion in the absence of the Wiskott-Aldrich syndrome protein

Abstract

Autoimmunity is a surprisingly common complication of primary immunodeficiencies, yet the molecular mechanisms underlying this clinical observation are not well understood. One widely known example is provided by Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder caused by mutations in the gene encoding the WAS protein (WASp) with a high incidence of autoimmunity in affected patients. WASp deficiency affects T-cell antigen receptor (TCR) signaling and T-cell cytokine production, but its role in TCR-induced apoptosis, one of the mechanisms of peripheral immunologic tolerance, has not been investigated. We find that WASp-deficient mice produce autoantibodies and develop proliferative glomerulonephritis with immune complex deposition as they age. We also find that CD4(+) T lymphocytes from WASp-deficient mice undergo reduced apoptosis after restimulation through the TCR. While Fas-induced cell death is normal, WASp deficiency affects TCR-induced secretion of Fas ligand (FasL) and other components of secretory granules by CD4(+) T cells. These results describe a novel role of WASp in regulating TCR-induced apoptosis and FasL secretion and suggest that WASp-deficient mice provide a good model for the study of autoimmune manifestations of WAS and the development of more specific therapies for these complications.

Figure 1

Autoantibody production WASp-deficient mice. (A) Fluorescent ANA titers in WASp-deficient and age-matched control mice on 129SvEv genetic background divided into 2 groups based on age (> 6 months and < 6 months). Statistical comparison was performed using the Fisher exact test. A total of 266 serum samples were tested. For the group less than or equal to 6 months old (n = 48), the mean age ± SD in days was WASp-deficient, 119 ± 35; WT controls, 104 ± 33 (P = .18). For the group more than 6 months old (n = 218), the mean age ± SD in days was WASp-deficient, 325 ± 98; WT controls, 303 ± 83 (P = .08). (B) Representative images of 129 background WASp ANA (serum dilution 1:640) with positive (B6.lpr/lpr 1:640) and negative 129 SvEv WT controls (serum dilution 1:40). Note the staining of mitotic figures by serum from the WASp-deficient mouse, suggesting the presence of anti-chromatin antibodies. (C) Kaplan-Meier analysis of the proportion of mice becoming ANA-positive at 1:640 cutoff titers over time (solid line, WT controls; dashed line, WASp-deficient mice). (D) Sera from ANA-positive mice tested for anti-dsDNA specificity by ELISA and expressed as a percent of the total tested samples per genotype. Mean age in days ± SD (not statistically significantly different) and number of mice per group are indicated (anti-dsDNA–negative, □; anti-dsDNA–positive, ■).

Figure 2

Immune complex deposition and mesangial cell proliferation in WASp-deficient mice. (A) Representative PAS-stained glomeruli from WASp-deficient and 129 SvEv control mice and immunofluorescence images of IgA, IgG, IgM, and C3 deposition in glomeruli from the same animals. (B) Quantitation of immunofluorescence results from mice of the indicated genotype more than 6 months old measured as in panel B. (C) Circulating immune complexes in mice of the indicated genotypes and ages. (D) Urinary albumin/creatinine ratios in a cohort of WASp-deficient and 129 age-matched control mice.

Figure 3

Impaired TCR-mediated apoptosis of activated WASp-deficient CD4⁺ T lymphocytes. (A) Specific cell death of activated CD4⁺ T cells from WT and WASp deficient mice restimulated with the indicated concentrations of plate-bound anti-CD3 mAb for 6 hours and measured by annexin V and PI staining. The data are average and SEM of 5 independent experiments with age- and sex-matched mice on the 129 backgrond. Apoptosis measurements were performed in triplicate for each sample. Results of unpaired t test comparisons of cell death at each dose of anti-CD3 are shown as *P < .05, **P < .005, ***P < .001. (B) Surface TCR, Fas on activated CD4⁺ T cells from WASp-deficient and control mice activated as in panel A. (C) Proliferation of WT and WASp-deficient T cells activated in the presence of exogenous IL-2 as in panel A measured by ³H-thymidine incorporation at 72 hours. (D) Cell death of WASp-deficient and control T-cell blasts after addition of the indicated concentrations of Mega-FasL (an oligomerized biologically active form of soluble FasL). (E) Viable cell number during IL-2 cytokine deprivation (cRPMI) or with IL-2 measured by trypan blue exclusion. (F) Cell viability measured by PI and DiOC of WASp-deficient and control CD4⁺ T cells during IL-2 cytokine withdrawal. (E-F) Data are representative of 2 independent experiments.

Figure 4

Normal up-regulation of FasL mRNA and surface expression in WASp-deficient T cells. (A) Induction of FasL mRNA measured by real-time quantitative polymerase chain reaction (RT-qPCR) after exposure of activated CD4⁺ T lymphocytes to 1 μg/mL plate-bound anti-CD3 antibody for 6 hours. Induction of mRNA is shown relative to stimulation with isotype control antibodies. Average and SD of mRNA induction is shown. Similar results were observed in 2 independent experiments. (B) WT and WASp-deficient T cells were restimulated with anti-CD3 for the indicated number of hours, and surface FasL was quantitated by FACS. The mean change in geometric mean fluorescence is shown for WT and WASp-KO T cells restimulated for the indicated periods of time with anti-CD3. The data are the average ± SEM of 3 independent experiments.

Figure 5

Reduced bioactive FasL and granule secretion by WASp-deficient T cells. (A) Secreted FasL measured by ELISA in supernatants after 6 hours of stimulation of activated CD4⁺ T lymphocytes with the indicated concentrations anti-CD3 antibody. Values are the average ± SEM of data from 2 mice per group, and similar results were obtained in 3 independent experiments. (B) Supernatants from CD4⁺ T cells restimulated for 6 hour with anti-CD3 were fractionated by centrifugation through 100-kDa cutoff membranes, and FasL was quantitated in each fraction by ELISA. No FasL was detected in the < 100-kDa fraction when purified vesicular FasL was filtered through identical membranes. (C) The indicated concentrations of purified vesicular and soluble FasL were added to WEHI-279 cells, and cytotoxicity was quantitated by a luminescent cell viability assay. Anti-FasL antibody was added to demonstrate specificity of this assay for bioactive FasL. Specific cell death was quantitated as described in the methods. (D) FasL-dependent apoptosis-inducing activity of the indicated fractions of supernatants collected from cells WT and WASp-KO T cells. Supernatants from cells restimulated in C were assayed on WEHI-279 cells for apoptosis-inducing activity. Anti-FasL was added to the indicated samples to neutralize FasL activity. Asterisks mark the results of comparisons of WASp-KO with the identical WT cell supernatant fractions, and anti-FasL–treated samples compared with the same samples without anti-FasL. Results of P values from comparisons using Student unpaired t test are denoted as *P < .05, **P < .005, ***P < .001. (E) β-Hexosaminadase release from activated WASp-deficient and control T cells restimulated with the indicated concentrations of anti-CD3 mAb. The curve of percent specific release was significantly different in WASp-deficient mice for both CD4 and CD8 than controls (P < .001, 2-way analysis of variance).