Phosphoinositide 3-kinase δ gene mutation predisposes to respiratory infection and airway damage

Abstract

Genetic mutations cause primary immunodeficiencies (PIDs) that predispose to infections. Here, we describe activated PI3K-δ syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M, and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

Fig. 1. Families with the E1021K p110δ mutation

(A) ○ and □ - unaffected; ● and ■ - affected; and - available data indicate recurrent infections. Age at the time of death is shown for patients who died ≤30 years of age. PIK3CD genotype is shown if known: wt, wild type allele encoding glutamic acid (E1021); mut, mutant allele encoding lysine (K1021). (B) Sequence chromatogram showing heterozygous mutation c.3061G>A in the PIK3CD gene leading to the E1021K amino-acid change in p110δ. CpG dinucleotide is underlined.

Fig. 2. In vitro activity and structure of p110δ

(A) Basal and pY-stimulated PI3K activity at 20 nM concentration. Graphs are mean ± SD of 3 independent experiments. P-values were calculated by two-tailed t-test. (B) Inhibition of mutant and wild-type p110δ/p85α as a function of IC87114 or GS-1101 concentration (data are mean ± SD, N=3). (C) Domain organization of p110δ. (D) Structural model of the p110δ/p85α heterodimer. p110δ catalytic subunit (pale green), nSH2 and iSH2 domains of the p85 regulatory subunit (cyan), cSH2 domain (magenta), p110δ activation loop (thick chocolate tube beneath kα12), residue E1021 of p110δ (green spheres) and the analogous residue in H1047R mutant of p110α (cyan spheres). The IC87114 inhibitor bound in the active site is shown in stick representation. (E) Membrane binding of p110δ. FRET between the PI3K complex and Dansyl-PS-containing membrane vesicles in the absence (solid lines) or presence (dashed lines) of the pY peptide (data are mean ± SD, N=3).

Fig. 3. Functional analyses of T cells in patients with APDS

(A) Intracellular PIP₃ levels in CD4+ and CD8+ T lymphocytes of patients (red squares, N=6) and controls (blue circles, N=5) at indicated times after anti-CD3/anti-CD28 stimulation in the presence or absence of IC87114. The data are expressed as the ratio of the quantity of PIP₃ divided by that of the internal standard (ISD) and normalized according to the cell number. The data show mean +/− SEM. P-values were calculated using two-way ANOVA with Bonferroni correction. (B) Representative (N=3) Western blot showing levels of p110δ, AKT and phospho-AKT (pAKT) proteins in CD4+ T cells isolated from fresh blood samples of a healthy control (C) and a patient (P) without stimulation (−) or after 10 min stimulation (+) with anti-CD3 and anti-CD28 antibodies. (C) Representative (N=2) Western blot showing levels of p110δ, and pAKT proteins in CD4+ T cell blasts of a p110δ knockout mouse transduced with retroviral constructs expressing either GFP or wild-type p110δ (p110δ^WT) or kinase dead p110δ (p110δ^D911A) or p110δ^E1021K without stimulation (−) or after stimulation (+) with anti-CD3 antibodies and anti-CD28 antibodies. (D) Quantification of surviving CD4+ and CD8+ T cells as indicated by % of cells excluding viability dye. Cells of patients (red, N=4) and controls (blue, N=7) were studied without stimulation and after stimulation with anti-CD3/anti-CD28 antibodies and in the presence of IC87114. Each subject was studied in triplicate. The data show mean +/− SEM. P-values were calculated using a two-way ANOVA with Sidak’s multiple comparisons test.