Enhanced AKT Phosphorylation of Circulating B Cells in Patients With Activated PI3Kδ Syndrome

Abstract

Activated PI3Kδ syndrome (APDS) is a primary immunodeficiency characterized by recurrent respiratory tract infections, lymphoproliferation, and defective IgG production. Heterozygous mutations in PIK3CD, PIK3R1, or PTEN, which are related to the hyperactive phosphoinositide 3-kinase (PI3K) signaling, were recently presented to cause APDS1 or APDS2 (APDSs), or APDS-like (APDS-L) disorder. In this study, we examined the AKT phosphorylation of peripheral blood lymphocytes and monocytes in patients with APDSs and APDS-L by using flow cytometry. CD19⁺ B cells of peripheral blood in APDS2 patients showed the enhanced phosphorylation of AKT at Ser473 (pAKT) without any specific stimulation. The enhanced pAKT in CD19⁺ B cells was normalized by the addition of a p110δ inhibitor. In contrast, CD3⁺ T cells and CD14⁺ monocytes did not show the enhanced pAKT in the absence of stimulation. These findings were similarly observed in patients with APDS1 and APDS-L. Among CD19⁺ B cells, enhanced pAKT was prominently detected in CD10⁺ immature B cells compared with CD10^- mature B cells. Enhanced pAKT was not observed in B cells of healthy controls, patients with common variable immunodeficiency, and hyper IgM syndrome due to CD40L deficiency. These results suggest that the enhanced pAKT in circulating B cells may be useful for the discrimination of APDS1, APDS2, and APDS-L from other antibody deficiencies.

Keywords: AKT phosphorylation; activated PI3 kinase delta syndrome; catalytic subunit p110δ of phosphatidylinositol 3-kinase; flow cytometry; immunodeficiency; regulatory subunit p85α of phosphatidylinositol 3-kinase.

Figure 1

The effect of splice site mutation identified in P1. (A) A germline heterozygous mutation, (B) 1425 + 2 T > A, in PIK3R1 was identified by whole exome sequencing and confirmed by Sanger sequencing. (C) mRNA was extracted from peripheral blood mononuclear cells from P1 and his mother. Complementary DNA was then synthesized to assess the significance of the nucleotide substitution, 1425 + 2 T > A, on splicing. (C,D) The RT-PCR fragment was cloned into a pGEM-T easy vector. Loss of exon 10 in patient (C) but not mother (D) was revealed by Sanger sequencing.

Figure 2

Flow-cytometry-based AKT phosphorylation (pAKT) assay of PMBCs. (A) The level of pAKT in the presence or absence of p110δ inhibitor treatment was assessed by flow cytometry in total peripheral blood mononuclear cells (PBMCs), CD3⁺ T cells, CD14⁺ monocytes, and CD16⁺CD56⁺ natural killer cells from activated PI3Kδ syndrome 2 (APDS2) (P1), common variable immunodeficiency (CVID) (P8), or hyper IgM syndrome (HIGM) (P9). The number of events analyzed was >5,000. Red bold line: no treatment and black dotted line: p110δ inhibitor treatment. (B) The level of pAKT in negatively selected B cells from PBMCs from P1 and one healthy control was compared with pAKT in CD19⁺ B cells. Red solid line: no treatment and black dotted line: p110δ inhibitor treatment. (C) A summary of difference in mean fluorescence intensity of whole lymphocytes derived from 24 adult healthy controls or all of the patients with APDS2 or CVID/HIGM. There is no statistical significant among this three groups (control vs. CVID; p = 0.24, APDS2 vs. CVID; p = 0.13, and control vs. APDS2; p = 0.52).

Figure 3

Enhanced AKT phosphorylation (pAKT) in CD19⁺ B cells from patients with activated PI3Kδ syndromes (APDSs) or APDS-like (APDS-L). (A) Total AKT expression in CD19⁺ B cells from APDS2 patients (P1) was measured by flow-cytometry. Red bold line: activated PI3Kδ syndrome 2 (APDS2) patient and black dotted line: healthy control. (B) The phosphorylation and protein expression of AKT was investigated by immunoblotting. (C) pAKT (pS473) in the presence of p110δ inhibitor was analyzed by flow cytometry in CD19⁺ B cells from healthy controls and patients with APDS1, APDS2, or APDS-L, CVID/HIGM. The number of events analyzed was >5,000. (D) A summary of difference in mean fluorescence intensity of CD19⁺ B cells derived from healthy controls or patients with APDS1 (four patients), APDS2 (P1, P2, P3, and P4), APDS-L (four patients), or CVID/HIGM (P5, P6, P7, P8, and P9). *p < 0.05 and **p < 0.01.

Figure 4

(A) AKT phosphorylation (pAKT) expression in activated T cells, (B) pAKT expression CD19⁺ B cells stimulated with CD40L and IL-4. (A) The level of pAKT in T cells was assessed by flow cytometry in activated PI3Kδ syndrome 2 patient (P1) and healthy controls (representative result from two controls is shown). The total number of events analyzed was 2,000. Red bold line: no treatment and black dotted line: p110δ inhibitor treatment. (B) pAKT expression in CD19⁺ B cells from (P1) and healthy control following stimulation with CD40L and IL-4. Blue line: isotype control, black and red solid line: no treatment, and black and red dot line: CD40L and IL-4 treatment.

Figure 5

Enhanced AKT phosphorylation (pAKT) in activated PI3Kδ syndrome 2 (APDS2) patients was pronounced in a flow-cytometry assay of CD10⁺CD19⁺ B cells. (A,B) The enhancement of pAKT observed in CD19⁺ B cells was further investigated by separating them into CD10⁺CD19⁺ immature B cells and CD10⁻CD19⁺ mature B cells from P2 (representative histogram of three experiments) (A) and healthy controls (B). The number of events analyzed was >5,000. As an exception, CD10⁺CD19⁺ immature B cells analyzed in healthy control were 800 events. Red solid line: no treatment and black dotted line: p110δ inhibitor treatment. (C,D,E) A summary of difference in mean fluorescence intensity of CD19⁺ B cells (C), CD10⁻CD19⁺ mature B cells (D), and CD10⁺CD19⁺ immature B cells (E) from healthy controls or patients with APDS1 (three patients), APDS2 (P1, P2, and P4), or APDS-like (APDS-L) (three patients). *p < 0.05 and **p < 0.01.