Evaluation of B‐cell intracellular signaling by monitoring the PI3K‐Akt axis in patients with common variable immunodeficiency and activated phosphoinositide 3‐kinase delta syndrome

Abstract

Background: Primary antibody deficiencies (PADs) are characterized by hypogammaglobulinemia and impaired B-cell differentiation. Patients with common variable immunodeficiency (CVID) present severe reductions in at least 2 serum immunoglobulins and impaired terminal differentiation of B cells. Most patients with CVID do not appear to present monogenic defects. Activated phosphoinositide 3-kinase delta syndrome (APDS), caused by gain-of-function mutations in the PIK3CD gene (p110δ), can present in patients with a CVID-like phenotype. Memory B-cell differentiation requires the orchestrated activation of numerous intracellular signaling pathways, which promote transcriptional programs required for long-term B-cell survival. The aim of this study was to develop a flow cytometry assay to trace the PI3K-Akt-mTOR pathway, a critical component of B-cell homeostasis, and analyze its status in PADs. Methods: We analyzed the intracellular expression of Akt and S6 by flow cytometry and their phosphorylation status in both baseline conditions and upon B-cell receptor activation with anti-IgM in various primary B-cell subsets of patients with CVID and APDS. Results: B cells from CVID patients showed reduced phosphorylation in Akt and S6 proteins after anti-IgM stimulation. Constitutive high baseline B-cell levels of Akt and S6 phosphorylation in a patient with APDS were reduced once m-TOR inhibition therapy was initiated.Conclusions: Intracellular flow cytometry can be routinely employed to explore alterations in the PI3K-Akt-mTOR pathway in B cells from patients with PADs. AKT and S6 phosphorylation levels are informative biomarkers that could be employed as mTOR inhibitors for monitoring therapies targeting this pathway.

Dysregulated PI3K Signaling in B Cells of CVID Patients

“…Here, we demonstrated that basal as well as induced PI3K signaling, which regulates the metabolic state, survival, activation and differentiation of this lymphocyte lineage, is severely altered in the naïve B cells of CVID patients. In addition to previous reports [68,69], we found increased baseline but impaired BCR-mediated PI3K pathway activation, especially in the CD21 low B cells of CVID patients, providing potential therapeutic options to target these cells by specific inhibitors of PI3Kδ or mTOR. Altered PI3K signaling may well also contribute to the immunodeficiency in CVID as PI3K signal strength, and the dynamic regulation of transcription factors, such as FOXO1 and HIF1α, determines the selection into short-lived plasmablasts or germinal center B-cell fate, the induction of somatic hyper mutation and class switch recombination and finally the integrity of memory and plasma cell formation [54,55,62,70,71].…”

Activated phosphoinositide 3-kinase delta syndrome misdiagnosed as anti-neutrophil cytoplasmic antibody-associated vasculitis: a case report

“… 1 , 2 , 4 Elevated phosphorylation of AKT (pAKT) and S6K (pS6K) have been found in these patients and are considered valuable indicators of the pathogenic variant in APDS and the hyperactivated PIK/AKT/mTOR/S6K pathway. 6 , 7 , 9 Moreover, in previous studies, patients with APDS who were treated with rapamycin or leniolisib (inhibitors of the PIK/AKT/mTOR/S6K pathway) showed decreased levels of pAKT and pS6K 5 , 9 , 10 Therefore, the pAKT or pS6K levels of the patient in the present case need to be followed up to monitor the efficacy of rapamycin, which may also be a partial supplement for the functional validation of E525G.…”

“…A previous study revealed that PIK3CD GOF mutations may result in sustained and exaggerated activation of polyclonal CD8+ T cells; thus, many patients with APDS have increased frequencies of CD8+ T cells. However, these CD8+ T cells seem to exhibit premature immunosenescence and exhaustion, 9 , 12 which reduces their cytotoxicity. PIK3CD GOF mutations also dysregulate the expression of key regulatory molecules and reduce the cytotoxic function of NK cells.…”

Activated phosphoinositde 3-kinase (PI3Kδ) syndrome: an Italian point of view on diagnosis and new advances in treatment

“…Today, several techniques are available in the literature to assess PI3Kδ functionality, including flow cytometry evaluation of intracellular phosphorylation of AKT and S6 in vitro on PBMC, T, B cells and cell lines [ 13 , 21 ]. These functional tests, as well as the analysis of protein levels by means of Western Blot, could also be used during the follow-up of patients to assess the efficacy of the therapy [ 22 , 23 ].…”