Establishing reference ranges for molecular signaling pathways in primary immunodeficiencies

Yılmaz, K., Establishing Reference Ranges for Signaling Molecules in Primary Immunodeficiencies, Hacettepe University Graduate School of Health Sciences, Immunology Program, Master's Thesis, Ankara, 2025. This thesis aimed to determine age-specific reference values in healthy control individuals for the phospho-flow cytometric diagnosis of Inborn Errors of Immunity (IEI) associated with STAT1, STAT3, and PI3K signaling pathways. Expression levels and stimulation responses of signaling-related molecules were evaluated under both unstimulated and stimulated conditions in T and B lymphocytes and peripheral blood mononuclear cells (PBMCs) from selected individuals. For STAT1 phosphorylation, significant differences were observed in basal levels in B lymphocytes between the "0–2 years" and "11–17 years" groups (p=0.005 and p=0.004, respectively), and in T lymphocytes under IFN-γ stimulation between the "3–6 years" and "11–17 years" groups compared to the "18 years and older" group (p=0.004 and p=0.000, respectively). Basal STAT3 expression in B lymphocytes was significantly higher in the "0–2 years," "3–6 years," and "11–17 years" groups compared to the "18 years and older" group (p=0.005, p=0.000, and p=0.000, respectively), and was also significantly higher in the PBMCs of the "3–6 years" group (p=0.000). Upon IL-6 stimulation, STAT3 expression was significantly higher in both PBMCs (p=0.000 and p=0.003) and B lymphocytes (p=0.003 and p=0.001) in the "3–6 years" and "11–17 years" groups compared to the adult group. In IL-6-stimulated T lymphocytes, STAT3 expression differed significantly between patients and healthy controls (p=0.017). Likewise, stimulation responses based on STAT3 expression in B lymphocytes also differed significantly between patients and healthy individuals (p=0.014). In the PI3K signaling pathway analyses, basal expression of phosphorylated AKT (p-AKT) in PBMCs was significantly higher in the "3–6 years" group compared to the "0–2 years" group (p=0.004). In CD40-stimulated PBMCs, phosphorylated mTOR (p-mTOR) expression was significantly higher in the "0–10 years" group compared to the "11–17 years" group (p=0.019). Basal p-mTOR expression in PBMCs also differed significantly between healthy and patient groups (p=0.017). Significant differences were also observed in basal p-AKT levels in B lymphocytes (p=0.024), p-mTOR expression in PBMCs stimulated with PMA-Ionomycin and IL-4 (p=0.028), and p-AKT expression in B lymphocytes (p=0.027) between healthy individuals and patients. Keywords: Immune Functional Analysis, Inborn Errors of Immunity, Intracellular Signaling Pathways, Phospho-flow Cytometry BAP Rapid Support Project / Project ID: 21708