From Validation to Clinical Interpretation: A Flow Cytometric Th17-Cell Assay for STAT1- and STAT3-Related Inborn Errors of Immunity.

Th17-cells are crucial for defence against fungal and bacterial infections and deficiency characterises certain inborn errors of immunity (IEI), including STAT3-related Hyper IgE syndrome (HIES) and STAT1-related Chronic Mucocutaneous Candidiasis (CMC). Standardised methods for clinical Th17-cell quantification are limited. We developed a flow cytometric assay for quantifying Th17-cells in peripheral blood based on PMA/ionomycin stimulation and intracellular IL-17 labeling and evaluated its precision, robustness and longitudinal stability. A reference interval was established in 51 healthy adults and the impact of age and sex was assessed. The assay was applied to 31 patients carrying STAT1 or STAT3 variants or presenting phenotypes for which Th17-cell quantification is informative. Diagnostic performance was analysed using receiver operating characteristic (ROC) curves and Matthew's Correlation Coefficients (MCC). The assay showed high intra- and inter-assay precision (CV 5.1% and 10%) and Th17-cell fractions remained stable after 72 h of post-fixation storage. Delayed pre-fixation reduced precision. Age and sex did not significantly influence Th17-cell fractions, supporting a unified Th17-cell reference interval of T-helper cells [0.21%-1.7%]. Compared with healthy controls, Th17-cell fractions were significantly lower in patients with STAT3 variants, patients with HIES or with both. ROC and MCC analyses indicated that Th17-cell fractions above 0.44% reliably excluded pathogenic variants or associated phenotypes in all patient groups. This validated Th17-cell assay is robust, reproducible and suitable for clinical use. It can guide genetic testing in IEI by identifying patients with Th17-cell deficiencies and excluding variants or phenotypes unlikely to be causative.