Disease‐causing STAT3 variants can be discriminated by a functional flow cytometry test

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BACKGROUND: Heterozygous STAT3 variants acting by dominant negative (DN), gain-of-function (GOF), or haploinsufficiency (HI) mechanisms that have been associated with different inborn errors of immunity. However, the biological impact of STAT3 variants is complex to determine and it generally involves vector-generation, transfection, and multiple iterations for definition. OBJECTIVE: To establish a single, reliable test to functionally characterize putative disease-causing STAT3 variants in patient primary cells. METHODS: We recruited patients carrying previously published/validated STAT3-DN (n = 13), STAT3-GOF (n = 4), and STAT3-HI (n = 5) variants. We functionally evaluated the IL-10-mediated/STAT3-dependent inhibition ratio of LPS-induced TNF-α production by peripheral blood mononuclear cells (PBMC) using flow cytometry to evaluate the biologic impact of STAT3 variants. Optimization and threshold definition were performed by bootstrapping and k-fold cross-validation of Receiver Operating Characteristic (ROC)-area under the curve (AUC). RESULTS: The inhibition ratio of TNF-α production was significantly diminished in STAT3-DN (-3.44-fold, p < .0001) and STAT3-HI samples (-2.28-fold, p < .0001), while significantly augmented in STAT3-GOF samples (+1.47-fold, p = .002) when compared to healthy controls (HC, n = 16). Optimizing the combinations of IL-10 concentrations discriminated between HC and STAT3-DN (ROC-AUC = 1), and STAT3-GOF (ROC-AUC = 0.89). Further test optimization allowed discrimination between STAT3-HI variants when compared to HC (ROC-AUC = 1) and to STAT3-DN (ROC-AUC = 0.92). CONCLUSION: Determination of the IL-10 mediated, STAT3-dependent TNFα inhibition ratio in PBMC-derived monocytes by flow cytometry is a sensitive and specific method to functionally assess and discriminate STAT3-DN, -GOF, and -HI variants. This test can be used in research and clinical laboratory settings.

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