Disease-Causing STAT3 Variants Can Be Discriminated by a Functional Flow Cytometry Test
Year: 2026
Type: article
Abstract: Background Heterozygous STAT3 protein variants acting by dominant-negative (DN), gain-of-function (GOF), or haploinsufficiency (HI) mechanisms have been associated with different clinical/immunological phenotypes. However, the biological impact of STAT3 protein variants is complex to determine, and it generally requires vector generation, transfection, and multiple iterations for definition. Objective To establish a single, reliable test to functionally characterize putative disease-causing STAT3 protein 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 of LPS-induced TNF-a production by peripheral blood mononuclear cell (PBMC) monocytes using flow cytometry to assess the biological impact of STAT3 protein variants. Optimization and threshold definition were performed by bootstrapping and k-fold cross-validation of the receiver operating characteristic (ROC)-area under the curve (AUC). Results The inhibition ratio of TNF-a production was significantly diminished in STAT3-DN (−3.44-fold, p < 0.0001) and STAT3-HI patient samples (−2.28-fold, p < 0.0001), while significantly augmented in STAT3-GOF patient samples (+1.47-fold, p = 0.002) when compared to healthy controls (HC, n = 16). Optimization of the combinations of IL-10 concentrations discriminated between HC from STAT3-DN (ROC-AUC = 1) and STAT3-GOF (ROC-AUC = 0.89). Further test optimization allowed discrimination of 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 TNF-α inhibition after LPS stimulation ratio in PBMC monocytes using 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.
Source: Journal of Human Immunity
Authors: Ana Esteve-Sole, Alexandra F. Freeman, Amy Hsu, Tiphanie P. Vogel, Lisa R. Forbes Satter, Jenna R.E. Bergerson, Taura Webb, Joseph Pechacek, Kristen McDaniels, Megan A. Cooper, Carolina Bouso, Matias Oleastro, Silvia Denielian, Luis Ignacio Gonzalez-Granado, Michail S. Lionakis, Steven M. Holland, Thomas A. Fleisher, HyeSun Kuehn, Sergio Rosenzweig
Institutions: National Institutes of Health Clinical Center, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Texas Children's Hospital, Washington University in St. Louis, Garrahan Hospital, Universidad Complutense de Madrid, Research Institute Hospital 12 de Octubre, Hospital Universitario 12 De Octubre
Language: English
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: diamond
OpenAlex ID: W7159987237
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