Abstract Text: Thymus hypoplasia is evident in individuals with autosomal recessive, compound heterozygous and single allelic Forkhead Box N1 (FOXN1) mutations. FOXN1 regulates thymic epithelial cell (TEC) development and function, which is essential for T cell development. The impact of diverse human FOXN1 mutations on TECs and thymopoiesis remains unclear for most variants. We determined the consequence of different FOXN1 variants on protein activity (luciferase reporter assays), localization (cell imaging), and dominant negative capabilities (co-expression studies). With these assays, we mapped the nuclear localization signal in FOXN1, identified de novo sequences in human FOXN1 that mediate dominant negative functions and uncovered domains modulating transcriptional activities. Both loss-, partial loss- and gain- of function FOXN1 variants were uncovered. To determine their impact on thymopoiesis, we modified reaggregate thymus organ cultures (RTOC) by incorporating flow sorting to isolate and recombine different cell subsets. In this system, only FOXN1 sufficient TECs will support thymopoiesis when combined with hematopoietic, mesenchymal, and endothelial cell subsets. Murine TECs from “nude” embryos (lacking FOXN1) are unable to support this thymus growth. The flow sorted “nude” TECs were reconstituted with purified TAT-Foxn1 fusion proteins prior to reaggregation. The transduction of wildtype TAT-Foxn1 re-established RTOC growth, thymopoiesis and T cell development within a 10-day culture period. This RTOC strategy can enable one to define the FOXN1 variants of unknown significance (VUS) as benign, partially, or fully attenuated in their capacity to support thymopoiesis. Taken together, our findings establish FOXN1 genotype-phenotype relationships and reveal a novel hymopoietic screening strategy.
