FOXN1 variant regulation of T cell production - Project Summary The thymic tissue is the only organ supporting the development of the T cells of the immune system. The quintessential stromal cell type in this tissue needed for thymopoiesis is the thymic epithelial cell (TEC). TECs govern the positive and negative selection of T cells in the thymus. The master transcriptional regulator of TECs is the Forkhead Box N1 (FOXN1) gene. Three distinct autosomal recessive FOXN1 mutations were identified several decades ago, resulting in a nude and T-B+NK+ severe combined immunodeficiency phenotype. The T cell lymphopenia resulted from an athymia. In such cases, an allogeneic thymic tissue transplant, depleted of hematopoietic cells, remains the best clinical strategy for restoring some T cell development. Recent widespread use of genome sequencing for patients who have low T cell receptor excision circles (TRECs), which is a measure of low T cell output from the thymus, has increased the number of human FOXN1 variants to >500. As described in our prior JCI and JACI publications, we categorized the diverse FOXN1 variants based on their impact on protein function and thymopoiesis. Interestingly, most patients with low TRECs who had single allelic FOXN1 variants normalize their peripheral T cell numbers over time. This presents a major conundrum as to how such patients should be managed clinically. Thus, a serious scientific gap exists in our understanding as to why most single allelic FOXN1 variants result in a transient T cell lymphopenia. In this exploratory grant, we will determine if a previously unconsidered mechanism involving monoallelic FOXN1 expression accounts for the transient T cell lymphopenia. This will be done with mouse models genocopying selected human FOXN1 variants. Comparing the allelic expression of Foxn1 in TECs at embryonic, postnatal, and aged stages will establish whether monoallelic gene expression predominates at specific developmental stages. Since certain human FOXN1 mutations are more damaging than others, understanding their impact on thymopoiesis is also needed for appropriate clinical management. Our proposed studies will include modifications to thymus organoid procedures that enable a comparison of different epithelial cells for their capacity to support tissue growth. This will include the use TAT-FOXN1 fusion proteins that can be transduced into Foxn1-deficient TECs, prior to reaggregate thymic organ culture assembly, to compare the different human variants. With our strong expertise in thymus tissue specification and organ cultures, we will identify novel strategies to improve thymus functionality.
