Once Upon Athymia: The T-Cell Pool of a Patient with FOXN1 Mutation, 17 Years Post-Thymic Transplant

FOXN1 deficiency, characterized by alopecia totalis, nail dystrophy, and severe T-cell immunodeficiency, leads to nude/severe combined immunodeficiency (SCID) phenotype. FOXN1 gene is expressed by the thymic epithelium and mutations in this gene lead to developmental defects of thymic epithelial cells resulting in lack of T-cell development. Our lab has been following since birth an 18-year-old patient with FOXN1 deficiency, that despite the lack of thymus presents circulating T-cells with a significant population with an aberrant phenotype (double negative abT-cells (DN)). She was submitted to thymic transplantation at 14 months of age, with effective immunological reconstitution being achieved, despite HLA-mismatch. Thymic transplantation was preceded by immunosuppressive treatment, clearing all circulating T-cells, except the DNab T-cell population. Allogenic thymic graft functionality increased progressively reaching levels comparable to those found in healthy children, followed by a sharp decline at 4-year post-transplant. In this study we aim to characterize for the first time, to our knowledge, at a single-cell level the circulating T-cell compartment upon thymus transplantation. Specifically, we aim to investigate: 1) the mechanisms underlying the maintenance of naïve T-cells given the evidence of failure of the allogenic thymic graft, and 2) the aberrant DNabT-cells known to be present before thymic transplantation. 

Circulating T-cells were evaluated by high-dimensional spectral flow cytometry. In parallel, naïve CD4 and CD8 T-cells as well as DNab T-cells were enriched through FACS sorting and complemented with other CD3 T-cells. This enriched population was used to perform scRNAseq combined with TCR and cell surface protein sequencing (CITE-Seq). For the CITE-seq analysis, we selected a naïve marker (CD45RA) and CD31. 

From our flow cytometry analysis, we observed two major findings: 1) the persistence of the DNab T-cell population, and 2) a major contraction of the naïve T-cell compartment. Furthermore, the naïve CD4 T-cells display a unique aberrant phenotype with high levels of TCF1 expression. The scRNAseq dataset produced is currently being analyzed. 

With our analysis we intend to uncover homeostatic mechanisms underlying the maintenance of these T-cell populations in this clinical setting. Ultimately, we expect to provide new insights on human CD4 T-cell biology and identify pathways for immune reconstitution.