Autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED) is an autoimmune disease targeting multiple organs. It is caused by a loss-of-function mutation in the AIRE gene, the principal transcription factor regulating the expression of self-antigens by the thymus’ epithelial cells (TECs), controlling the elimination of self-reactive T lymphocytes and the selection of regulatory T cells (Tregs). Both mechanisms are essential in establishing self-tolerance, and dysregulated in APECED patients. Our main goal is to re-establish the production of mature T cells and Tregs in vitro, within thymic organoids derived from induced pluripotent stem cells (iPScs) reprogrammed from APECED patients and corrected for the AIRE mutation.

We first set up and conducted the directed differentiation of APECED patient-derived iPSc lines (corrected or not for the AIRE mutation) towards a hematopoietic stem and progenitor cell (HSPC) identity, and obtained reproductible results showing the emergence of a population characterized by the hematopoietic marker CD34. A large fraction of these cells also expresses the markers CD43, CD44, CD45 and CD7, presenting a phenotype resembling the hematopoietic progenitors that enter the thymus in vivo. We then conducted the directed differentiation of iPScs towards a TEC precursor identity (EPCAM+ CD205+), and aggregated these cells with the CD34+ HSPCs to form iPSc-derived thymic organoids, cultivated over a five-week period. Flow cytometry analysis revealed the emergence of a T lymphocyte population (CD45+ CD3+) comprising double negative and simple positive (SP) CD4+ and CD8+ cells on day 28. On day 35, the generated T cell compartment becomes more abundant and almost exclusively comprises SP CD8+ cells, that are for the large majority TCR positive, highlighting their mature state and functional potential.

These results demonstrate the differentiation potential of iPSc-derived HSPCs towards mature and functional T cells within an iPSc-derived thymic organoid system, and open new research perspectives towards cell based therapies in APECED, including the introduction of various strategies such as lentiviral-mediated FoxP3 expression to enhance Treg differentiation.