Thymic stromal defects and immune dysregulation in hypomorphic recombinase activating gene (RAG) deficiency:...

Thymic epithelial cells (TECs) are essential for positive and negative selection during T-cell development. Hypomorphic recombinase activating gene (RAG) deficiency is characterized by T-cell lymphopenia and an increased risk of infections and autoimmunity. However, whether abnormalities of TEC composition and function also contribute to these clinical features is unknown.

Using a Rag1 hypomorphic (Rag1^mut) mouse model, we examined composition and function of the TEC compartment, both before and after hematopoietic stem cell transplantation.

Flow cytometry, immunofluorescent imaging, and single-cell RNA sequencing were used to characterize the TECs and thymocytes. Bone marrow transplantation studies were performed to define level of donor chimerism required to fully correct thymic abnormalities.

The thymus of Rag1^mut mice is small, with an increased proportion of cortical and a severe reduction of medullary TECs, as well as reduced expression of tissue-restricted antigens. Transplantation of Rag1^mut hematopoietic stem cells into wild-type mice phenocopied the altered thymic architecture of Rag1^mut mice. Transplantation of wild-type hematopoietic stem cells into Rag1^mut mice fully corrected thymocyte development and normalized the TEC phenotype. However, in competitive settings, lower levels of wild-type hematopoietic stem cell engraftment were associated with persistence of mutant-derived double-negative thymocytes and of a low number of Itgβ4-positive, medullary TEC-low cells, incomplete restoration of the TEC compartment, and autoantibody production.

Impaired thymocyte development in Rag1^mut mice leads to abnormal TEC composition and immune dysregulation, whose correction requires robust levels of donor chimerism after hematopoietic stem cell transplantation.