Thymic stromal defects and immune dysregulation in hypomorphic recombinase activating gene (RAG) deficiency: Implications for hematopoietic cell transplantation.

Abstract

Background: 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.

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.