Development of Thymic Organoids Heterotopically to Educate and Induce T Lymphocytes

Background: Vascularized composite allotransplantation (VCA) is a potential treatment for extensive injuries that replaces defects like-with-like, however allografts are immune-rejectable.

Methods: This study developed in vitro thymic organoids and examined whether donor-derived HSCs could be educated in vivo into T lymphocytes via central tolerance. TECs, TMCs, and HSCs from C57BL/7 (CD45.2⁺) or SJL/L (CD45.1⁺) mice were labeled with cell surface markers and examined by flow cytometry. Co-culturing three cell lines in vitro created thymic aggregates. Aggregates transplanted to C57BL/7 (CD45.2⁺) mice's inguinal regions developed thymic organoids. Immunorejection genes were identified bioinformatically. Western blot, immunofluorescence, and flow cytometry were utilized to measure rejection-related protein levels and T cell surface markers in thymic organoids to determine T cell inducement and immunomodulation.

Results: In vitro, TECs, TMCs, and HSCs created thymic aggregates, which became thymic organoids after in vivo transplantation and produced CD8⁺ and CD4⁺ Tregs. Bioinformatics showed high correlations between transplanted rejection and IFNG, IL2RG, FCGR3A, and ICAM1 genes. Immunofluorescence and Western blot showed increased protein expression of IFNG, IL2RG, FCGR3A (immunomodulation biomarker), and decreased protein expression of CK8, CK14, and ICAM1 (TEC biomarker) in thymic organoids.

Conclusion: Thymic organoids heterotopically implanted in vivo can promote heterologous HSC-derived T cell development.

T lymphocytes; hematopoietic stem cells; thymic epithelial cells; thymic mesenchymal cells; vascularized composite allotransplantation.