Abstract
Thymus-dependent de novo T-cell generation is essential for maintaining T-cell diversity and immune competence. Although thymic involution after puberty reduces thymic output, residual thymic function remains important for immune surveillance and immune reconstitution following hematopoietic stem cell transplantation (HSCT). However, the cellular features that distinguish thymus-derived naïve T cells from those maintained through peripheral homeostatic expansion remain poorly understood. Here, we employed a congenic GFP-labeled mouse transplantation model combined with longitudinal single-cell transcriptomic and T-cell receptor repertoire analyses to define the origin and dynamics of naïve T-cell regeneration after HSCT. By distinguishing thymus-derived de novo generated T cells from peripherally expanded T cells, we found that recent thymic emigrants and both CD4⁺ and CD8⁺ naïve T cells preferentially reconstitute through a thymus-dependent pathway. We further identified distinct naïve T-cell states enriched within the thymus-derived compartment, including CD61-associated and Ly6C-associated naïve T-cell states. These populations displayed dynamic changes during immune reconstitution and were associated with thymic functional status in transplantation and physiological thymic involution mouse models. In human datasets and primary peripheral blood samples, CD61-associated features correlated with age-related thymic activity, sjTREC levels, and preliminary measures of thymic recovery after allogeneic HSCT. Collectively, our study defines cellular features of thymus-dependent naïve T-cell regeneration after HSCT and identifies CD61-associated naïve T-cell states as candidate peripheral features linked to thymic recovery. These findings provide new insights into thymus-dependent immune reconstitution and establish a framework for evaluating peripheral signatures of thymic regeneration after HSCT.