Thymic dysfunction promotes the emergence of pathogenic peripheral CD4⁺CD8⁺ T cells and induces tissue injury in acute gvhd Free

Acute graft-versus-host disease (aGVHD) is one of the most severe complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT), primarily caused by donor-derived alloreactive T cells that attack recipient tissues, leading to systemic inflammation and multi-organ damage. While the effector role of donor T cells is well recognized, their developmental origin, activation dynamics, and persistence remain poorly understood. The thymus is the central organ for T-cell development and immune tolerance, yet its role in aGVHD pathogenesis remains underexplored.

In preliminary experiments using a murine aGVHD model, we observed marked thymic atrophy, along with a significant reduction in total thymocyte numbers. Flow cytometric analysis revealed a near-complete absence of CD4⁺CD8⁺ double-positive (DP) thymocytes, with only a small population of single-positive (SP) CD4⁺ and CD8⁺ cells remaining. These findings suggest that thymic architecture and function are severely compromised during aGVHD, potentially interfering with both positive and negative selection processes. This impairment may lead to the premature release of incompletely developed T cells into the peripheral circulation.

Unexpectedly, we identified a substantial accumulation of DP T cells in peripheral target organs, including the colon, liver, and skin, in aGVHD mice. These cells were virtually absent in bone marrow–transplanted, T cell–depleted (BM-TCD) controls. Given that DP cells are typically restricted to the thymus and are rarely found in the periphery under normal conditions, their presence in peripheral tissues suggests a pathological process of premature egress from the thymus due to structural and functional failure. This observation raised the hypothesis that these peripheral DP cells may be developmentally abnormal and functionally involved in mediating tissue injury.

To investigate the biological properties of these DP cells, we performed both single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic profiling of three representative tissues in aGVHD mice: the colon (a representative target organ), the thymus (the central lymphoid organ), and the spleen (a peripheral lymphoid organ). The aGVHD model was established using GFP-labeled C57BL/6 donor cells transplanted into lethally irradiated BALB/c recipients. The BM-TCD group served as the negative control.

scRNA-seq analysis revealed that peripheral DP cells in the aGVHD colon expressed high levels of cell cycle–related genes such as Birc5, Cdca8, Pclaf, and Mki67, indicating strong proliferative potential. In contrast, expression of lineage-defining transcription factors and effector molecules typically associated with SP T cells, including Cd83, Tnfsf8, and Gata3, was minimal. These data suggest that the peripheral DP cells are in a non-canonical, undifferentiated state distinct from conventional effector T cells. Spatial transcriptomic data confirmed that these DP cells were enriched in subepithelial regions of the colon, which colocalized with areas of epithelial disruption and immune infiltration, implicating them in direct tissue damage.

We further compared DP cells from the thymus of BM-TCD mice with splenic DP cells from aGVHD mice. Thymic DP cells exhibited high expression of normal developmental markers including Rag1, Rag2, Rorc, Ccr9, and Sox4, consistent with ongoing T-cell maturation within a healthy thymic environment. In contrast, splenic DP cells from aGVHD mice expressed elevated levels of activation and cytotoxicity-associated genes such as Gzmb, Il2rb, and Cd38, suggesting that they had transitioned into an activated and potentially pathogenic state. This phenotypic shift supports the concept that the inflammatory milieu of aGVHD promotes extrathymic activation and reprogramming of aberrantly exported DP cells.

Our findings reveal that thymic dysfunction in aGVHD permits the release of immature DP T cells into the periphery, where they acquire pathogenic features and accumulate in target tissues. This thymus–periphery axis may be a key driver of ongoing inflammation and damage in aGVHD. Therapeutic strategies that restore thymic function or eliminate pathogenic DP cells may provide novel avenues to alleviate disease severity.