Donor T-cell Infiltration Drives Lasting Thymic Dysfunction after MHC-Haploidentical Hematopoietic Cell Transplantation Even in Recipients of Post-Transplantation Cyclophosphamide

Mature donor T cells provide early immune protection after allogeneic HCT, controlling both malignancy and infection. Yet, mature donor T cells have limited diversity, contributing to heightened infectious risk post-HCT; recovery of T-cell diversity requires a functional thymus. Despite success in preventing severe GVHD, post-transplant cyclophosphamide (PTCy) results in post-HCT immune defects, including limited T-cell diversity and impaired pathogen control, but whether these defects are due to effects on the thymus or mature donor T cells is unknown. To investigate thymic recovery, we used our MHC-haploidentical murine HCT model (B6C3F1 H2b/k →B6D2F1 H2b/d ). Lethally irradiated (10.5 Gy) 7-week-old female B6D2F1 were transplanted with either T-cell-depleted B6C3F1 bone marrow (TD) alone or B6C3F1 splenocytes and TD marrow (T-cell-replete TR). Thymic regeneration was rapid in TD HCT ± PTCy, with normalization of thymocyte differentiation and cellularity by day +21. By contrast, TR HCT was associated with extensive infiltration of mature donor T cells, leading to profound thymic atrophy and delayed recovery. Higher intensity conditioning with either irradiation or busulfan further increased T-cell infiltration and injury. PTCy partially rescued thymic differentiation, with numerical recovery by day +90, but functional analyses revealed persistent defects in negative selection. Indeed, bone marrow-derived Vβ6 + alloreactive CD4 + T cells continued to emerge at frequencies comparable to syngeneic B6C3F1→B6C3F1 HCT controls, indicating impaired intrathymic clonal deletion. Moreover, in TD HCT, alloreactive Vβ6 + CD4 + T cells were skewed toward a regulatory (Treg) fate, whereas this skewing was absent after TR HCT regardless of PTCy treatment. Tracking of thymocyte clonality showed greater clonal expansion of alloreactive Vβ6 + T-cell clones after TD HCT. Mechanistically, TR allogeneic HCT resulted in impaired differentiation of medullary thymic epithelium cells (mTECs), with selective loss of mTEC IIIa and mTEC IIIb subsets. High-dimensional imaging (Phenocycler) showed transient numerical alterations in thymic cell clusters at day +21 that normalized by day +150. Yet, spatial neighborhood interactions among stromal and hematopoietic cells remained profoundly altered long-term. Infiltration of mature donor T cells after MHC-haploidentical HCT causes lasting thymic injury and defective mTEC maturation, leading to persistent failure of intrathymic clonal deletion. Although PTCy mitigates structural damage and restores thymic cellularity, it does not fully rescue central tolerance or thymic function. These results identify a previously unrecognized mechanism of immune reconstitution failure after TR HCT and highlights the need for strategies that preserve thymic integrity and mTEC-mediated tolerance.