Preserved Treg cell numbers and function in patients developing aGVHD after posttransplant cyclophosphamide. aGVHD is characterized by preserved hallmarks of Treg functionality. (A) aGVHD after PTCy is not associated with loss of Treg/effector T cell balance. In both HLA-matched donor (MD) and haplo-BMT, Treg ratios are preserved at day 28 after transplantation, irrespective of aGVHD development. (B-D) GVHD and GVHD-free matched donor alloBMT patient samples were collected on day 28 and day 180 and processed for RNA-Seq analysis of lymphocyte subsets, including CD8⁺ T cells, conventional T cells (Tconv), and Treg. (B) Experimental workflow schema. (C) Tregs arising during post-PTCy aGVHD upregulate genes essential for Treg function. Hierarchical clustering analysis shows the top 60 differentially regulated genes between Tregs isolated from aGVHD (n = 4) and GVHD-free patients (n = 7) at day 28. IFNG and cytolytic effectors GZMA/GZMK, essential for Treg function, are highlighted in patients with aGVHD. (D) Enrichment analysis of Tregs on day 28 and day 180 from patients with aGVHD prior to aGVHD onset and after aGVHD control shows significant and positive enrichment for gene signatures identified as defining highly suppressive Tregs, with this phenotype persisting after aGVHD resolution.