Composition of immune cells in peripheral blood of patients with leukemia in remission after stem cell transplantation (HSCT) with or without graft-versus-host disease (GvHD): a comparative analysis with healthy donors

Abstract

Background Allogeneic hematopoietic stem cell transplantation (HSCT) is potentially curative for many hematologic malignancies. Clinical outcomes are critically determined by the balance between graft-versus-malignancy effects and graft-versus-host disease (GvM and GvHD, respectively). We hypothesized that integrated immune phenotyping can distinguish immune tolerance from clinically relevant alloreactivity in patients with hematological neoplasia who are in remission after HSCT. These findings may have implications for GvHD screening and monitoring and could support treatment decision-making. Methods Multi-color flow cytometry of peripheral blood mononuclear cells profiled myeloid, adaptive, and innate compartments in healthy donors (n = 10) and post-HSCT patients in remission: without GvHD [NoGvHD (n = 19)], with chronic (c) GvHD (n = 18, including n = 4 overlap-a/cGvHD), and with acute (a) GvHD (n = 6). Frequencies of immune cell subsets were compared between cohorts and stratified by clinical features [e.g., severity, organ involvement, donor lymphocyte infusion (DLI), GvHD development, and cGvHD progression]. Results NoGvHD patients (clinically asymptomatic and whose disease were in remission) showed immune-cell composition differing substantially from healthy donors: CD14+ monocytes (Mo), M-MDSCs, and especially checkpoint molecule (cpm)-expressing Mo (TIM3, 2B4, and PDL1) were reduced, while CTLA-4+ polymorphonuclear myeloid-derived suppressor cells (CTLA-4+-PMN-MDSCs) were elevated, as well as T-effector memory (Tem) cells and cpm-expressing T cells (PD1, LAG3, 2B4, TIM3, and TIGIT). cGvHD showed eroded regulatory myeloid subsets and skewed T cells toward effector-memory dominance with increased frequencies of inhibitory cpm-expressing T cells. aGvHD showed marked late-proliferating T-cell expansion (Tprol-late/T3+ fold change 9.92 vs. Healthy), Treg depletion, and loss of regulatory NK56bri populations. Overlap a/cGvHD displayed chronic T-cell stimulation with altered tissue homing (Intβ7+ enriched), and DLI-induced GvHD showed effector differentiation with insufficient regulatory compensation. Conclusion NoGvHD is not “Healthy minus disease” but a distinct post-HSCT immune state. Multi-compartment immune signature profiling by flow cytometry distinguishes preserved tolerance (Healthy) from post-HSCT remodeling (NoGvHD), progressive dysregulation (cGvHD), and acute hyperactivation (aGvHD), and refines GvHD subgroups by clinical context.