Decitabine Conditioning Improves Natural Killer Cell Maturation and Limits CD8⁺ T Cell Terminal Differentiation...

Allogeneic stem cell transplantation (alloSCT) can be curative for acute myeloid leukemia (AML) patients due to graft-versus-tumor immune responses. However, relapse post-alloSCT remains a major clinical challenge, underscoring the necessity for adjuvant treatments that both reduce tumor load before alloSCT and strengthen natural killer (NK) cell and T cell immunity post-transplantation. The hypomethylating agent decitabine (DAC) holds promise as it is well-tolerated and has direct antiproliferative and proapoptotic effects on leukemic cells. In addition, DAC modulates tumor immunogenicity and exhibits immunomodulatory properties. Yet integral insight into the impact of adding DAC to the pre-alloSCT conditioning on the immunophenotype and function of NK and T cells post-alloSCT is lacking. Here we investigated the immunomodulatory effects of adding DAC to the pre-alloSCT conditioning on the NK and T cell landscape and function in AML patients post-transplantation. This retrospective cohort study included AML patients who underwent alloSCT with DAC (n = 30) or without DAC (n = 22) as part of their conditioning regimen. Peripheral blood samples were collected at 3 months and 6 months post-alloSCT. High-dimensional flow cytometry was used to assess the phenotypic profiles, whereas proliferation, cytokine production, and cytotoxicity assays were performed to study their function. Irrespective of the conditioning regimen, checkpoint molecules on NK cells, including PD-1, TIM-3, LIGHT, and OX40, were low post-alloSCT. Notably, DAC conditioning significantly increased the abundance of mature NK cell clusters coexpressing KIRah, KIRb, CD69, CD57, and DNAM-1. Interestingly, NK cells from DAC-conditioned patients retained their cytotoxic and cytokine production capacity, with mature NK cells demonstrating significantly enhanced responsiveness to K562 stimulation compared to immature NK cells. Within the CD4⁺ T cells, DAC conditioning reduced the frequencies of T_H1/T_H17 cells and a proportion of T_H17 cells. Interestingly, DAC treatment led to a diminished abundance of CD8⁺ T cell clusters coexpressing immune checkpoint molecules, including PD-1, TIGIT, KLRG1, TIM-3, and CD57, implying a less terminally differentiated phenotype. Functionally, CD4⁺ and CD8⁺ T cells from DAC-conditioned patients demonstrated robust proliferation in response to anti-CD3 stimulation and exhibited polyfunctionality, producing IFN-γ, TNF-α, and IL-2. Notably, the higher abundance of CD8⁺ T cells co-expressing inhibitory checkpoint molecules in non-DAC-conditioned patients was inversely associated with IL-2 production on polyclonal stimulation. Overall, the addition of DAC to the conditioning regimen was associated with favorable immunomodulatory effects on NK cells and T cells post-alloSCT in AML patients. These findings suggest that DAC may enhance donor NK and T cell-mediated graft-versus-tumor responses, supporting its further clinical evaluation as an adjuvant prior to alloSCT. © 2025 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.