Abstract
ObjectivePrevious studies showed that hypomethylating agents (HMAs) could alleviate acute graft-versus-host disease (aGvHD), but affect engraftment after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The combination of two different HMAs in lower doses might overcome this problem. This study aimed to evaluate the treatment effect of the combination of two HMAs—azacitidine (5-Aza) and histone H3K27 methyltransferase inhibitor 3-deazaneplanocin (DZNep)—for the prophylaxis of aGvHD after allo-HSCT and to explore the possible mechanisms.MethodsWe first optimized the concentrations of individual and combinational 5-Aza and DZNep treatments to ensure no obvious toxicities on activated T cells by evaluating T-cell proliferation, viability, and differentiation. A mouse model of aGvHD was then established to assess the prophylactic efficacy of 5-Aza, DZNep, and their combination on aGvHD. The immunomodulatory effect on T cells and the hematopoietic reconstruction were assessed. Additionally, RNA sequencing (RNA-seq) was performed to identify the underlying molecular mechanisms.ResultsCompared with single treatments, the in vitro application of 5-Aza with DZNep could more powerfully reduce the production of T helper type 1 (Th1)/T cytotoxic type 1 (Tc1) cells and increase the production of regulatory T cells (Tregs). In an allo-HSCT mouse model, in vivo administration of 5-Aza with DZNep could enhance the prophylactic effect for aGvHD compared with single agents. The mechanism study demonstrated that the combination of 5-Aza and DZNep in vivo had an enhanced effect to inhibit the production of Th1/Tc1, increase the proportions of Th2/Tc2, and induce the differentiation of Tregs as in vitro. RNA-seq analysis revealed the cytokine and chemokine pathways as one mechanism for the alleviation of aGvHD with the combination of 5-Aza and DZNep.ConclusionThe combination of 5-Aza and DZNep could enhance the prophylactic effect for aGvHD by influencing donor T-cell differentiation through affecting cytokine and chemokine pathways. This study shed light on the effectively prophylactic measure for aGvHD using different epigenetic agent combinations.
Highlights
Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic stem cell transplantation, which limits its success [1]
DZNep was dissolved in sterilized water to prepare a 50-mM stock solution. Both drugs were diluted in phosphate-buffered saline (PBS) before use, so that the final concentration of dimethyl sulfoxide (DMSO) was below 2% in all the experiments
T helper 1 (Th1) is characterized as CD4+IFNg+ T cells, while Tc1 is characterized as CD8+IFNg+ T cells
Summary
Graft-versus-host disease (GvHD) remains a major complication after allogeneic hematopoietic stem cell transplantation (alloHSCT), which limits its success [1]. Immunosuppression therapy, including anti-thymus globulin (ATG), cyclosporin A (CsA), methotrexate (MTX), and mycophenolate mofetil (MMF), is currently widely used to prevent and treat GvHD. Since immunosuppression therapy mainly affects the donorderived T cells, it increases the risks of infection, relapse, and long-term adverse reactions [2]. Identifying alternative strategies to prevent and treat GvHD is of great significance to improve the survival of allo-HSCT recipients. Acute graft-versus-host disease (aGvHD) is a complicated sequential immune response when donor immune cells recognize recipient antigens [3]. Given that a variety of cytokines and immune cells contribute to the occurrence and development of aGvHD, therapies that regulate the functions of relevant cytokines and immune cells might be effective in alleviating aGvHD [4]
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