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Basic biology of acute GvHD
inhibitors, rather than pan-HDAC inhibitors, may be appropriate targets for further study.
Sensitization of non-hematopoietic cells
Emerging evidence indicates the importance of non- hematopoietic cells in aGvHD.17 Koyama et al. demon- strated that antigen presentation from non-hematopoietic cells could induce lethal aGvHD independently of T-cell interactions with hematopoietic APC.2 Microbiota in the gastrointestinal tract can secrete IL-12 to induce major histocompatibility complex class II upregulation on intes- tinal epithelial cells (IEC), initiating lethal aGvHD.6 In addition, fibroblastic stromal cells in the lymph nodes have been shown to drive aGvHD through the presenta- tion of Delta-like Notch ligands, DLL1 and DLL4 specifi- cally.5 Inhibition of the Notch ligands and receptors con- ferred protection against GvHD in murine models.5 However, we have a limited understanding of the epige- netic effects these non-hematopoietic cells have on aGvHD. In the light of these striking findings, this repre- sents an important avenue for future investigation.
Epigenetic control of alloreactive T cells
Upon encountering allogeneic host APC, infused donor T cells are activated and undergo robust proliferation and differentiation into effector T cells (Figure 1), which include IFN-γ-producing CD4+ Th1 cells, IL-4-producing CD4+ Th2 cells, IL-17-producing CD4+ Th17 cells and cytotoxic CD8+ T cells.1 Effector T cells mediate tissue injury during aGvHD. Alloantigen-sensitized donor T cells can also become memory T cells that mediate per- sistent host tissue injury. Over the past two decades, much research has been undertaken to understand the molecular mechanisms that control the generation and maintenance of alloreactive effector and memory T cells during the induction phase of aGvHD.
Epigenetic programming of effector T-cell responses
EZH2
Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that catalyzes histone 3 lysine 27 trimethylation (H3K27me3), which primarily silences genes,24 and is a core component of the polycomb repres- sive complex-2 (PRC2).24 Evidence suggests that EZH2 is involved in Th1 and Th2 polarization25 as well as the pro- liferation and differentiation of hematopoietic stem cells.26 EZH2 is also involved in cancer development and progres- sion,24 which has stimulated efforts to develop methods of inhibiting the enzyme.
EZH2 plays an essential role in T-cell immune respons- es. Studies by our group27-30 and others31 have demonstrat- ed the functional relevance of EZH2 in regulating antigen- driven T-cell responses. Using experimental murine mod- els, we discovered EZH2’s role in regulating allogeneic T- cell proliferation, differentiation and function.27,28 Conditional loss of Ezh2 in donor T cells inhibited aGvHD in mice. Although EZH2-deficient T cells could be activat- ed and underwent initial proliferation, their ability to undergo continual proliferation and expansion became defective during the later stage of aGvHD induction.28 Unexpectedly, as a gene silencer, EZH2 was required to promote the expression of transcription factors T-bet and STAT4, which are critical for effector differentiation.27
Subsequent studies revealed that EZH2 regulation of tran- scription factor expression and function depends on the differentiation stage of antigen-driven T cells.32 Ezh2 knockout in T cells impaired their differentiation into IFN- γ-producing effector cells.28 However, Ezh2 ablation, EZH2 protein inhibition and EZH2 protein destabilization all did not affect graft-versus-leukemia activity, leading to improved overall survival in recipients.28,30,33 Thus, target- ing EZH2 may represent an effective therapeutic strategy for aGvHD prevention and treatment.
HDAC1, HDAC6 and HDAC11
HDAC are important for regulating the proliferative and cytotoxic capabilities of activated T cells. Pharmacological inhibition of HDAC by SAHA has been shown to suppress T-cell-receptor-mediated T-cell prolif- eration through the induction of apoptosis.34 Hdac1 knockout in a murine allergic asthma model showed a significant increase in airway inflammation and Th2 cytokine production.35 Upon Hdac1 deletion, in vitro stud- ies noted an enhanced induction of Th1 and Th2 cells.35 Thus, HDAC1 plays a negative regulatory role for the functions of Th1 and Th2 subsets.
In T cells, HDAC11 may suppress the graft-versus-host reaction. Hdac11 knockout resulted in increased T-cell pro- liferation and release of pro-inflammatory cytokines asso- ciated with upregulation of eomesodermin (EOMES) and T-bet which are important in effector differentiation.36 Indeed, decreased expression of HDAC11 exacerbated aGvHD in mice.36
HDAC6 can deacetylate non-histone proteins such as heat shock protein 90 (HSP90).37 Acetylation disrupts HSP90’s chaperone function and inhibits LCK phosphory- lation.38 In a GvHD-like model involving OT-I T-cell trans- plants to K14-mnOVA mice, control mice developed mucosal and skin lesions, while inhibition of HDAC6 using a specific inhibitor, ACY-1215, prevented similar lesions from forming for 14 days after transplantation. This protective effect was accompanied by dramatically decreased production of CD8+ effector T cells that secret- ed high levels of IL-2 and IFN-γ.38 Further studies of these HDAC should be conducted in GvHD models to defini- tively validate their roles in driving or mitigating aGvHD.
SIRT3
SIRT3 is a mitochondrial HDAC that regulates meta- bolic enzyme acetylation.39 SIRT3 is expressed in meta- bolically stressed cells such as alloreactive T cells.40 Loss of SIRT3 in donor T cells led to decreased GvHD severity in mice. The protective effect associated with Sirt3 dele- tion was associated with a reduction in reactive oxygen species and decreased activation and expression of chemokine receptor CXCR3.40
DNMT
Because DNA methyltransferases (DNMT) – DNMT1, DNMT3A, DNMT3B and DNMT3L – can enact global transcription suppression, they have been widely studied in the context of immunity. DNMT1 is the principal enzyme that maintains methylation across DNA replica- tion.41 DNMT3A and DNMT3B contribute to methylation maintenance and are also responsible for de novo DNA methylation.41 DNMT inhibitors such as 5-azacytidine (Aza) have been shown to impair T-cell activation, expan- sion and cytokine release early in culture via downregula-
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