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A. Li et al.
tion of cell cycle- and cytokine-related genes.42 Sánchez- Abarca et al. treated mice undergoing allo-HSCT with Aza and found that early treatment prevented aGvHD develop- ment without increasing regulatory T cells (Treg); researchers speculated that this was likely due to Aza inhi- bition of T-cell expansion, which had been demonstrated in vitro.42 In a humanized murine allo-HSCT, xenogeneic GvHD model, Aza treatment was also noted to decrease the frequency of IFN-γ-secreting CD4+ human T cells and granzyme B- and perforin 1-secreting CD8+ human T cells in vivo.43 Using Dnmt3a conditional knockout mice, a recent study by Youngblood et al. revealed the impor- tance of DNMT3A in the regulation of T-cell exhaustion.44 Moving forward, similar genetic approach- es will be useful in understanding the precise mecha- nisms of the effect of DNMT inhibition on aGvHD.
Epigenetic programming of alloreactive memory T cells
A hallmark of aGvHD is cytopathic injury mediated by persistent alloreactive effector T cells, which can occur within weeks and persist for years after transplanta- tion.1,7-9,29 Data from our8,9,29 studies suggest that memory T cells that develop during aGvHD sustain alloreactive effector cells.8,9,29 These alloantigen-sensitized memory T cells differ from naturally-occurring T cells because the ability of memory T cells to mediate aGvHD is limited by their T-cell receptor repertoires.45 Memory T cells are generated during the primary immune response from proliferating T cells upon APC activation.46 After re- encountering antigens, they undergo rapid and robust proliferation and elaboration of effector function. They have stem cell-like self-renewal properties, are distin- guishable from both naïve and effector T cells and are resistant to existing immunosuppressive agents.47,48 In fact, whether or not their resilience contributes to the low response rates to current aGvHD therapies (~40%) is the subject of ongoing debate.1, 7-9
EZH2
EZH2 is required for the development of memory pre- cursors early after antigenic priming, for the maturation of memory T cells and for the recall response of mature memory T cells.32 EZH2 deficiency in activated CD8+ T cells caused significant skewing toward central memory precursors and drastically increased the relative propor- tion of terminally differentiated effector cells that were unable to contribute to further expansion.32 EZH2 repressed the expression of Blimp-1, ID2 and EOMES, which promote effector differentiation, and promoted and sustained expression of ID3, a gatekeeper critical for memory formation and survival.32 Given EZH2’s crucial roles in the regulation of alloreactive T cells, EZH2-medi- ated memory formation may be responsible for the gen- eration and maintenance of alloreactive memory T cells during aGvHD.
Other regulators
Histone methyltransferase SUV39H1 may play a role in repressing memory genes. Upon infection with Listeria monocytogenes, SUV39H1-defective CD8+ T cells demon- strated enhanced “long-term memory reprogramming,” allowing them to persist in mice.49 The Mixed-Lineage Leukemia gene encodes histone lysine methyltransferase 2A and may be a regulator of memory Th2 cells.50 Protein arginine methyltransferase 5 (PRMT5) has also been
implicated as a supporter of memory T-cell reactivation; its inhibition suppressed memory Th1 responses in experimental autoimmune encephalitis.51 We anticipate continued studies of these epigenetic regulators that con- sider their relevance to aGvHD.
Epigenetic regulation of regulatory T cells
Treg with CD4+CD25+FOXP3+ phenotype can suppress immune responses via cytokine- and contact-dependent mechanisms.1,52 Stable Forkhead box P3 (FOXP3) expres- sion has been considered a critical determinant of Treg identity and activity, but some suggest this characteriza- tion may be incomplete, pointing to the importance of independent epigenetic alterations.53 Natural Treg (nTreg) and induced Tregs (iTreg) are the most closely- studied Treg subsets in aGvHD; nTreg develop within the thymus while iTreg arise from activated CD4+ T cells in the periphery.54 Because they can repress T-cell prolif- eration and survival, Treg have been established as an important cell population in reducing aGvHD.55,56 Indeed, infusion of nTreg into allo-HSCT mice abrogated aGvHD lethality,57 and iTreg have been shown to sup- press aGvHD in allogeneic models.58,59 While nTreg use has achieved preliminarily promising results in clinical trials,56 the potential of iTreg is less clear. Each has disad- vantages; there are few nTreg in the peripheral blood and their use requires ex vivo expansion.60 On the other hand, iTreg, with unstable FOXP3 expression, are often unable to maintain a suppressive phenotype.61-63
DNMT
Researchers have attempted to stabilize FOXP3 expression by maintaining demethylation of the Foxp3 locus.64,65 As DNMT are involved in maintaining methy- lation, they are thought to contribute to Foxp3 suppres- sion. Indeed, without demethylation of a CpG island in the Foxp3 locus, cells’ FOXP3 expression and suppressive ability are limited.66 The use of DNMT inhibitors to sus- tain Treg stability and abrogate aGvHD has achieved some success.
Choi et al. showed that treatment with the DNMT inhibitors decitabine and Aza induced Treg from CD4+CD25- cells.67 Transplantation of decitabine- and Aza-treated cells into mice undergoing allo-HSCT reduced clinical aGvHD and improved survival.67 Directly treating mice with Aza after allo-HSCT resulted in similar effects. Interestingly, these suppressive effects of iTreg were found to be maintained even in Foxp3 knockout cells, suggesting that their anti-GvHD activity may be downstream or independent of FOXP3 expres- sion.67 In a humanized murine allo-HSCT, xenogeneic GvHD model, in vivo Aza treatment was associated with longer survival and lower xenogeneic GvHD scores.43 Researchers suggested that Aza treatment induced Il2 promoter hypomethylation, leading to increased IL-2 expression and augmented Treg proliferation.43
EZH2
EZH2 is known to co-localize with FOXP368 and has also been implicated in the maintenance of Treg identity after activation.69 Tumes et al. noted that iTreg differenti- ation is impaired without EZH2.31 Ablation of Ezh2 led to autoimmunity associated with a faulty FOXP3-depen-
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haematologica | 2020; 105(11)