Memory T cells sustain effector T-cell production while self-renewing in reaction to persistent antigen; yet, excessive expansion reduces memory potential and impairs antitumor immunity. Epigenetic mechanisms are thought to be important for balancing effector and memory differentiation; however, the epigenetic regulator(s) underpinning this process remains unknown. Herein, we show that the histone methyltransferase Ezh2 controls CD8+ T memory precursor formation and antitumor activity. Ezh2 activates Id3 while silencing Id2, Prdm1 and Eomes, promoting the expansion of memory precursor cells and their differentiation into functional memory cells. Akt activation phosphorylates Ezh2 and decreases its control of these transcriptional programs, causing enhanced effector differentiation at the expense of T memory precursors. Engineering T cells with an Akt-insensitive Ezh2 mutant markedly improves their memory potential and capability of controlling tumor growth compared to transiently inhibiting Akt. These findings establish Akt-mediated phosphorylation of Ezh2 as a critical target to potentiate antitumor immunotherapeutic strategies.

Key Points Ezh2 is specifically required to induce effector cells producing IFN-γ and expansion of T cells late upon alloantigen activation. Genetic inactivation of Ezh2 function reduces GVHD but preserves antitumor effects in mice after allogeneic BMT.

Graft-versus-host disease (GVHD) reflects an exaggerated inflammatory allogeneic T-cell response in hosts receiving allogeneic hematopoietic stem cell transplantation (HSCT). Inhibition of pan-Notch receptor signaling in donor T cells causes reduction of GVHD. However, which Notch ligand(s) in what antigen-presenting cells are important for priming GVH reaction remains unknown. We demonstrate that δ-like ligand-4 (Dll4) and Dll4-positive (Dll4hi) inflammatory dendritic cells (i-DCs) play important roles in eliciting allogeneic T-cell responses. Host-type Dll4hi i-DCs occurred in the spleen and intestine of HSCT mice during GVHD induction phase. These Dll4hi i-DCs were CD11c+B220+PDCA-1+, resembling plasmacytoid DCs (pDCs) of naïve mice. However, as compared to unstimulated pDCs, Dll4hi i-DCs expressed higher levels of costimulatory molecules, Notch ligands Jagged1 and Jagged2 and CD11b and, produced more Ifnb and Il23 but less Il12. In contrast, Dll4-negative (Dll4lo) i-DCs were CD11c+B220−PDCA-1−, and had low levels of Jagged1. In vitro assays showed that Dll4hi i-DCs induced significantly more IFN-γ- and IL-17-producing effector T cells (3- and 10-fold, respectively) than Dll4lo i-DCs. This effect could be blocked by anti-Dll4 antibody. In vivo administration of Dll4 antibody reduced donor alloreactive effector T cells producing IFN-γ and IL-17 in GVHD target organs, leading to reduction of GVHD and improved survival of mice after allogeneic HSCT. Our findings indicate that Dll4hi i-DCs represent a previously uncharacterized i-DC population distinctive from steady state DCs and Dll4lo i-DCs. Furthermore, Dll4 and Dll4hi i-DCs may be beneficial targets for modulating allogeneic T-cell responses, and could facilitate the discovery of human counterparts of mouse Dll4hi i-DCs.

Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep IntroductionPathogenic T-cell responses can be detrimental to the host. For example, GVHD is a life-threatening complication after allogeneic BM transplantation (BMT). 1-3 GVHD is caused by donor T cells that attack normal tissues of the recipient. 1-3 Standard immunosuppressive therapy for GVHD lacks efficacy and impairs the antitumor activity. 1,2,4 New approaches are needed to control GVHD.Epigenetic modifications are thought to be important for T-cell immune responses. 5,6 These modifications include histone methylation, DNA methylation, and histone acetylation. 7-10 Histone methylation is the modification of certain amino acids of a histone by adding methyl groups. [8][9][10] Depending on the site and degree of methylation, histone methylation can be activating or repressive. For example, trimethylation of histone H3 at lysine 4 (H3K4me3), H3K36me3, and H3K79me3 are associated with transcriptional activation, whereas H3K9me3, H3K27me3, and H4K20me3 are related to gene repression. 8,9 Recent studies have reported that histone methylation may play important roles in regulating the expression of genes associated with survival, proliferation, and differentiation of Ag-activated T cells. 11,12 Unlike histone methylation, DNA methylation results in global silencing of gene expression, 10 whereas histone acetylation is associated with a relaxing chromatin structure that facilitates transcription. 10 It has been shown that the DNA methylation inhibitor 5-Aza-2-deoxycytidine (5-AzaC) and the histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) may prevent GVHD in mice through a mechanism of modulating regulatory T cells (Tregs) 13,14 and APCs, 15-17 respectively. However, global modifications of DNA and chromatin structures are found to be associated with toxicities and adverse effects. 10 Thus, novel epigenetic approaches capable of targeting a specific set of genes in alloreactive T cells are desirable for controlling GVHD while minimizing adverse effects.3-Deazaneplanocin A (DZNep) possesses the potent ability to selectively inhibit some histone methylation, such as H3K27me3, H3K4me3, and H4K20me3. 18,19 DZNep is an inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. AdoHcy hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine. 20,21 When this enzyme is inhibited, AdoHcy accumulates in cells, leading to inhibition of the histone methyltransferease (HMT) activity and the subsequent histone methylation inhibition. 20 DZNep acts through a different pathway than DNA methylation inhibitors and HDAC inhibitors. 10,18,19 An Inside Blood analysis of this article appears at the fron...

Acquired aplastic anemia (AA) is a potentially fatal bone marrow (BM) failure syndrome. IFN-γ-producing T helper (Th)1 CD4+ T cells mediate the immune destruction of hematopoietic cells, and are central to the pathogenesis. However, the molecular events that control the development of BM-destructive Th1 cells remain largely unknown. Ezh2 is a chromatin-modifying enzyme that regulates multiple cellular processes primarily by silencing gene expression. We recently reported that Ezh2 is crucial for inflammatory T cell responses after allogeneic BM transplantation. To elucidate whether Ezh2 mediates pathogenic Th1 responses in AA and the mechanism of Ezh2 action in regulating Th1 cells, we studied the effects of Ezh2 inhibition in CD4+ T cells using a mouse model of human AA. Conditionally deleting Ezh2 in mature T cells dramatically reduced the production of BM-destructive Th1 cells in vivo, decreased BM-infiltrating Th1 cells, and rescued mice from BM failure. Ezh2 inhibition resulted in significant decrease in the expression of Tbx21 and Stat4 (which encode transcription factors T-bet and STAT4, respectively). Introduction of T-bet but not STAT4 into Ezh2-deficient T cells fully rescued their differentiation into Th1 cells mediating AA. Ezh2 bound to the Tbx21 promoter in Th1 cells, and directly activated Tbx21 transcription. Unexpectedly, Ezh2 was also required to prevent proteasome-mediated degradation of T-bet protein in Th1 cells. Our results identify T-bet as the transcriptional and post-translational Ezh2 target that acts together to generate BM-destructive Th1 cells, and highlight the therapeutic potential of Ezh2 inhibition in reducing AA and other autoimmune diseases.