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.

Despite widespread use of the anti-CD20 monoclonal antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. A better understanding of rituximabmediated killing mechanisms is essential to develop more effective therapeutic agents. In this study, we modulated the binding property of rituximab by introducing several point mutations in its complementarity-determining regions. The data showed that changing the binding avidity of rituximab in the range from 10 ؊8 to 10 ؊10 M could regulate its antibodydependent cellular cytotoxicity but not affect its complement-dependent cytotoxicity and apoptosis-inducing activity in B-lymphoma cells. Contradictory to previous findings, we found that the complementdependent cytotoxicity potency of CD20 mAb was independent of the off-rate. Despite still being a type I CD20 mAb, a rituximab triple mutant (H57DE/H102YK/ L93NR), which had a similar binding avidity to a double mutant (H57DE/H102YK), was unexpectedly found to have extremely potent apoptosis-inducing activity. Moreover, this triple mutant, which was demonstrated to efficiently initiate both caspase-dependent and -independent apoptosis, exhibited potent in vivo therapeutic efficacy, even in the rituximabresistant lymphoma model, suggesting that it might be a promising therapeutic agent for B-cell lymphomas. (Blood. 2009; 114:5007-5015) IntroductionThe CD20 molecule is a 30-to 35-kDa integral membrane protein expressed by B lymphocytes in early stages of differentiation and by most B-cell lymphomas. 1,2 CD20 is an ideal target for monoclonal antibodies (mAbs), as it is expressed at high levels on most B-cell malignancies but does not become internalized or shed from the plasma membrane after mAb treatment. 3,4 The mouse/human chimeric anti-CD20 antibody, rituximab, is the first therapeutic mAb approved for the treatment of relapsed/refractory low-grade or follicular B-cell non-Hodgkin lymphomas. 5,6 Previous studies have suggested that several mechanisms might be involved in providing therapeutic efficacy, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and the induction of apoptosis. 4,7 The relative contributions of these different mechanisms of action are still a matter of debate. 4,7 Anti-CD20 mAbs are usually defined as either type I or II, based on their ability to redistribute CD20 into lipid rafts. 8,9 Type I mAbs (rituximab and most anti-CD20 mAbs) are able to efficiently shift CD20 complexes into rafts, but the type II mAbs (B1 and 11B8) are not. The in vitro assays further indicate that type I mAbs usually exhibit potent CDC activity and relatively low level of apoptosis unless extensively cross-linked by antibody, 8 whereas type II mAbs are relatively inactive in complement activation but tend to promote more apoptosis. 9,10 Both types of mAb are equally potent in ADCC with FcR-bearing myeloid effectors.Although rituximab has been widely used in the treatment of lymphoma, only 48% of patients respond to the treatme...

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.