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Graft-versus-host disease (GVHD) induced by donor-derived T cells remains the major limitation of allogeneic bone marrow transplantation (allo-BMT). We previously reported that the pan-Notch inhibitor dominant-negative form of Mastermind-like 1 (DNMAML) markedly decreased the severity and mortality of acute GVHD mediated by CD4(+) T cells in mice. To elucidate the mechanisms of Notch action in GVHD and its role in CD8(+) T cells, we studied the effects of Notch inhibition in alloreactive CD4(+) and CD8(+) T cells using mouse models of allo-BMT. DNMAML blocked GVHD induced by either CD4(+) or CD8(+) T cells. Both CD4(+) and CD8(+) Notch-deprived T cells had preserved expansion in lymphoid organs of recipients, but profoundly decreased IFN-γ production despite normal T-bet and enhanced Eomesodermin expression. Alloreactive DNMAML T cells exhibited decreased Ras/MAPK and NF-κB activity upon ex vivo restimulation through the TCR. In addition, alloreactive T cells primed in the absence of Notch signaling had increased expression of several negative regulators of T cell activation, including Dgka, Cblb, and Pdcd1. DNMAML expression had modest effects on in vivo proliferation but preserved overall alloreactive T cell expansion while enhancing accumulation of pre-existing natural regulatory T cells. Overall, DNMAML T cells acquired a hyporesponsive phenotype that blocked cytokine production but maintained their expansion in irradiated allo-BMT recipients, as well as their in vivo and ex vivo cytotoxic potential. Our results reveal parallel roles for Notch signaling in alloreactive CD4(+) and CD8(+) T cells that differ from past reports of Notch action and highlight the therapeutic potential of Notch inhibition in GVHD.

Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines, but normal in vivo proliferation, increased accumulation of regulatory T cells, and preserved anticancer effects. Here, we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells, but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models, we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD, with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD, but led to treatment-limiting toxicity. In contrast, Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell-mediated human disorders.

Graft-versus-host disease (GVHD) remains the major barrier to the success of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is caused by donor T cells that mediate host tissue injury through multiple inflammatory mechanisms. Blockade of individual effector molecules has limited efficacy in controlling GVHD. Here, we report that Notch signaling is a potent regulator of T-cell activation, differentiation, and function during acute GVHD. Inhibition of canonical Notch signaling in donor T cells markedly reduced GVHD severity and mortality in mouse models of allogeneic HSCT. Although Notch-deprived T cells proliferated and expanded in response to alloantigens in vivo, their ability to produce interleukin-2 and inflammatory cytokines was defective, and both CD4(+) and CD8(+) T cells failed to up-regulate selected effector molecules. Notch inhibition decreased the accumulation of alloreactive T cells in the intestine, a key GVHD target organ. However, Notch-deprived alloreactive CD4(+) T cells retained significant cytotoxic potential and antileukemic activity, leading to improved overall survival of the recipients. These results identify Notch as a novel essential regulator of pathogenic CD4(+) T-cell responses during acute GVHD and suggest that Notch signaling in T cells should be investigated as a therapeutic target after allogeneic HSCT.