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Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1- mediated O -glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O -glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O -glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4 + and CD8 + effector T cells and in Foxp3 + regulatory T cells. CD43 core-2 O -glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4 + and CD8 + T cells with high cytokine-producing ability. Gcnt1 -deficient T cells still drove lethal alloreactivity, showing that core-2 O -glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O -glycosylation as a marker of Notch signaling, we identified Ccl19-Cre + fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O -glycosylation also reported Notch signaling in CD8 + T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O -glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic cell transplantation. Notch signals delivered during the first 48 h after transplantation drive proinflammatory cytokine production in conventional T cells (Tconv) and inhibit the expansion of regulatory T cells (Tregs). Short-term Notch inhibition induces long-term GVHD protection. However, it remains unknown whether Notch blockade blunts GVHD through its effects on Tconv, Tregs, or both and what early Notch-regulated molecular events occur in alloantigen-specific T cells. To address these questions, we engineered T cell grafts to achieve selective Notch blockade in Tconv versus Tregs and evaluated their capacity to trigger GVHD in mice. Notch blockade in Tconv was essential for GVHD protection as GVHD severity was similar in the recipients of wild-type Tconv combined with Notch-deprived versus wild-type Tregs. To identify the impact of Notch signaling on the earliest steps of T cell activation in vivo, we established a new acute GVHD model mediated by clonal alloantigen-specific 4C CD4 + Tconv. Notch-deprived 4C T cells had preserved early steps of activation, IL-2 production, proliferation, and Th cell polarization. In contrast, Notch inhibition dampened IFN-γ and IL-17 production, diminished mTORC1 and ERK1/2 activation, and impaired transcription of a subset of Myc-regulated genes. The distinct Notch-regulated signature had minimal overlap with known Notch targets in T cell leukemia and developing T cells, highlighting the specific impact of Notch signaling in mature T cells. Our findings uncover a unique molecular program associated with the pathogenic effects of Notch in T cells at the earliest stages of GVHD., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid organs nor initial T cell activation was affected by Dll1/4 loss. Thus, we have uncovered a pathogenic function for fibroblastic stromal cells in alloimmune reactivity that can be dissociated from their homeostatic functions. Our results reveal what we believe to be a previously unrecognized Notch-mediated immunopathogenic role for stromal cell niches in secondary lymphoid organs after allo-BMT and define a framework of early cellular and molecular interactions that regulate T cell alloimmunity.