GCNT1-Mediated O-Glycosylation of the Sialomucin CD43 Is a Sensitive Indicator of Notch Signaling in Activated T Cells

Abstract

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.

Therapeutic afucosylated monoclonal antibody and bispecific T-cell engagers for T-cell acute lymphoblastic leukemia

“…The heavily glycosylated protein CD43 exists in many variants 25 that are differentially expressed by T-cell during ontogenesis and activation, or with aberrant expression in cancer cells. 26 27 This explains why, even if the UMG1-epitope is expressed on CD43 core protein structure, its pattern of expression significantly differs from other epitopes recognized by available anti-CD43 mAbs. 28–30 Recently, other anti-CD43 antibodies have been described for potential use in the treatment of acute myeloid leukemia (AML).…”

Genome-wide association study of high-sensitivity C-reactive protein, D-dimer, and interleukin-6 levels in multiethnic HIV+ cohorts

“…Another novel locus, GCNT1, is a member of the β-1,6-N-acetylglucosaminyltransferase gene family and GCNT1 knockout mice have been shown to have increased susceptibility to Mycobacterium tuberculosis infection and the complete deficiency of GCNT1 was associated with increased lung expression of the neutrophil chemoattractant CXCL2 [ 51 ]. Moreover, Notch signaling regulates Gcnt1 -mediated core-2 O -glycosylation in activated T cells and the core-2 O -glycoform of CD43 could be a sensitive indicator of Notch signaling [ 52 ]. Furthermore, GCNT1 expression in prostate cancer positively correlates with cancer progression and prostate-specific antigen recurrence [ 53 ] (Supplemental Table 2).…”

Therapeutic Targeting of Notch Signaling: From Cancer to Inflammatory Disorders

“…For Notch ligands, initial studies have focused on the role of their expression in professional antigen-presenting cells, such as conventional dendritic cells (Amsen et al, 2004). However, recent work highlighted critical immunological roles for Dll1 and Dll4 Notch ligands expressed by non-hematopoietic fibroblastic stromal cell niches in secondary lymphoid organs (Fasnacht et al, 2014;Chung et al, 2017;Perkey et al, 2020). Other genetic strategies that block canonical Notch signaling include inactivation of Rbpj, encoding RBP-Jκ, and conditional expression of dnMAML, a truncated N-terminal fragment of Mastermind-like1 (MAML1) fused to GFP that exerts potent dominant negative activity downstream of all Notch receptors (Tanigaki et al, 2002;Maillard et al, 2004).…”

“…In one report, protective effects were reported upon Notch1/2 inactivation only in regulatory T cells (Tregs) (Charbonnier et al, 2015). In terms of Notch ligands, Dll1 and Dll4 in the host accounted for all the effects of Notch signaling in GVHD, with a dominant role for Dll4 (Mochizuki et al, 2013;Tran et al, 2013;Chung et al, 2019;Perkey et al, 2020). Mechanistically, Notch inhibition blunted the production of multiple inflammatory cytokines in alloreactive T cells, including IFNγ, TNFα and IL-17, while leading to increased expansion of preexisting Tregs, enhanced Treg function and decreased accumulation of T cells in the gut (Zhang et al, 2011;Sandy et al, 2013a;Tran et al, 2013;Charbonnier et al, 2015).…”

“…Conversely, short-term Notch inhibition even with a single dose of anti-Dll1/4 antibodies was sufficient to confer long-term protection from GVHD. During early days after allo-HCT, T cells were found to interact with Dll1/4 ligands expressed by specialized niches of non-hematopoietic fibroblastic reticular cells lineage traced with a Ccl19-Cre transgene in secondary lymphoid organs (Chung et al, 2017;Perkey et al, 2020). The dominant role of non-hematopoietic cells as a source of Notch ligands in this context came as a surprise, as dendritic cells as well as other professional antigen-presenting cells had been considered previously as the likely source of ligands.…”