Notch1 specifically upregulates expression of the cytokine interferon‐γ in peripheral T cells through activation of NF‐κB. However, how Notch mediates NF‐κB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF‐κB induction during T‐cell activation. NF‐κB activation occurs within minutes of T‐cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used γ‐secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF‐κB activation, but did not affect the initial activation of NF‐κB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF‐κB and competes with IκBα, leading to retention of NF‐κB in the nucleus. Additionally, we show that N1IC can directly regulate IFN‐γ expression through complexes formed on the IFN‐γ promoter. Taken together, these data suggest that there are two ‘waves’ of NF‐κB activation: an initial, Notch‐independent phase, and a later, sustained activation of NF‐κB, which is Notch dependent.

For many years, researchers have focused on the contribution of Notch signalling to lymphoid development. Only recently have investigators begun to ask what role, if any, Notch has during the activation and differentiation of naive CD4(+) T cells in the periphery. As interest in this issue grows, it is becoming increasingly clear that the main role of Notch signalling, to regulate cell-fate decisions, might also be influential in peripheral T cells.

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IntroductionNotch proteins are a family of ligand-activated large (300 kDa) single-pass transmembrane heterodimeric receptors. 1 Notch controls multiple cell fate decisions and differentiation processes during lymphocyte development and function and is required at various stages of T-cell development. 2,3 Deregulated Notch signaling during T-cell development leads to malignant transformation, including the cancer most closely associated with aberrant Notch expression in humans, acute T-cell acute lymphoblastic leukemia (T-ALL), which constitutes approximately 15% to 20% of ALLs seen in adults and children. 4,5 The oncogenic potential of Notch was first identified in (t7;9) chromosomal rearrangement in approximately 2% of human T-ALL, whereby intracelluar Notch1 is translocated to the T-cell receptor (TCR) ␤ gene. 6 More than 50% of human T-ALLs bear mutations in Notch1, indicating a prominent role for Notch in this T-cell malignancy. 7 Inhibitors of Notch signaling abrogate the growth of human and murine T-ALL cell lines bearing Notch1 gain-of-function mutations, indicating Notch is required in established tumors. 8,9 In vertebrates, 4 notch receptors (Notch 1-4) are activated by 5 different Notch ligands expressed on various cell types: Jagged1, Jagged2, and Delta-like (DL)1, DL3, and DL4. 2,3 After ligandbinding, proteolytic cleavage by ␥-secretase releases the signalingcompetent intracellular domain of Notch (N IC ). 10-12 N IC is composed of a RAM domain, ankyrin repeats (ANK) that mediate protein-protein interactions, nuclear localization sequences, a transactivation domain (TAD), and a C-terminal PEST domain regulating protein turnover. Human T-ALL cases frequently bear activating mutations in the extracellular heterodimerization domain and/or the C-terminal PEST domain of Notch1, resulting in ligand-independent activation. 7 During canonical Notch signaling, N IC translocates to the nucleus, engages its nuclear binding protein CSL (CBF-1, mammals; suppressor of hairless, Drosophila melanogaster; Lag-1, Caenorhabditis elegans) and transcribes downstream target genes, including the HES family of transcriptional repressors. 13,14 In the absence of N IC , CSL recruits repressor complexes to the regulatory regions of Notch/CSL target genes, inhibiting transcription. N IC interaction with CSL acts as a switch that promotes the assembly of CSL coactivator complexes. [15][16][17] ␥-Secretase inhibitors (GSIs) block proteolytic cleavage of Notch receptors, thereby preventing activation of Notch. Use of GSI in activated T cells results in down-regulation of nuclear factor (NF)-B activity, cytokine (interleukin-2 [IL-2] and interferon-␥ [IFN-␥]) production, and cell proliferation. 18 In T-cell lymphomas, context-specific putative target genes have been identified through which Notch1 may promote transformation by altering cell-growth kinetics. [19][20][21] The D-type cyclins (cyclins D1, D2, and D3) are the first cyclins to be induced as cells enter the G 1 phase of the cell cycle, [22][23][24] and, thus, if regulat...

The maturation of naive CD8+ T cells into effector CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator eomesodermin (EOMES), which is thought to regulate expression of two key effector molecules, perforin and granzyme B. Although EOMES is important for effector CTL development, the precise mechanisms regulating CD8+ effector cell maturation remains poorly understood. In this study, we show that Notch1 regulates the expression of EOMES, perforin, and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL activity through direct regulation of EOMES, perforin, and granzyme B.

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T helper17 (Th17) cells are known to play a critical role in adaptive immune responses to several important extracellular pathogens. Additionally, Th17 cells are implicated in the pathogenesis of several autoimmune and inflammatory disorders as well as in cancer. Therefore, it is essential to understand the mechanisms that regulate Th17 differentiation. Notch signaling is known to be important at several stages of T cell development and differentiation. Here we report that Notch1 is activated in both mouse and human in-vitro polarized Th17 cells and blockade of Notch signaling significantly down-regulates the production of Th17 associated cytokines suggesting an intrinsic requirement for Notch during Th17 differentiation in both species. We also present evidence, using promoter reporter assays, knockdown studies as well as chromatin immunoprecipitation, that IL-17 and RORγt are direct transcriptional targets of Notch signaling in Th17 cells. Finally, in-vivo inhibition of Notch signaling reduced IL-17 production and Th17 mediated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Thus, this study highlights the importance of Notch signaling, in Th17 differentiation and indicates that selective targeted therapy against Notch may be an important tool to treat autoimmune disorders, including multiple sclerosis.