Notch1 signaling sustains the proinflammatory behavior of Th1 cells, implicated in the development of aplastic anemia in humans and mice.

• NF-kB differentially regulates CXCR4 expression on naïve and pathogenic CD8 1 T cells.• CXCR4 expression on pathogenic T cells facilitates their trafficking to the BM in a mouse model of AA.Aplastic anemia (AA) is a disease characterized by T-cell-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells. Physiologically, T cells migrate to the BM in response to chemokines, such as SDF-1a, the ligand for CXCR4. However, how T cells traffic to the BM in AA is poorly understood. CXCR4 is aberrantly expressed in immune-mediated diseases and its regulation by nuclear factor-kB (NF-kB) in cancer models is well documented. In this study, we show that CXCR4 is highly expressed on BM-infiltrating CD4 Studies of AA patients and animal models of BMF suggest acquired AA is an immune-mediated disease.3,4 Aberrant responses mediated by T helper type-1 (Th1), Th17, and cytotoxic CD8 1 T cells, together with impaired function of regulatory T cells, 5-10 culminate in BM destruction. Although the pathophysiology of AA is well defined, the molecular mechanisms responsible for T-cell infiltration into the BM during AA progression are poorly understood.Small populations of mature CD4 1 and CD8 1 T cells reside in the BM. It is a priming site for antigen-specific T cells, 11-13 as well as a homing site for memory T cells.14-16 Physiologically, T cells migrate to the BM in response to chemokines, such as stromal-cell derived factor-1a (SDF-1a) which is highly expressed by BM stromal cells.17,18 SDF-1a, also known as CXCL12, is the natural ligand for the chemokine receptor, CXCR4.19 SDF-1a-CXCR4 interactions initiate multiple signaling pathways that augment T cell co-stimulation, proliferation, cytokine production, migration, and survival. [20][21][22][23][24][25] In T cells, activation through the T-cell receptor, polyclonal stimulation, SDF-1a interaction, and IFN-g are stimuli that downregulate CXCR4, whereas signaling through IL-2, IL-4, IL-7, and IL-15 upregulates its expression. 26-31The nuclear factor-kB (NF-kB) family of transcription factors consists of five subunits, RelA (p65), RelB, c-Rel, NF-kB1 (p50), and NF-kB2 (p52), that function as homo-or heterodimers. NF-kB signaling plays a central role in T-cell activation, proliferation, differentiation, and survival. 32Dysregulated CXCR4 and NF-kB signaling pathways contribute to disease pathology in multiple immune-mediated diseases including multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, and type 1 diabetes. [33][34][35][36][37][38][39][40][41] Both signaling pathways have also been associated with hematopoietic and nonhematopoietic malignancies. [42][43][44] Moreover, NF-kB-mediated regulation of CXCR4 expression and function in breast, pancreatic, gastric, prostatic, and ovarian cancers is well documented. [45][46][47][48][49][50][51] However, the contribution of CXCR4 and NF-kB signaling pathways to the pathology of acquired AA has not previously been explored.Through pharmacologic and genetic approaches, we demonstrate tha...

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB–DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.

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