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Background-Giant cell arteritis is a granulomatous vasculitis of the aorta and its branches that causes blindness, stroke, and aortic aneurysm. CD4 T cells are key pathogenic regulators, instructed by vessel wall dendritic cells to differentiate into vasculitic T cells. The unique pathways driving this dendritic cell-T-cell interaction are incompletely understood, but may provide novel therapeutic targets for a disease in which the only established therapy is long-term treatment with high doses of corticosteroids. Methods and Results-Immunohistochemical and gene expression analyses of giant cell arteritis-affected temporal arteries revealed abundant expression of the NOTCH receptor and its ligands, Jagged1 and Delta1. Cleavage of the NOTCH intracellular domain in wall-infiltrating T cells indicated ongoing NOTCH pathway activation in large-vessel vasculitis. NOTCH activation did not occur in small-vessel vasculitis affecting branches of the vasa vasorum tree. We devised 2 strategies to block NOTCH pathway activation: ␥-secretase inhibitor treatment, preventing nuclear translocation of the NOTCH intracellular domain, and competing for receptor-ligand interactions through excess soluble ligand, Jagged1-Fc.In a humanized mouse model, NOTCH pathway disruption had strong immunosuppressive effects, inhibiting T-cell activation in the early and established phases of vascular inflammation. NOTCH inhibition was particularly effective in downregulating Th17 responses, but also markedly suppressed Th1 responses. Conclusions-Blocking

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Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and often succumb within months because effective therapy is lacking. A novel therapeutic approach has been suggested by the discovery that UM cell lines driven by mutant constitutively active Gq or G11 can be targeted by FR900359 (FR) or YM-254890, which are bioavailable, selective inhibitors of the Gq/11/14 subfamily of heterotrimeric G proteins. Here, we have addressed the therapeutic potential of FR for UM. We found that FR inhibited all oncogenic Gq/11 mutants reported in UM. FR arrested growth of all Gq/11-driven UM cell lines tested, but induced apoptosis only in a few. Similarly, FR inhibited growth of, but did not efficiently kill, UM tumor cells from biopsies of primary or metastatic tumors. FR evoked melanocytic redifferentiation of UM tumor cells with low (class 1), but not high (class 2), metastatic potential. FR administered systemically below its LD 50 strongly inhibited growth of PDX-derived class 1 and class 2 UM tumors in mouse xenograft models and reduced blood pressure transiently. FR did not regress xenografted UM tumors or significantly affect heart rate, liver function, hematopoiesis, or behavior. These results indicated the existence of a therapeutic window in which FR can be explored for treating UM and potentially other diseases caused by constitutively active Gq/11.

Immune-mediated damage to medium-sized arteries results in wall remodeling with intimal hyperplasia, luminal stenosis and tissue ischemia. In the case of the aorta, vasculitis may result in dissection, aneurysm or rupture. The response-to-injury program of the blood vessel is a concerted action between the immune system and wall-resident cells, involving the release of growth and angiogenic factors from macrophages and giant cells and the migration and hyperproliferation of vascular smooth muscle cells. Innate immune cells, specifically, dendritic cells (DC) positioned in the vessel wall, have been implicated in the earliest steps of vasculitis. Pathogen-derived molecular patterns are capable of activating vascular DC and initiating adaptive immune responses. The pattern of the emerging vessel wall inflammation is ultimately determined by the initial insult. Ligands to toll-like receptor (TLR) 4, such as lipopolysaccharides, facilitate the recruitment of CD4 T cells that invade deep into the wall and distribute in a panarteritic pattern. Conversely, ligands for TLR5 condition vascular DC to support perivasculitic infiltrates. In essence, both innate and adaptive immune reactions collaborate to render the arterial wall susceptible to inflammatory damage. Unique features of the tissue microenvironment, including specialized DC, shape the course of the inflammatory response. Differences in vascular damage pattern encountered in different patients may relate to distinct instigators of vasculitis.

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Dendritic cells (DCs) shape T-cell response patterns and determine early, intermediate, and late outcomes of immune recognition events. They either facilitate immunostimulation or induce tolerance, possibly determined by initial DC activation signals, such as binding Toll-like receptor (TLR) ligands. Here, we report that DC stimulation through the TLR3 ligand dsRNA [poly(I:C)] limits CD4 T-cell proliferation, curtailing adaptive immune responses. CD4+ T cells instructed by either lipopolysaccharide (LPS) or poly(I:C)-conditioned DCs promptly upregulated the activation marker CD69. Whereas LPS-pretreated DCs subsequently sustained T-cell clonal expansion, proliferation of CD4+ T cells exposed to poly(I:C)-pretreated DCs was markedly suppressed. This proliferative defect required DC-T cell contact, was independent of IFN-alpha, and was overcome by exogenous IL-2, indicating T-cell anergy. Coinciding with the downregulation, CD4+ T cells expressed the inhibitory receptor PD-1. Antibodies blocking the PD-1 ligand PD-L1 restored proliferation. dsRNA-stimulated DCs preferentially induced PD-L1, whereas poly(I:C) and LPS both upregulated the costimulatory molecule CD86 to a comparable extent. Poly(dA-dT), a ligand targeting the cytoplasmic RNA helicase pattern-recognition pathway, failed to selectively induce PD-L1 upregulation, assigning this effect to the TLR3 pathway. Poly(I:C)-conditioned DCs promoted accumulation of phosphorylated SHP-2, the intracellular phosphatase mediating PD-1 inhibitory effects. The ability of dsRNA to bias DC differentiation toward providing inhibitory signals to interacting CD4+ T cells may be instrumental in viral immune evasion. Conversely, TLR3 ligands may have therapeutic value in silencing pathogenic immune responses.

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