Objective. Transient B cell depletion with the monoclonal anti-CD20 antibody rituximab has resulted in favorable clinical responses in patients with rheumatoid arthritis (RA). However, little is known about the regeneration profile of different peripheral B cell subpopulations. The aim of this study was to delineate the regeneration profile of different B cell subsets in the peripheral blood after selective anti-CD20-mediated B cell depletion.Methods. Seventeen patients with RA refractory to standard therapy were treated with rituximab. Patients 1-6 received 4 weekly infusions of rituximab at a dose of 375 mg/m 2 , and patients 7-17 received 2 infusions of rituximab (1,000 mg), 2 weeks apart. Four-color staining was performed at several time points, using CD38, IgD, and CD27 in addition to other cell surface markers. In one patient, the mutational status of the immunoglobulin receptor was examined.Results

Chronic inflammation has been implicated in the pathogenesis of many severe autoimmune disorders, as well as in diabetes, pulmonary diseases, and cancer. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only curative therapeutic approach currently available. In the present work, we investigated the role of the major proinflammatory cytokine tumor necrosis factor A (TNFA) in the malignancy of PDAC cells in vitro and in vivo. In vitro, TNFA strongly increased invasiveness of Colo357, BxPc3, and PancTuI cells and showed only moderate antiproliferative effect. TNFA treatment of mice bearing orthotopically growing PDAC tumors led to dramatically enhanced tumor growth and metastasis. Notably, we found that PDAC cells themselves secrete TNFA. Although inhibition of TNFA with infliximab or etanercept only marginally affected proliferation and invasiveness of PDAC cells in vitro, both reagents exerted strong antitumoral effects in vivo. In severe combined immunodeficient mice with orthotopically growing Colo357, BxPc3, or PancTuI tumors, human-specific anti-TNF antibody infliximab reduced tumor growth and metastasis by about 30% and 50%, respectively. Importantly, in a PDAC resection model performed with PancTuI cells, we found an even stronger therapeutic effect for both anti-TNF compounds. Infliximab and etanercept reduced the number of liver metastases by 69% and 42%, respectively, as well as volumes of recurrent tumors by 73% and 51%. Thus, tumor cell-derived TNFA plays a profound role in malignancy of PDAC, and inhibition of TNFA represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy.

Tumor necrosis factor (TNF) elicits its biological activities by stimulation of two receptors, TNFR1 and TNFR2, both belonging to the TNF receptor superfamily. Whereas TNFR1-mediated signal transduction has been intensively studied and is understood in detail, especially with respect to activation of the classical NFB pathway, cell death induction, and MAP kinase signaling, TNFR2-associated signal transduction is poorly defined. Here, we demonstrate in various tumor cell lines and primary T-cells that TNFR2, but not TNFR1, induces activation of the alternative NFB pathway. In accord with earlier findings demonstrating that only membrane TNF, but not soluble TNF, properly activates TNFR2, we further show by use of TNFR1-and TNFR2-specific mutants of soluble TNF and membrane TNF that soluble ligand trimers fail to activate the alternative NFB pathway. In accord with the known inhibitory role of TRAF2 in the alternative NFB pathway, TNFR2-, but not TNFR1-specific TNF induced depletion of cytosolic TRAF2. Thus, we identified activation of the alternative NFB pathway as a TNF signaling effect that can be specifically assigned to TNFR2 and membrane TNF. Tumor necrosis factor (TNF)2 is a highly pleiotropic cytokine and the prototypic member of the phylogenetically conserved TNF ligand family (1, 2). TNF, as well as other members of this cytokine family, is a type II transmembrane protein, which selfassembles into noncovalently bound trimers (1, 2). TNF occurs also as a soluble trimeric protein, which is derived from the transmembrane form by limited proteolysis (1, 2). TNF is mainly released from activated macrophages and T-cells, but it can also be produced by a variety of other cell types, especially after contact with bacterial products. TNF interacts with two receptors, TNFR1 and TNFR2, which both belong to the TNF receptor superfamily. Whereas TNFR1 is constitutively expressed in most cell types, TNFR2 is typically found on immune and endothelial cells (3). Remarkably, soluble and transmembrane TNF differ in their capability to stimulate signaling via TNFR1 and TNFR2. Whereas transmembrane TNF (memTNF) triggers signaling potently via both TNF receptors, soluble TNF trimers (sTNF) only activate TNFR1 robustly and show none or only limited activity on TNFR2 (4).TNFR1 contains a death domain (DD) and utilizes this protein-protein interaction domain to recruit intracellular signaling proteins involved in the activation of proinflammatory pathways, but also in cell death induction. For example, activation of the classical NFB pathway and the various MAP kinases by TNFR1 rely on recruitment of the DD-containing serine-threonine kinase RIP, the DD-containing adaptor protein TRADD, and a complex of the TRADD-interacting TRAF2 protein with cIAP1 and cIAP2 (3,5,6). Notably, RIP has also been implicated in TNFR1-induced necrosis, and TRADD, together with FADD and caspase-8, is crucially involved in TNFR1-mediated apoptosis (3). In general, the signaling mechanisms utilized by TNFR1 are biochemically well understood and the i...

Material Supplementary 5.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.090355

Soluble TNF-like weak inducer of apoptosis (TWEAK) trimers induce, in a variety of cell lines, translocation of cytosolic tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) to a triton X-100-insoluble compartment without changes in the total cellular TRAF2 content. TWEAK-induced TRAF2 translocation is paralleled by a strong increase in nuclear factor kappaB 2 (NFkappaB2)/p100 processing to p52, indicating that TRAF2 redistribution is sufficient for activation of the alternative NFkappaB pathway. In accordance with the crucial role of TRAF2 in proinflammatory, anti-apoptotic TNF receptor-1 (TNFR1) signaling, we observed that TWEAK-primed cells have a reduced capacity to activate the classical NFkappaB pathway or JNK (cJun N-terminal kinase) in response to TNF. Furthermore, TWEAK-primed cells are sensitized for the TNFR1-mediated induction of apoptotic and necrotic cell death. Notably, the expression of the NFkappaB-regulated, TRAF2-interacting TRAF1 protein can attenuate TWEAK-induced depletion of the triton X-100-soluble TRAF2 fraction and improve TNFR1-induced NFkappaB signaling in TWEAK-primed cells. Taken together, we demonstrate that soluble TWEAK desensitizes cells for proinflammatory TNFR1 signaling and thus identify TWEAK as a modifier of TNF signaling.