Lihe Su

TL1A–DR3 interaction regulates Th17 cell function and Th17-mediated autoimmune disease

T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A–DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A−/− dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A−/− animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A–DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.

Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Independent of Its B Cell Survival Function

Herein we demonstrate that B cell-activating factor of the TNF family (BAFF), a B cell survival factor, also regulates CD21/35 and CD23 expression. BAFF blockade in wild-type mice down-modulates CD21/35 and CD23 on B cells while survival remains intact, and BAFF exposure causes elevated CD21/35 and CD23 expression. Similar down-modulation is observed in bcl-2-transgenic mice treated with a BAFF inhibitor. This is the first evidence that BAFF has a function independent of B cell survival. Reports using CD21/35 and CD23 expression to assess splenic B cell subsets in BAFF-null mice concluded a lack of B cells beyond the immature stage. Since CD21/35 and CD23 are inadequate for delineating B cell subpopulations in BAFF-null mice, we used expression of BAFF-R and several B cell markers to identify more mature splenic B cells in these mice. These data broaden our understanding of BAFF function and correct the view that BAFF-null mice lack mature B cells.

Proinflammatory Effects of Tweak/Fn14 Interactions in Glomerular Mesangial Cells

TNF-like weak inducer of apoptosis, or TWEAK, is a relatively new member of the TNF-ligand superfamily. Ligation of the TWEAK receptor Fn14 by TWEAK has proinflammatory effects on fibroblasts, synoviocytes, and endothelial cells. Several of the TWEAK-inducible cytokines are important in the pathogenesis of kidney diseases; however, whether TWEAK can induce a proinflammatory effect on kidney cells is not known. We found that murine mesangial cells express cell surface TWEAK receptor. TWEAK stimulation of mesangial cells led to a dose-dependent increase in CCL2/MCP-1, CCL5/RANTES, CXCL10/IFN-γ-induced protein 10 kDa, and CXCL1/KC. The induced levels of chemokines were comparable to those found following mesangial cell exposure to potent proinflammatory stimuli such as TNF-α + IL-1β. CXCL11/interferon-inducible T cell α chemoattractant, CXCR5, mucosal addressin cell adhesion molecule-1, and VCAM-1 were up-regulated by TWEAK as well. TWEAK stimulation of mesangial cells resulted in an increase in phosphorylated Iκ-B, while pretreatment with an Iκ-B phosphorylation inhibitor significantly blocked chemokine induction, implicating activation of the NF-κB signaling pathway in TWEAK-induced chemokine secretion. Importantly, the Fn14-mediated proinflammatory effects of TWEAK on kidney cells were confirmed using mesangial cells derived from Fn14-deficient mice and by injection in vivo of TWEAK into wild-type vs Fn14-deficient mice. Finally, TWEAK-induced chemokine secretion was prevented by treatment with novel murine anti-TWEAK Abs. We conclude that TWEAK induces mesangial cells to secrete proinflammatory chemokines, suggesting a prominent role for TWEAK in the pathogenesis of renal injury. Our results support Ab inhibition of TWEAK as a potential new approach for the treatment of chemokine-dependent inflammatory kidney diseases.

TWEAK Is a Novel Arthritogenic Mediator

TNF-like weak inducer of apoptosis (TWEAK) is a TNF family member with pleiotropic effects on a variety of cell types, one of which is the induction of proinflammatory cytokines by synovial fibroblasts derived from rheumatoid arthritis (RA) patients. In this study, we report that the serum TWEAK level was dramatically elevated during mouse collagen-induced arthritis (CIA) and blocking TWEAK by a neutralizing mAb significantly reduced the clinical severity of CIA. Histological analyses also revealed that TWEAK inhibition diminished joint inflammation, synovial angiogenesis, as well as cartilage and bone erosion. Anti-TWEAK treatment proved efficacious when administered just before the disease onset but not during the priming phase of CIA. Consistent with this, TWEAK inhibition did not affect either cellular or humoral responses to collagen. In contrast, TWEAK inhibition significantly reduced serum levels of a panel of arthritogenic mediators, including chemokines such as MIP-1β (CCL-4), lymphotactin (XCL-1), IFN-γ-inducible protein 10 (IP-10) (CXCL-10), MCP-1 (CCL-2), and RANTES (CCL-5), as well as the matrix metalloprotease-9. Exploring the possible role of the TWEAK/Fn14 pathway in human RA pathogenesis, we showed that TWEAK can target human primary chondrocytes and osteoblast-like cells, in addition to synovial fibroblasts. We further demonstrated that TWEAK induced the production of matrix metalloproteases in human chondrocytes and potently inhibited chondrogenesis and osteogenesis using in vitro models. These results provide evidence for a novel cytokine pathway that contributes to joint tissue inflammation, angiogenesis, and damage, as well as may inhibit endogenous repair, suggesting that TWEAK may be a new therapeutic target for human RA.

Urinary TWEAK as a biomarker of lupus nephritis: a multicenter cohort study

IntroductionTNF-like weak inducer of apoptosis (TWEAK) has been implicated as a mediator of chronic inflammatory processes via prolonged activation of the NF-κB pathway in several tissues, including the kidney. Evidence for the importance of TWEAK in the pathogenesis of lupus nephritis (LN) has been recently introduced. Thus, TWEAK levels may serve as an indication of LN presence and activity.MethodsMulticenter cohorts of systemic lupus erythematosus (SLE) patients and controls were recruited for cross-sectional and longitudinal analysis of urinary TWEAK (uTWEAK) and/or serum TWEAK (sTWEAK) levels as potential biomarkers of LN. The performance of TWEAK as a biomarker for nephritis was compared with routinely used laboratory tests in lupus patients, including anti-double stranded DNA antibodies and levels of C3 and C4.ResultsuTWEAK levels were significantly higher in LN patients than in non-LN SLE patients and other disease control groups (P = 0.039). Furthermore, uTWEAK was better at distinguishing between LN and non-LN SLE patients than anti-DNA antibodies and complement levels, while high uTWEAK levels predicted LN in SLE patients with an odds ratio of 7.36 (95% confidence interval = 2.25 to 24.07; P = 0.001). uTWEAK levels peaked during LN flares, and were significantly higher during the flare than at 4 and 6 months prior to or following the flare event. A linear mixed-effects model showed a significant association between uTWEAK levels in SLE patients and their disease activity over time (P = 0.008). sTWEAK levels, however, were not found to correlate with the presence of LN or the degree of nephritis activity.ConclusionsHigh uTWEAK levels are indicative of LN, as opposed to non-LN SLE and other healthy and disease control populations, and reflect renal disease activity in longitudinal follow-up. Thus, our study further supports a role for TWEAK in the pathogenesis of LN, and provides strong evidence for uTWEAK as a candidate clinical biomarker for LN.

Alefacept, an Immunomodulatory Recombinant LFA-3/IgG1 Fusion Protein, Induces CD16 Signaling and CD2/CD16-Dependent Apoptosis of CD2+ Cells

Alefacept, an immunomodulatory recombinant fusion protein composed of the first extracellular domain of LFA-3 fused to the human IgG1 hinge, CH2, and CH3 domains, has recently been shown in phase II and III clinical trials to safely reduce disease expression in patients with chronic plaque psoriasis. Alefacept modulates the function of and selectively induces apoptosis of CD2+ human memory-effector T cells in vivo. We have sought to gain further understanding of the mechanisms of action that influence the biological activity of alefacept and may contribute to its efficacy and patient responsiveness. Specifically evaluated is the ability of alefacept to activate intracellular signals mediated via CD2 and/or FcγRIII (CD16). Experimentation using isoforms of alefacept engineered to have amino acid substitutions in the IgG1 CH2 domain that impact FcγR binding indicate that alefacept mediates cognate interactions between cells expressing human CD2 and CD16 to activate cells, e.g., increase extracellular signal-regulated kinase phosphorylation, up-regulate cell surface expression of the activation marker CD25, and induce release of granzyme B. In the systems used, this signaling is shown to require binding to CD2 and CD16 and be mediated through CD16, but not CD2. Experimentation using human CD2-transgenic mice and isoforms of alefacept confirmed the requirement for FcγR binding for detection of the pharmacological effects of alefacept in vivo. Thus alefacept acts as an effector molecule, mediating cognate interactions to activate FcγR+ cells (e.g., NK cells) to induce apoptosis of sensitive CD2+ target cells.

A Novel Role for Tumor Necrosis Factor–Like Weak Inducer of Apoptosis (TWEAK) in the Development of Cardiac Dysfunction and Failure

Background— Tumor necrosis factor–like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, is a multifunctional cytokine known to regulate cellular functions in contexts of injury and disease through its receptor, fibroblast growth factor–inducible molecule 14 (Fn14). Although many of the processes and downstream signals regulated by the TWEAK/Fn14 pathway have been implicated in the development of cardiac dysfunction, the role of TWEAK in the cardiovascular system is completely unknown. Methods and Results— Herein, we demonstrate that mouse and human cardiomyocytes express the TWEAK receptor Fn14. Furthermore, we determine that elevated circulating levels of TWEAK, induced via transgenic or adenoviral-mediated gene expression in mice, result in dilated cardiomyopathy with subsequent severe cardiac dysfunction. This phenotype was mediated exclusively by the Fn14 receptor, independent of tumor necrosis factor-α, and was associated with cardiomyocyte elongation and cardiac fibrosis but not cardiomyocyte apoptosis. Moreover, we find that circulating TWEAK levels were differentially upregulated in patients with idiopathic dilated cardiomyopathy compared with other forms of heart disease and normal control subjects. Conclusions— Our data suggest that TWEAK/Fn14 may be important in regulating myocardial structural remodeling and function and may play a role in the pathogenesis of dilated cardiomyopathy.

Discovery and Investigation of Misincorporation of Serine at Asparagine Positions in Recombinant Proteins Expressed in Chinese Hamster Ovary Cells

Misincorporation of amino acids in proteins expressed in Escherichia coli has been well documented but not in proteins expressed in mammalian cells under normal recombinant protein production conditions. Here we report for the first time that Ser can be incorporated at Asn positions in proteins expressed in Chinese hamster ovary cells. This misincorporation was discovered as a result of intact mass measurement, peptide mapping analysis, and tandem mass spectroscopy sequencing. Our analyses showed that the substitution was not related to specific protein molecules or DNA codons and was not site-specific. We believe that the incorporation of Ser at sites coded for Asn was due to mischarging of tRNAAsn rather than to codon misreading. The rationale for substitution of Asn by Ser and not by other amino acids is also discussed. Further investigation indicated that the substitution was due to the starvation for Asn in the cell culture medium and that the substitution could be limited by using the Asn-rich feed. These observations demonstrate that the quality of expressed proteins should be closely monitored when altering cell culture conditions.

Heteromultimeric Complexes of CD40 Ligand Are Present on the Cell Surface of Human T Lymphocytes

CD40 ligand (CD40L), a 33-kDa type II membrane glycoprotein expressed primarily on activated CD4+ T lymphocytes, is responsible for the helper function of T cells on resting B cells in a non-antigen-dependent, non-major histocompatability complex-restricted fashion. Interaction of CD40L with its receptor CD40 induces proliferation of and isotype switching in B lymphocytes. Recently we solved the x-ray structure of recombinant soluble CD40L and showed that, similar to other members of the tumor necrosis factor family, CD40L indeed exists as a trimer. We now report that, under normal physiological conditions, CD40L molecules exist as heteromultimeric complexes. These CD40L complexes, made of the full length and smaller fragments of CD40L, are present on the cell surface of T lymphocytes and are capable of interacting with CD40 molecule. A prominent fragment with a mass of 31 kDa accounts for as much as half of the CD40L on the surface of Jurkat cells. N-terminal sequence data revealed that this fragment lacks the cytoplasmic tail. A minor 18-kDa fragment of CD40L was also characterized which lacks the cytoplasmic tail, transmembrane region, and stalk region of the extracellular domain. The presence of CD40L heteromultimeric variants implies an additional regulation of the functional activity of this ligand complex.

Development of an Fn14 agonistic antibody as an anti-tumor agent

TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.