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.
The Ku autoantigen is a heterodimer of 70 (p70) and ϳ80 kDa (p80) subunits that is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) complex involved in DNA repair and V(D)J recombination. Binding to DNA ends is critical to the function of DNA-PK, but how Ku interacts with DNA is not completely understood. To define the role of p70 and p80 and their dimerization in DNA binding, heterodimers were assembled by co-expressing the subunits using recombinant baculoviruses. Two p70 dimerization sites, amino acids 1-115 and 430 -482, respectively, were identified. Binding of p70 to linear double-stranded DNA could be demonstrated by an immunoprecipitation assay, and required the C-terminal portion (amino acids 430 -609), but not interaction with p80. The p70 mutants 1-600, 1-542, 1-115, and 430 -600 did not bind DNA efficiently. However, DNA binding of 1-600, 1-542, and 1-115, but not 430 -600, was restored by dimerization with p80, indicating that p70 has two DNA binding sites, each partially overlapping one of the dimerization sites. The C-terminal domain can bind DNA by itself, but the Nterminal domain requires dimerization with p80. These observations could be relevant to the multiple functional activities of Ku and explain controversies regarding the role of dimerization in DNA binding.The Ku (p70/p80) antigen was first identified and characterized as a DNA binding autoantigen recognized by antibodies present in the sera of certain patients with scleroderma-polymyositis overlap syndrome or systemic lupus erythematosus (SLE) (1). It was later shown to be the DNA binding component of a DNA-dependent protein kinase that phosphorylates several chromatin-bound proteins in vitro and is involved in V(D)J recombination and double-stranded (ds) 1 DNA break repair (2, 3). Ku antigen, a heterodimer of 70-(p70) and ϳ80-kDa (p80) subunits, binds to dsDNA termini, nicks, or single to double strand transitions (reviewed in Ref. 1). Sequence-specific DNA binding also has been reported (1, 4). Using DNA immunoprecipitation and Southwestern blot assays, the p70 protein has been shown to interact with DNA without requiring p80 (5-7), and a DNA binding domain was mapped to the C-terminal region (p70, amino acids 536 -609) (7,8). However, in gel shift assays, dimerization of p70 with p80 is required for DNA binding (9, 10). Determining how p70 and p80 interact with each other and with DNA termini or with specific sequences is critical to understanding the functions of Ku in DNA repair, V(D)J recombination, and transcriptional activation. To better understand the role of p70/p80 dimerization in DNA binding, p70 deletion mutants expressed in Sf9 cells using recombinant baculoviruses were used to identify functional sites of the p70 subunit. EXPERIMENTAL PROCEDURESCells and Viruses-K562 (human erythroleukemia, from the American Type Culture Collection, ATCC, Rockville, MD) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/ streptomycin. The Sf9 (Spodoptera frugiperda ovary) cell line...
SummaryAntinudear antibodies (ANAs) reactive with a limited spectrum of nuclear antigens are characteristic of systemic lupus erythematosus (SLE) and other collagen vascular diseases, and are also associated with certain viral infections. The factors that initiate ANA production and determine ANA specificity are not well understood. In this study, high titer ANAs specific for the p53 tumor suppressor protein were induced in mice immunized with purified complexes of murine p53 and the Simian virus 40 large T antigen (SVT), but not in mice immunized with either protein separately. The autoantibodies to p53 in these mice were primarily of the IgG1 isotype, were not cross-reactive with SVT, and were produced at titers up to 1:25,000, without the appearance of other autoantibodies. The high levels of autoantibodies to p53 in mice immunized with p53/SVT complexes were transient, but low levels of the autoantibodies persisted. The latter may have been maintained by self antigen, since the anti-p53, but not the SVT, response in these mice could be boosted by immunizing with murine p53. Thus, once autoimmunity to p53 was established by immunizing with p53/SVT complexes, it could be maintained without a requirement for SVT. These data may be explained in at least two ways. First, altered antigen processing resulting from the formation of p53/SVT complexes might activate autoreactive T helper cells specific for cryptic epitopes of routine p53, driving anti-p53 autoantibody production. Alternatively, SVT-responsive T cells may provide intermolecular-intrastructural help to B cells specific for murine p53. In a second stage, these activated B cells might themselves process self p53, generating p53-responsive autoreactive T cells. The induction of autoantibodies during the course of an immune response directed against this naturally occurring complex of self and nonself antigens may be relevant to the generation of specific autoantibodies in viral infections, and may also have implications for understanding the pathogenesis of ANAs in SLE. In particular, our results imply that autoimmunity can be initiated by a "hit and run" mechanism in which the binding of a viral antigen to a self protein triggers an immune response that subsequently can be perpetuated by self antigen.
Anti-DEK autoantibodies are less specific for JRA than previously believed. They are produced in association with a variety of inflammatory conditions, many of which are associated with granuloma formation and/or predominant Thl cytokine production. Anti-DEK antibodies may be a marker for a subset of autoimmunity associated with interferon-gamma production rather than a particular disease subset.
Ku autoantigen, a heterodimer of 70-and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair. Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion. The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent. Both must be intact for end binding activity in gel shift assays. To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses. We show by several criteria that amino acids 371-510 of p80 interact with p70. Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region. In DNA immunoprecipitation assays, amino acids 179 -510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179 -732 were necessary. Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases.Ku antigen was identified and characterized using autoantibodies from the sera of patients with systemic autoimmune diseases (reviewed in Ref. 1). Later, it was shown to be associated with a DNA-dependent protein kinase that phosphorylates chromatin-bound proteins in vitro (2) and is involved in double-stranded (ds) 1 DNA break repair (DSBR), V(D)J recombination, and isotype switching (3-5), as well as telomeric length maintenance and silencing (6). Ku is a heterodimer of 70-kDa (p70) and ϳ80-kDa (p80) subunits that binds dsDNA ends (1, 7). Sequence-specific DNA binding also has been reported (1,8). Defining the contribution of p70 and p80 to DNA binding is of interest because of the dual sequence-and endspecific binding of Ku and because of uncertainty as to its precise role in DNA repair. In DNA immunoprecipitation and Southwestern blot assays, p70 binds DNA in the absence of p80 (7, 9, 10), but in gel shift assays, only the dimer can bind (11,12). Although p80 alone does not bind to DNA, DSBR mutants in the XRCC5 complementation group have defects in the p80 gene (13)(14)(15).Defining the mechanism of p70-p80 dimerization may help to explain the role of p80 in sequence-specific DNA binding, DNA end binding, and/or DSBR. The p70 subunit contains two p80 interaction domains, amino acids 1-115 and 430 -482, respectively, as well as two regions involved in DNA binding, each partially overlapping one of the interaction domains (16). A p80-independent DNA binding site is located on the C terminus (amino acids 536 -609) (16, 17), whereas the N-terminal region must bind p80 in order to bind DNA. The goal of this study was to define the dimerization-dependent DNA binding site. Using p70 and p80 mutants, a large central domain of p80 involved in both dimerization and DNA binding was identified. This site, like the p80-independent DNA binding site, preferentially recognized DNA ends, suggesting that a s...
A patient was identified with an unusual autoimmune syndrome consisting of systemic lupus erythematosus and sarcoidosis. Her serum contained extremely high levels of autoantibodies to the DEK protooncogene product. The patient's serum was used to clone a dek complementary DNA, which was expressed as a histidine-tagged fusion protein in Escherichia coli. Using affinity-purified recombinant DEK protein, anti-DEK autoantibodies were found in the patient's serum at a titer of 1:10(6) by enzyme-linked immunosorbent assay (ELISA). Longitudinal studies revealed marked variations in anti-DEK autoantibody levels over time. Although it has been suggested that anti-DEK autoantibodies are a marker for pauciarticular juvenile rheumatoid arthritis with iridocyclitis, the present data suggest that they may be associated with other disease subsets as well. The quantitative ELISA technique will be useful for defining these subsets further and for examining the relationship between anti-DEK titers and disease activity.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.