Laura L. Stunz

CD40 and Autoimmunity: The Dark Side of a Great Activator

CD40 is a tumor necrosis factor receptor superfamily member expressed by immune and non-immune cells. CD40:CD154 interactions mediate T-dependent B cell responses and efficient T cell priming. Thus, CD40 is a likely candidate to play roles in autoimmune diseases in which activated T and B cells cause pathology. Diseases in which CD40 plays a pathogenic role include autoimmune thyroiditis, type 1 diabetes, inflammatory bowel disease, psoriasis, multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus. This review discusses the role of CD40:CD154 interaction in human and mouse autoimmunity, human polymorphisms associated with disease incidence, and disrupting CD40:CD154 interactions as an autoimmune therapy.

CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-κB activation

Bacterial DNA and CpG-oligodeoxyribonucleotides (ODN) are powerful B cell activators, inducing apoptosis protection, cell cycle entry, proliferation, costimulatory molecule expression, immunoglobulin (Ig) and interleukin-6 (IL-6) secretion. However, proximal events in B cell activation by ODN are only partially characterized, including the translocation of NF-kappaB to the nucleus. In this paper, we provide evidence that CpG-ODN-induced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-kappaB activation. NF-kappaB activation occurred within 30 minutes of stimulation of murine B cells with a phosphorothioate (S) CpG-ODN and persisted for up to 40 hours, with p50, p65, and c-Rel as the major components. Similar to other NF-kappaB inducers, CpG-ODN caused an early IkappaBalpha and IkappaBbeta degradation plus cleavage of the p50 precursor and subsequent NF-kappaB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-kappaB DNA binding and transcriptional activation. These inhibitory S-ODN differed from stimulatory S-ODN by having 2-3 G substitutions in the central motif. As inhibitory S-ODN did not directly interfere with the NF-kappaB DNA binding but prevented CpG-induced NF-kappaB nuclear translocation of p50, p65, and c-Rel and blocked p105, IkappaBalpha, and IkappaBbeta degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.

TRAF Proteins in CD40 Signaling

The tumor necrosis factor receptor (TNFR) superfamily molecule CD40 is expressed by a wide variety of cell types following activation signals, and constitutively on B lymphocytes, macrophages, and dendritic cells. CD40 signals to cells stimulate kinase activation, gene expression, production of a antibody and a variety of cytokines, expression or upregulation of surface molecules, and protection or promotion of apoptosis. Initial steps in CD40-mediated signal cascades involve the interactions of CD40 with various members of the TNFR-associated factor (TRAF) family of cytoplasmic proteins. This review summarizes current understanding of the nature of these interactions, and how they induce and regulate CD40 functions.

TNF Receptor-Associated Factor 3 Is Required for T Cell-Mediated Immunity and TCR/CD28 Signaling

We recently reported that TNFR-associated factor (TRAF)3, a ubiquitously expressed adaptor protein, promotes mature B cell apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. In this article, we report the generation and characterization of T cell-specific TRAF3−/− mice, in which the traf3 gene was deleted from thymocytes and T cells. Ablation of TRAF3 in the T cell lineage did not affect CD4 or CD8 T cell populations in secondary lymphoid organs or the numbers or proportions of CD4+,CD8+ or double-positive or double-negative thymocytes, except that the T cell-specific TRAF3−/− mice had a 2-fold increase in FoxP3+ T cells. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3-deficient mice exhibited defective IgG1 responses to a T-dependent Ag, as well as impaired T cell-mediated immunity to infection with Listeria monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon costimulation and that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity.

Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions

Summary:  A large and diverse group of receptors utilizes the family of cytoplasmic signaling proteins known as tumor necrosis factor receptor (TNFR)‐associated factors (TRAFs). In recent years, there has been a resurgence of interest and exploration of the roles played by TRAF3 and TRAF5 in cellular regulation, particularly in cells of the immune system, the cell types of focus in this review. This work has revealed that TRAF3 and TRAF5 can play diverse roles for different receptors even in the same cell type, as well as distinct roles in different cell types. Evidence indicates that TRAF3 and TRAF5 play important roles beyond the TNFR‐superfamily (SF) and viral mimics of its members, mediating certain innate immune receptor and cytokine receptor signals, and most recently, signals delivered by the T‐cell receptor (TCR) signaling complex. Additionally, much research has demonstrated the importance of TRAF3‐mediated cellular regulation via its cytoplasmic interactions with additional signaling proteins. In particular, we discuss below evidence for the participation by TRAF3 in a number of the regulatory post‐translational modifications involving ubiquitin that are important in various signaling pathways.

A Novel Mechanism for TNFR-Associated Factor 6-Dependent CD40 Signaling

Members of the TNFR family play critical roles in the regulation of the immune system. One member of the family critical for efficient activation of T-dependent humoral immune responses is CD40, a cell surface protein expressed by B cells and other APC. The cytoplasmic domain of CD40 interacts with several members of the TNFR-associated factor (TRAF) family, which link CD40 to intracellular signaling pathways. TRAF2 and 6 appear to play particularly important roles in CD40 signaling. Previous studies suggest that the two molecules have certain overlapping roles in signaling, but that unique roles for each molecule also exist. To better define the roles of TRAF2 and TRAF6 in CD40 signaling, we used somatic cell gene targeting to generate TRAF-deficient mouse B cell lines. A20.2J cells deficient in TRAF6 exhibit marked defects in CD40-mediated JNK activation and the up-regulation of CD80. Our previous experiments with TRAF2-deficient B cell lines suggest that TRAF6 and TRAF2 may have redundant roles in CD40-mediated NF-κB activation. Consistent with this hypothesis, we found CD40-mediated activation of NF-κB intact in TRAF6-deficient cells and defective in cells lacking both TRAF2 and TRAF6. Interestingly, we found that TRAF6 mutants defective in CD40 binding were able to restore CD40-mediated JNK activation and CD80 up-regulation in TRAF6-deficient cells, indicating that TRAF6 may be able to contribute to certain CD40 signals without directly binding CD40.

Enhanced Toll‐like receptor (TLR) responses of TNFR‐associated factor 3 (TRAF3)‐deficient B lymphocytes

The key role of TRAF6 in TLR signaling pathways is well known. More recent evidence has implicated TRAF3 as another TRAF family member important to certain TLR responses of myeloid cells. Previous studies demonstrate that TRAF3 functions are highly context‐dependent, displaying receptor and cell‐type specificity. We thus examined the TLR responses of TRAF3−/−mouse B lymphocytes to test the hypothesis that TRAF3 plays distinct roles in such responses, depending on cell type. TRAF3−/− DC are known to have a defect in type 1 IFN production and here, showed diminished production of TNF and IL‐10 and unaltered IL‐6. In marked contrast, TRAF3−/− B cells made elevated amounts of TNF and IL‐6 protein, as well as IL‐10 and IP‐10 mRNA, in response to TLR ligands. Also, in contrast to TRAF3−/− DC, the type 1 IFN pathway was elevated in TRAF3−/− B cells. Increased early responses of TRAF3−/− B cells to TLR signals were independent of cell survival or proliferation but associated with elevated canonical NF‐κB activation. Additionally, TRAF3−/− B cells displayed enhanced TLR‐mediated expression of AID and Ig isotype switching. Thus, TRAF3 plays varied and cell type‐specific, biological roles in TLR responses.

Roles of TRAF molecules in B lymphocyte function

Tumor necrosis factor receptor associated factors (TRAFs) play a variety of interesting and important roles in the regulation of B lymphocyte function. They act both as cytoplasmic regulatory molecules, and as signal transducers for receptors involved in both innate and adaptive humoral immune responses. In this brief review, we highlight the current state of knowledge of the diverse roles of TRAF molecules in the functions of B lymphocytes.

The adaptor TRAF3 restrains the lineage determination of thymic regulatory T cells by modulating signaling via the receptor for IL-2

The number of Foxp3+ regulatory T cells (Treg cells) must be tightly controlled for efficient suppression of autoimmunity with no impairment of normal immune responses. Here we found that the adaptor TRAF3 was intrinsically required for restraining the lineage determination of thymic Treg cells. T cell–specific deficiency in TRAF3 resulted in a two- to threefold greater frequency of Treg cells, due to the more efficient transition of precursors of Treg cells into Foxp3+ Treg cells. TRAF3 dampened interleukin 2 (IL-2) signaling by facilitating recruitment of the tyrosine phosphatase TCPTP to the IL-2 receptor complex, which resulted in dephosphorylation of the signaling molecules Jak1 and Jak3 and negative regulation of signaling via Jak and the transcription factor STAT5. Our results identify a role for TRAF3 as an important negative regulator of signaling via the IL-2 receptor that affects the development of Treg cells.

Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1)

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.