TRAF3 in T Cells Restrains Negative Regulators of LAT to Promote TCR/CD28 Signaling.

The adaptor protein TNFR-associated factor 3 (TRAF3) is required for in vivo T cell effector functions and for normal TCR/CD28 signaling. TRAF3-mediated enhancement of TCR function requires engagement of both CD3 and CD28, but the molecular mechanisms underlying how TRAF3 interacts with and impacts TCR/CD28-mediated complexes to enhance their signaling remains an important knowledge gap. We investigated how TRAF3 is recruited to, and regulates, CD28 as a TCR costimulator. Direct association with known signaling motifs in CD28 was dispensable for TRAF3 recruitment; rather, TRAF3 associated with the CD28-interacting protein linker of activated T cells (LAT) in human and mouse T cells. TRAF3-LAT association required the TRAF3 TRAF-C domain and a newly identified TRAF2/3 binding motif in LAT. TRAF3 inhibited function of the LAT-associated negative regulatory protein Dok1, which is phosphorylated at an inhibitory tyrosine residue by the tyrosine kinase breast tumor kinase (Brk/PTK6). TRAF3 regulated Brk activation in T cells, limiting the association of protein tyrosine phosphatase 1B (PTP1B) with the LAT complex. In TRAF3-deficient cells, LAT complex-associated PTP1B was associated with dephosphorylation of Brk at an activating tyrosine residue, potentially reducing its ability to inhibit Dok1. Consistent with these findings, inhibiting PTP1B activity in TRAF3-deficient T cells rescued basal and TCR/CD28-mediated activation of Src family kinases. These results reveal a new mechanism for promotion of TCR/CD28-mediated signaling through restraint of negative regulation of LAT by TRAF3, enhancing the understanding of regulation of the TCR complex.

Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR.

Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) plays context-specific roles in multiple receptor-mediated signaling pathways in different cell types. Mice lacking TRAF3 in T cells display defective T-cell-mediated immune responses to immunization and infection and demonstrate defective early signaling via the TCR complex. However, the role of TRAF3 in the function of GITR/TNFRSF18, an important costimulatory member of the TNFR superfamily, is unclear. Here we investigated the impact of T cell TRAF3 status on both GITR expression and activation of specific kinases in the GITR signaling pathway in T cells. Our results indicate that TRAF3 negatively regulates GITR functions in several ways. First, expression of GITR protein was elevated in TRAF3-deficient T cells, resulting from both transcriptional and posttranslational regulation that led to greater GITR transcript levels, as well as enhanced GITR protein stability. TRAF3 associated with T cell GITR in a manner dependent upon GITR ligation. TRAF3 also inhibited several events of the GITR mediated early signaling cascade, in a manner independent of recruitment of phosphatases, a mechanism by which TRAF3 inhibits signaling through several other cytokine receptors. These results add new information to our understanding of GITR signaling and function in T cells, which is relevant to the potential use of GITR to enhance immune therapies.

TRAF3, ubiquitination, and B-lymphocyte regulation.

The signaling adapter protein tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) is both modified by and contributes to several types of ubiquitination events. TRAF3 plays a variety of context-dependent regulatory roles in all types of immune cells. In B lymphocytes, TRAF3 contributes to regulation of signaling by members of both the TNFR superfamily and innate immune receptors. TRAF3 also plays a unique cell type-specific and critical role in the restraint of B-cell homeostatic survival, a role with important implications for both B-cell differentiation and the pathogenesis of B-cell malignancies. This review focuses upon the relationship between ubiquitin and TRAF3, and how this contributes to multiple functions of TRAF3 in the regulation of signal transduction, transcriptional activation, and effector functions of B lymphocytes.

Systems-wide analysis of BCR signalosomes and downstream phosphorylation and ubiquitylation.

B-cell receptor (BCR) signaling is essential for the development and function of B cells; however, the spectrum of proteins involved in BCR signaling is not fully known. Here we used quantitative mass spectrometry-based proteomics to monitor the dynamics of BCR signaling complexes (signalosomes) and to investigate the dynamics of downstream phosphorylation and ubiquitylation signaling. We identify most of the previously known components of BCR signaling, as well as many proteins that have not yet been implicated in this system. BCR activation leads to rapid tyrosine phosphorylation and ubiquitylation of the receptor-proximal signaling components, many of which are co-regulated by both the modifications. We illustrate the power of multilayered proteomic analyses for discovering novel BCR signaling components by demonstrating that BCR-induced phosphorylation of RAB7A at S72 prevents its association with effector proteins and with endo-lysosomal compartments. In addition, we show that BCL10 is modified by LUBAC-mediated linear ubiquitylation, and demonstrate an important function of LUBAC in BCR-induced NF-κB signaling. Our results offer a global and integrated view of BCR signaling, and the provided datasets can serve as a valuable resource for further understanding BCR signaling networks.

The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression.

Genetic studies involving zebrafish and mice have demonstrated that the protein Gon4l (Gon4-like) is essential for hematopoiesis. These studies also suggested that Gon4l regulates gene expression during hematopoietic development, yet the biochemical function of Gon4l has not been defined. Here, we describe the identification of factors that interact with Gon4l and may cooperate with this protein to regulate gene expression. As predicted by polypeptide sequence conservation, Gon4l interacted and co-localized with the DNA-binding protein YY1 (Yin Yang 1). Density gradient sedimentation analysis of protein lysates from mouse M12 B cells showed that Gon4l and YY1 co-sediment with the transcriptional co-repressor Sin3a and its functional partner histone deacetylase (HDAC) 1. Consistent with these results, immunoprecipitation studies showed that Gon4l associates with Sin3a, HDAC1, and YY1 as a part of complexes that form in M12 cells. Sequential immunoprecipitation studies demonstrated that Gon4l, YY1, Sin3a, and HDAC1 could all associate as components of a single complex and that a conserved domain spanning the central portion of Gon4l was required for formation of this complex. When targeted to DNA, Gon4l repressed the activity of a nearby promoter, which correlated with the ability to interact with Sin3a and HDAC1. Our data suggest that Sin3a, HDAC1, and YY1 are co-factors for Gon4l and that Gon4l may function as a platform for the assembly of complexes that regulate gene expression.

Requirement for TRAF3 in signaling by LMP1 but not CD40 in B lymphocytes.

CD40, a member of the tumor necrosis factor receptor family, and the Epstein-Barr virus-encoded oncoprotein latent membrane protein 1 (LMP1) share several tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins for signaling. Among these, TRAF3 was the first identified to directly bind both receptors, yet its role remains a mystery. To address this, we generated B cell lines deficient in TRAF3 by homologous recombination. We found that CD40 signals were normal in the absence of TRAF3, with the exception of moderately enhanced c-Jun NH2-terminal kinase (JNK) activation and antibody secretion. In sharp contrast, LMP1 signaling was markedly defective in TRAF3-/- B cells. LMP1-induced activation of JNK and nuclear factor kappaB, up-regulation of CD23 and CD80, and antibody secretion were substantially affected by TRAF3 deficiency. Reconstitution of TRAF3 expression decreased CD40-induced JNK activation and antibody secretion, and fully restored LMP1 signaling. Although TRAF2 is widely believed to be important for LMP1 function, LMP1 signaling was intact in TRAF2-/- B cells. Our data reveal that CD40 and LMP1 unexpectedly use TRAF3 in different ways, and that TRAF3 is required for LMP1-mediated activation of B cells.

Nitric oxide utilizes NF-kappaB to signal its neuroprotective effect against alcohol toxicity.

Alcohol damages the developing brain and can lead to fetal alcohol syndrome. One of alcohol's most important neuropathologic effects is neuronal death. As neurons mature, they become less vulnerable to alcohol-induced death because they acquire a protective signaling pathway, mediated by nitric oxide (NO). This pathway is the NO-cGMP-cyclic GMP-dependent protein kinase G (NO-cGMP-PKG) pathway. The goal of the present studies was to determine whether nuclear factor kappa B (NF-kappaB) is the downstream effector through which the NO-cGMP-PKG pathway signals its neuroprotective effects against alcohol. An activator of NF-kappaB, tumor necrosis factor-alpha (TNF-alpha), protected immature cerebellar granule neuron cultures against alcohol-induced cell death in a dose-dependent fashion. The protective effect of TNF-alpha was similar in magnitude to the protective effects of NMDA and DETA-NONOate, both of which are NO-cGMP-PKG pathway activators. Blockade of the pathway at its first step with NAME, second step with LY83583, or third step with PKG inhibitor increased alcohol-induced cell death and the vulnerability of mature neurons to alcohol toxicity. TNF-alpha protected the neurons, even when the NO-cGMP-PKG pathway was blocked at upstream sites. NF-kappaB activation inhibitor (NFi) worsened alcohol-induced cell death and blocked the protective effects of NO-cGMP-PKG pathway activators and TNF-alpha. TNF-alpha reduced the alcohol vulnerability of immature neurons, while NFi increased the vulnerability of mature neurons. Both NMDA and TNF-alpha led to the phosphorylation and degradation of IkappaBalpha, demonstrating that both agents can activate NF-kappaB in cerebellar granule cells. Thus, NF-kappaB plays a critical role in the acquisition of alcohol resistance by maturing neurons and is a key downstream effector through which the NO-cGMP-PKG pathway signals its neuroprotective effects against alcohol.

The role of vasoactive intestinal peptide (VIP) in megakaryocyte proliferation.

Megakaryocytopoiesis is a multistage process that involves differentiation of hematopoietic stem cells through the myeloid lineage, ultimately producing megakaryocytes and platelets. Vasoactive intestinal peptide (VIP) stimulates adenylate cyclase and induces differentiation in multiple cell types; VIP is expressed in hematopoietic stem cells and in megakaryocytes, but its function in these cells has not yet been delineated. The present study was designed to investigate whether the type 1 VIP receptor, VPAC1, mediates VIP effects on megakaryocytopoiesis. The human megakaryoblastic leukemia cell line (CMK) was transfected with VPAC1 and the transgene expression was confirmed by qualitative polymerase chain reaction and immunohistochemistry. The rate of proliferation and the patterns of differentiation were then compared for CMK and CMK/VPAC1 through multiple growth cycles. Upregulation of VPAC1 expression resulted in a decreased proliferation rate (p = 0.0003) and enhanced differentiation with CMK/VPAC1 cells having twice the cell surface area of control CMK cells (p = 0.001), thus increasing potential for proplatelet formation. These results suggest that VIP acts in an autocrine fashion via VPAC1 to inhibit megakaryocyte proliferation and induce proplatelet formation.

Staphylococcal Superantigens Stimulate Epithelial Cells through CD40 To Produce Chemokines.

Mucosal and skin tissues form barriers to infection by most bacterial pathogens. Staphylococcus aureus causes diseases across these barriers in part dependent on the proinflammatory properties of superantigens. We showed, through use of a CRISPR-Cas9 CD40 knockout, that the superantigens toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxins (SEs) B and C stimulated chemokine production from human vaginal epithelial cells (HVECs) through human CD40. This response was enhanced by addition of antibodies against CD40 through an unknown mechanism. TSST-1 was better able to stimulate chemokine (IL-8 and MIP-3α) production by HVECs than SEB and SEC, suggesting this is the reason for TSST-1's exclusive association with menstrual TSS. A mutant of TSST-1, K121A, caused TSS in a rabbit model when administered vaginally but not intravenously, emphasizing the importance of the local vaginal environment. Collectively, our data suggested that superantigens facilitate infections by disruption of mucosal barriers through their binding to CD40, with subsequent expression of chemokines. The chemokines facilitate TSS and possibly other epithelial conditions after attraction of the adaptive immune system to the local environment.IMPORTANCE Menstrual toxic shock syndrome (TSS) is a serious infectious disease associated with vaginal colonization by Staphylococcus aureus producing the exotoxin TSS toxin 1 (TSST-1). We show that menstrual TSS occurs after TSST-1 interaction with an immune costimulatory molecule called CD40 on the surface of vaginal epithelial cells. Other related toxins, where the entire family is called the superantigen family, bind to CD40, but not with a high-enough apparent affinity to cause TSS; thus, TSST-1 is the only exotoxin superantigen associated. Once the epithelial cells become activated by TSST-1, they produce soluble molecules referred to as chemokines, which in turn facilitate TSST-1 activation of T lymphocytes and macrophages to cause the symptoms of TSS. Identification of small-molecule inhibitors of the interaction of TSST-1 with CD40 may be useful so that they may serve as additives to medical devices, such as tampons and menstrual cups, to reduce the incidence of menstrual TSS.

TRAF3 as a Multifaceted Regulator of B Lymphocyte Survival and Activation.

The adaptor protein TNF receptor-associated factor 3 (TRAF3) serves as a powerful negative regulator in multiple aspects of B cell biology. Early in vitro studies in transformed cell lines suggested the potential of TRAF3 to inhibit signaling by its first identified binding receptor, CD40. However, because the canonical TRAF3 binding site on many receptors also mediates binding of other TRAFs, and whole-mouse TRAF3 deficiency is neonatally lethal, an accurate understanding of TRAF3's specific functions was delayed until conditional TRAF3-deficient mice were produced. Studies of B cell-specific TRAF3-deficient mice, complemented by investigations in normal and malignant mouse and human B cells, reveal that TRAF3 has powerful regulatory roles that are unique to this TRAF, as well as functions context-specific to the B cell. This review summarizes the current state of knowledge of these roles and functions. These include inhibition of signaling by plasma membrane receptors, negative regulation of intracellular receptors, and restraint of cytoplasmic NF- κB pathways. TRAF3 is also now known to function as a resident nuclear protein, and to impact B cell metabolism. Through these and additional mechanisms TRAF3 exerts powerful restraint upon B cell survival and activation. It is thus perhaps not surprising that TRAF3 has been revealed as an important tumor suppressor in B cells. The many and varied functions of TRAF3 in B cells, and new directions to pursue in future studies, are summarized and discussed here.

Roles of TRAF6 in CD40 signaling.

CD40 provides signals crucial to the activation of antigen-presenting cells during humoral and cell-mediated immune responses. A complex cohort of proteins interacts with the cytoplasmic domain of CD40 and mediates signaling. One member of this cohort is TNF receptor associated factor six (TRAF6). TRAF6 contributes to the CD40-mediated activation of NF-kappaB, stress-activated protein kinases, and perhaps other signaling molecules. TRAF6 may have roles as an adapter molecule, an activator of mitogen-activated protein kinases, and as a repressor of certain signaling circuits. Establishing the significance and interplay of these roles will lead to a more complete understanding of mechanisms important to the CD40-mediated activation of the immune system and will reveal novel targets for the development of therapeutic agents.

The CD40-CD154 interaction in B cell-T cell liaisons.

The CD40 receptor is expressed constitutively on B lymphocytes, for which it provides important signals regulating clonal expansion, antibody production and isotype switching, as well as the development of humoral memory. The major source of CD154, the ligand for CD40, is activated T lymphocytes. Interactions between CD40 and CD154 provide a number of signals that play important roles in regulating the complex and multifactorial interactions between these two major cell types of the adaptive immune response. Understanding both the biological effects of this receptor-ligand interaction, as well as how CD40 signaling pathways are controlled, adds to our detailed picture of the complex interplay between B and T cells.

TRAF3 enhances TCR signaling by regulating the inhibitors Csk and PTPN22.

The adaptor protein TNF receptor associated factor (TRAF) 3 is required for effective TCR signaling and normal T cell effector functions, and associates with the CD3/CD28 complex upon activation. To determine how TRAF3 promotes proximal TCR signaling, we studied TRAF3-deficient mouse and human T cells, which showed a marked reduction in activating phosphorylation of the TCR-associated kinase Lck. The impact of TRAF3 on this very early signaling event led to the hypothesis that TRAF3 restrains one or both of two known inhibitors of Lck, C-terminal Src kinase (Csk) and protein tyrosine phosphatase N22 (PTPN22). TRAF3 associated with Csk, promoting the dissociation of Csk from the plasma membrane. TRAF3 also associated with and regulated the TCR/CD28 induced localization of PTPN22. Loss of TRAF3 resulted in increased amounts of both Csk and PTPN22 in T cell membrane fractions and decreased association of PTPN22 with Csk. These findings identify a new role for T cell TRAF3 in promoting T cell activation, by regulating localization and functions of early TCR signaling inhibitors.

The oncogenic membrane protein LMP1 sequesters TRAF3 in B-cell lymphoma cells to produce functional TRAF3 deficiency.

Loss-of-function mutations in genes encoding the signaling protein tumor necrosis factor receptor-associated factor 3 (TRAF3) are commonly found in human B-cell malignancies, especially multiple myeloma and B-cell lymphoma (BCL). B-cell TRAF3 deficiency results in enhanced cell survival, elevated activation receptor signaling, and increased activity of certain transcriptional pathways regulating expression of prosurvival proteins. A recent analysis of TRAF3 protein staining of ∼300 human BCL tissue samples revealed that a higher proportion of samples expressing the oncogenic Epstein-Barr virus-encoded protein latent membrane protein 1 (LMP1) showed low/negative TRAF3 staining than predicted. LMP1, a dysregulated mimic of the CD40 receptor, binds TRAF3 more effectively than CD40. We hypothesized that LMP1 may sequester TRAF3, reducing its availability to inhibit prosurvival signaling pathways in the B cell. This hypothesis was addressed via 2 complementary approaches: (1) comparison of TRAF3-regulated activation and survival-related events with relative LMP1 expression in human BCL lines and (2) analysis of the impact upon such events in matched pairs of mouse BCL lines, both parental cells and subclones transfected with inducible LMP1, either wild-type LMP1 or a mutant LMP1 with defective TRAF3 binding. Results from both approaches showed that LMP1-expressing B cells display a phenotype highly similar to that of B cells lacking TRAF3 genes, indicating that LMP1 can render B cells functionally TRAF3 deficient without TRAF3 gene mutations, a finding of significant relevance to selecting pathway-targeted therapies for B-cell malignancies.

Antigen-specific B-lymphocyte activation.

B lymphocytes comprise a major component of the adaptive mammalian immune system, having the exclusive ability to produce and secrete immunoglobulins (Ig) of various forms (isotypes). This property alone renders B-cell activation critical to immunity, but the fully activated B cell also functions in antigen presentation and the production of a variety of cytokines and chemokines. There are many signals that must be coordinated to achieve and properly regulate antigen-specific B-lymphocyte activation and the development of humoral memory. This review seeks to summarize these components, and to highlight recent advances in each area that contribute to a greater understanding of the complex processes involved in B-cell activation.

Mechanisms of TNF receptor-associated factor (TRAF) regulation in B lymphocytes.

A key component of signaling by members of the tumor necrosis factor receptor (TNF-R) family is interaction with the cytoplasmic adapter proteins known as TRAFs. Several proteins encoded by microbes also interact with TRAFs. A notable example is the CD40 receptor, expressed on antigen presenting cells and providing key activation signals in T cell-dependent B cell activation. CD40 signals to B cells are mimicked by a constitutively active viral protein produced by the Epstein-Barr virus. For each of these receptors, multiple mechanisms of TRAF regulation contribute to signal transduction and the ultimate effect on the B cell. Recent findings concerning these regulatory mechanisms are summarized in this overview.

CD40-Mediated Activation of the NF-κB2 Pathway.

CD40 is a critical stimulatory receptor on antigen-presenting cells of the immune system. CD40-mediated activation of B cells is particularly important for normal humoral immune function. Engagement of CD40 by its ligand, CD154, on the surface of activated T cells initiates a variety of signals in B cells including the activation of MAP kinases and NF-κB. The transcriptional regulator NF-κB is in reality a family of factors that can promote B cell activation, differentiation, and proliferation. Complex - and only partially understood - biochemical mechanisms allow CD40 to trigger two distinct NF-κB activation pathways resulting in the activation of canonical (NF-κB1) and non-canonical (NF-κB2) NF-κB. This brief review provides a summary of mechanisms responsible for activation of the latter, which appears to be particularly important for enhancing the viability of B cells at various stages in their life cycle and may also contribute to the development of B cell malignancies. CD40 is also expressed by various cell types in addition to B cells, including T cells, macrophages, dendritic cells, as well as certain non-hematopoietic cells. Here too, while perhaps less extensively studied than in B cells, the CD40-mediated activation of NF-κB2 also appears to have important roles in cellular physiology.

Sedimentation and immunoprecipitation assays for analyzing complexes that repress transcription.

Co-repressor proteins function as platforms for the assembly of multi-subunit complexes that mediate transcriptional repression. Common components of such complexes are histone deacetylases, which catalyze the removal of acetyl groups from the tails of histones within nucleosomes, resulting in chromatin compaction and gene repression. In addition, co-repressor complexes generally interact with sequence-specific DNA-binding proteins that direct association with regulatory elements in the genome. Thus, identifying proteins that stably associate with co-repressors can provide insights regarding the biochemical function and target gene specificity of these molecules. Here, we describe a density gradient fractionation method for determining whether a co-repressor is incorporated into high-molecular complexes within cells and for identifying potential constituents of these complexes. We also describe a co-immunoprecipitation assay for confirming and further studying interactions between co-repressors and other proteins that may represent functional partners.

HOIL-1L interacting protein (HOIP) is essential for CD40 signaling.

CD40 is a cell surface receptor important in the activation of antigen-presenting cells during immune responses. In macrophages and dendritic cells, engagement of CD40 by its ligand CD154 provides signals critical for anti-microbial and T cell-mediated immune responses, respectively. In B cells, CD40 signaling has a major role in regulating cell proliferation, antibody production, and memory B cell development. CD40 engagement results in the formation of a receptor-associated complex that mediates activation of NF-κB, stress-activated protein kinases, and other signaling molecules. However, the mechanisms that link CD40 to these signaling events have been only partially characterized. Known components of the CD40 signaling complex include members of the TNF receptor-associated factor (TRAF) family of proteins. We previously showed that the TRAF family member TRAF2 mediates recruitment of HOIL-1L-interacting protein (HOIP) to the cytoplasmic domain of CD40, suggesting that HOIP has a role in the CD40 signaling pathway. To determine the role of HOIP in CD40 signaling, we used somatic cell gene targeting to generate mouse B cell lines deficient in HOIP. We found that the CD40-induced upregulation of CD80 and activation of germline immunoglobulin epsilon transcription were defective in HOIP-deficient cells. We also found that the CD40-mediated activation of NF-κB and c-Jun kinase was impaired. Recruitment of IκB kinase proteins to the CD40 signaling complex was undetectable in HOIP-deficient cells, potentially explaining the defect in NF-κB activation. Restoration of HOIP expression reversed the defects in cellular activation and signaling. These results reveal HOIP as a key component of the CD40 signaling pathway.

Fyn binds to and phosphorylates T cell immunoglobulin and mucin domain-1 (Tim-1).

The gene encoding T cell immunoglobulin and mucin domain-1 (Tim-1) is linked to atopy and asthma susceptibility in mice and humans. Tim-1 is a transmembrane protein expressed on activated lymphocytes and appears to have a role as a co-stimulatory receptor in T cells. The protein has not been shown to have enzymatic activity but contains a site within its cytoplasmic tail predicted to be a target for tyrosine kinases. Here, we show that Tim-1 can associate with the kinase Fyn, a member of the Src family of tyrosine kinases. This association does not require Fyn's kinase activity and is independent of the phosphorylation of a conserved tyrosine present within the cytoplasmic tail of Tim-1. Fyn is necessary for phosphorylation of this tyrosine in Tim-1 and the phosphorylation of Tim-1 varies with the levels of Fyn present in cells. These data suggest a role for Fyn in the signaling downstream of Tim-1.