CD154 (CD40 ligand)

CD40 ligand, a type II transmembrane protein recently renamed CD154, was originally considered restricted to activated T lymphocytes, functioning as a mediator of T cell-dependent B cell activation, proliferation, and differentiation. However, the spectrum of CD154 expression and function has broadened considerably during recent years, establishing new roles as a central mediator of immunity and inflammation for this member of the tumor necrosis factor (TNF) gene superfamily. The emerging picture indicates that ligation of the receptor CD40 via CD154, most potently in its trimeric form, functions in two ways. CD154 modulates physiologic processes, such as T cell-mediated effector functions and general immune responses required for appropriate host defense, but also triggers the expression of pro-inflammatory mediators, such as cytokines, adhesion molecules, and matrix degrading activities, all of which are associated with the pathogenesis of chronic inflammatory diseases, e.g., autoimmune disorders, arthritis, atherosclerosis, and cancer. Accordingly, CD40/CD154 interactions have advanced as a potential therapeutic target for these diseases, whereby two opposing strategies, interruption as well as enhancement of CD40 signaling, are explored for beneficial outcomes.     

The TNF receptor and Ig superfamily members form an integrated signaling circuit controlling dendritic cell homeostasis

Dendritic cells (DC) constitute the most potent antigen presenting cells of the immune system, playing a key role bridging innate and adaptive immune responses. Specialized DC subsets differ depending on their origin, tissue location and the influence of trophic factors, the latter remain to be fully understood. Myeloid-associated lymphotoxin-beta receptor (LTbetaR) signaling is required for the local proliferation of lymphoid tissue DC. This review focuses on the LTbetaR signaling cascade as a crucial positive trophic signal in the homeostasis of DC subsets. The noncanonical coreceptor pathway comprised of the immunoglobulin (Ig) superfamily member, B and T lymphocyte attenuator (BTLA) and TNFR superfamily member, herpesvirus entry mediator (HVEM) counter regulates the trophic signaling by LTbetaR. Together both pathways form an integrated signaling circuit achieving homeostasis of DC subsets.     

Delineation of a novel pathway that regulates CD154 (CD40 ligand) expression

The expression of CD154 (CD40 ligand) by activated T lymphocytes plays a central role in humoral and cellular immunity. The fundamental importance of this protein in mounting an immune response has made it an attractive target for immunomodulation. Several studies have demonstrated that CD154 expression is regulated at the level of mRNA turnover in a manner distinct from other cytokine genes. We have purified, sequenced, and characterized the two major proteins that bind the CD154 3' untranslated region (3'UTR) as members of the polypyrimidine tract binding protein (PTB) family. One of these proteins is a previously unreported alternatively spliced PTB isoform, which we call PTB-T. These proteins interact with a polypyrimidine-rich region within the CD154 3'UTR that lacks any known cis-acting instability elements. The polypyrimidine-rich region of the CD154 3'UTR was both necessary and sufficient to mediate changes in reporter gene expression and mRNA accumulation, indicating the presence of a novel cis-acting instability element. The presence of a cis-acting instability element in the polypyrimidine-rich region was confirmed using a tetracycline-responsive reporter gene approach. The function of this cis-acting element appears to be dependent on the relative cytoplasmic levels of PTB and PTB-T. Cotransfection of vectors encoding PTB-T consistently decreased the CD154 3'UTR-dependent luciferase expression. In contrast, transfection of plasmids encoding PTB tended to increase CD154 3'UTR-dependent luciferase expression. Thus, the CD154 3'UTR contains a novel cis-acting element whose function is determined by the binding of PTB and PTB-T. These data identify a specific pathway that regulates CD154 expression that can potentially be selectively targeted for the treatment of autoimmune disease and allograft rejection.     

Differential induction of cytokine genes and activation of mitogen-activated protein kinase family by soluble CD40 ligand and TNF in a human follicular dendritic cell line.

Follicular dendritic cells (FDC)3 play crucial roles in germinal center (GC) formation and differentiation of GC B cells. Many aspects of FDC function are influenced by contact with B or T cells, and by cytokines produced in the GC, which involve stimulation of CD40 and TNF-alpha receptors on FDC. In this study, using an established FDC line, HK cells, we compared the effects of CD40 and TNF receptor triggering on cytokine induction and activation of mitogen-activated protein kinase family. We show that HK cells spontaneously produced IL-6, M-CSF, and G-CSF mRNA. Both the soluble form of CD40 ligand (sCD40L) and TNF increased the level of M-CSF and G-CSF mRNA. While TNF strongly induced IL-6 mRNA, its expression was not affected by sCD40L treatment, differing from the strong IL-6 induction in other cell types upon CD40 stimulation. In addition, sCD40L treatment resulted in activation of extracellular signal-related kinase 1 and 2 (ERK1/2) and p38 without significant increase in c-Jun N-terminal kinase (JNK) activity. Lack of JNK activation differs in that most B cells respond to CD40 stimulation by inducing JNK activity strongly, suggesting distinct characteristics of CD40 signaling in FDC. Compared with the effects of sCD40L, TNF was capable of inducing JNK activity in addition to the activation of ERK1/2 and p38. Furthermore, the proximal signaling elements activated by TNF differed from those activated by sCD40L, in that TNF did not require PMA-sensitive protein kinase C isoforms in the activation of ERK and p38, whereas sCD40L did. However, signals activated by these stimuli converged on cytokine gene expression in a synergistic manner, which may have implication in augmenting FDC function during GC reaction.     

Quantitative contributions of TNF receptor superfamily members to CD8+ T‐cell responses

T‐cell responses to infections and cancers are regulated by co‐signalling receptors grouped into the binary categories of co‐stimulation or co‐inhibition. The co‐stimulation TNF receptor superfamily (TNFRSF) members 4‐1BB, CD27, GITR and OX40 have similar signalling mechanisms raising the question of whether they have similar impacts on T‐cell responses. Here, we screened for the quantitative impact of these TNFRSFs on primary human CD8⁺ T‐cell cytokine production. Although both 4‐1BB and CD27 increased production, only 4‐1BB was able to prolong the duration over which cytokine was produced, and both had only modest effects on antigen sensitivity. An operational model explained these different phenotypes using shared signalling based on the surface expression of 4‐1BB being regulated through signalling feedback. The model predicted and experiments confirmed that CD27 co‐stimulation increases 4‐1BB expression and subsequent 4‐1BB co‐stimulation. GITR and OX40 displayed only minor effects on their own but, like 4‐1BB, CD27 could enhance GITR expression and subsequent GITR co‐stimulation. Thus, different co‐stimulation receptors can have different quantitative effects allowing for synergy and fine‐tuning of T‐cell responses.     

Oxidized phospholipids negatively regulate dendritic cell maturation induced by TLRs and CD40

Maturation of dendritic cells (DCs) induced by pathogen-derived signals via TLRs is a crucial step in the initiation of an adaptive immune response and therefore has to be well controlled. In this study, we demonstrate that oxidized phospholipids (ox-PLs), which are generated during infections, apoptosis, and tissue damage, interfere with DC activation, preventing their maturation. ox-PLs blocked TLR-3- and TLR-4-mediated induction of the costimulatory molecules CD40, CD80, CD83, and CD86, the cytokines IL-12 and TNF, as well as lymphocyte stimulatory capacity. CD40 and TLR-2-mediated cytokine production was also inhibited, whereas up-regulation of costimulatory molecules via these receptors was not affected by ox-PLs. Thus, formation of ox-PLs during the course of an inflammatory response may represent a negative-feedback loop preventing excessive and sustained immune reactions through regulating DC maturation.     

TNF receptor 1 mediates dendritic cell maturation and CD8 T cell response through two distinct mechanisms

TNF-α and its two receptors (TNFR1 and 2) are known to stimulate dendritic cell (DC) maturation and T cell response. However, the specific receptor and mechanisms involved in vivo are still controversial. In this study, we show that in response to an attenuated mouse hepatitis virus infection, DCs fail to mobilize and up-regulate CD40, CD80, CD86, and MHC class I in TNFR1(-/-) mice as compared with the wild-type and TNFR2(-/-) mice. Correspondingly, virus-specific CD8 T cell response was dramatically diminished in TNFR1(-/-) mice. Adoptive transfer of TNFR1-expressing DCs into TNFR1(-/-) mice rescues CD8 T cell response. Interestingly, adoptive transfer of TNFR1-expressing naive T cells also restores DC mobilization and maturation and endogenous CD8 T cell response. These results show that TNFR1, not TNFR2, mediates TNF-α stimulation of DC maturation and T cell response to mouse hepatitis virus in vivo. They also suggest two mechanisms by which TNFR1 mediates TNF-α-driven DC maturation, as follows: a direct effect through TNFR1 expressed on immature DCs and an indirect effect through TNFR1 expressed on naive T cells.     

Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation

A number of mammalian antimicrobial proteins produced by neutrophils and cells of epithelial origin have chemotactic and activating effects on host cells, including cells of the immune system. Eosinophil granules contain an antimicrobial protein known as eosinophil-derived neurotoxin (EDN), which belongs to the RNase A superfamily. EDN has antiviral and chemotactic activities in vitro. In this study, we show that EDN, and to a lesser extent human pancreatic RNase (hPR), another RNase A superfamily member, activates human dendritic cells (DCs), leading to the production of a variety of inflammatory cytokines, chemokines, growth factors, and soluble receptors. Human angiogenin, a RNase evolutionarily more distant to EDN and hPR, did not display such activating effects. Additionally, EDN and hPR also induced phenotypic and functional maturation DCs. These RNases were as efficacious as TNF-alpha, but induced a different set of cytokine mediators. Furthermore, EDN production by human macrophages could be induced by proinflammatory stimuli. The results reveal the DC-activating activity of EDN and hPR and suggest that they are likely participants of inflammatory and immune responses. A number of endogenous mediators in addition to EDN have been reported to have both chemotactic and activating effects on APCs, and can thus amplify innate and Ag-specific immune responses to danger signals. We therefore propose these mediators be considered as endogenous multifunctional immune alarmins.     

Cooperation of TNF family members CD40 ligand,receptor activator of NF-kappa B ligand,and TNF-alpha in the activation of dendritic cells and the expansion of viral specific CD8+ T cell memory responses in HIV-1-infected and HIV-1-uninfected individuals

Members of the TNF superfamily have been shown to be instrumental in enhancing cell-mediated immune responses, primarily through their interactions with dendritic cells (DCs). We systematically evaluated the ability of three TNF superfamily molecules, CD40 ligand (CD40L), receptor activator of NF-kappaB ligand (RANKL), and TNF-alpha, to expand ex vivo EBV-specific CTL responses in healthy human individuals and ex vivo HIV-1-specific CTL responses in HIV-1-infected individuals. In both groups of individuals, we found that all three TNF family molecules could expand CTL responses, albeit at differing degrees. CD40L treatment alone was better than RANKL or TNF-alpha alone to mature DCs and to expand CTL. In healthy volunteers, TNF-alpha or RANKL could cooperate with CD40L to maximize the ability of DCs to expand virus-specific CTL responses. In HIV-1 infection, cooperative effects between TNF-alpha or RANKL in combination with CD40L were variable. TNF-alpha and RANKL cooperated with CD40L via differing mechanisms, i.e., TNF-alpha enhanced IL-12 production, whereas RANKL enhanced survival of CD40L-stimulated DCs. These findings demonstrate that optimal maturation of DCs requires multiple signals by TNF superfamily members that include CD40L. In HIV-1 infection, DCs may only require CD40L to maximally expand CTL. Finally, CTL responses were higher in CD4(+) T cell-containing conditions even in the presence of TNF family molecules, suggesting that CD4(+) T cells can provide help to CD8(+) T cells independently of CD40L, RANKL, or TNF-alpha.     

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity.     

Hyperexpression of CD40 ligand (CD154) in inflammatory bowel disease and its contribution to pathogenic cytokine production.

CD40 ligand (CD40L or CD154), a type II membrane protein with homology to TNF, is transiently expressed on activated T cells and known to be important for B cell Ig production and for activation and differentiation of monocytes and dendritic cells. Both Crohn's disease and ulcerative colitis are characterized by local production of cytokines such as TNF and by an influx of activated lymphocytes into inflamed mucosa. Herein, we investigated whether CD40L signaling participates in immune responses in these diseases. Our results demonstrated that CD40L was expressed on freshly isolated lamina propria T cells from these patients and was functional to induce IL-12 and TNF production by normal monocytes, especially after IFN-gamma priming. The inclusion of a blocking mAb to CD40L or CD40 in such cocultures significantly decreased monocyte IL-12 and TNF production. Moreover, lamina propria and peripheral blood T cells from these patients, after in vitro activation with anti-CD3, showed increased and prolonged expression of CD40L as compared with controls. Immunohistochemical analyses indicated that the number of CD40+ and CD40L+ cells was significantly increased in inflamed mucosa, being B cells/macrophages and CD4+ T cells, respectively. These findings suggest that CD40L up-regulation is involved in pathogenic cytokine production in inflammatory bowel disease and that blockade of CD40-CD40L interactions may have therapeutic effects for these patients.     

The role of CD40-CD154 interaction in cell immunoregulation

CD40, a member of the nerve growth factor/tumor necrosis factor receptor superfamily, and its ligand, CD154, play essential roles in cell immune responses. The results of many studies have indicated that CD40-CD154 interaction can upregulate costimulatory molecules, activate antigen-presenting cells (APCs), influence T-cell priming and T-cell-mediated effector functions as well as participate in the pathogenic processing of chronic inflammatory diseases, such as autoimmune diabetes, graft rejection, atherosclerosis, and cancer. Ligation of CD40 on cancer cells was also found to produce a direct growth-inhibitory effect through cell cycle blockage and/or apoptosis with no overt side effects on normal cells and treatment with CD154 can heighten tumor rejection immune response as well. However, systemic treatment with CD154 has some potential risks. Therefore, searching for efficient and safe strategies of CD154-based cancer therapy has been a hot topic in human cancer research. This review focuses on the latest discovered functions of CD40-CD154 interaction in cell immune responses and on the new findings of CD154-based human cancer therapy.     

Cutting edge: selective impairment of CD8+ T cell function in mice lacking the TNF superfamily member LIGHT

Interactions of LIGHT and its receptors, herpesvirus entry mediator on T cells and lymphotoxin beta receptor on stromal cells, are implicated in the regulation of lymphoid organogenesis, costimulation of T cells, and activation of dendritic cells. In this work we report that LIGHT-deficient mice had normal lymphoid organs with T cells and APCs that normally responded to Ag stimulation and normally stimulated T cells. Although the number of Vbeta8(+) T cells in naive LIGHT(+/+) and LIGHT(-/-) mice was identical, Vbeta8(+)CD8(+) T cell proliferation in response to staphylococcal enterotoxin B was significantly lower in LIGHT(-/-) mice. Consistently, induction and cytokine secretion of CD8(+) CTL to MHC class I-restricted peptide was also reduced in LIGHT(-/-) mice. However, the proliferative response of Vbeta8(+)CD4(+) T cells to staphylococcal enterotoxin B was comparable in LIGHT(-/-) and LIGHT(+/+) mice. Our results suggest that LIGHT is required for activation of normal CD8(+) T cells but not CD4(+) T cells.     

Cytotoxic activity of human dendritic cells is differentially regulated by double-stranded RNA and CD40 ligand

The main function of dendritic cells (DCs) is to induce adaptive immune response through Ag presentation and specific T lymphocyte activation. However, IFN-alpha- or IFN-gamma-stimulated CD11c+ blood DCs and IFN-beta-stimulated monocyte-derived DCs were recently reported to express functional TNF-related apoptosis-inducing ligand (TRAIL), suggesting that DCs may become cytotoxic effector cells of innate immunity upon appropriate stimulation. In this study, we investigate whether dsRNA and CD40 ligand (CD40L), that were characterized as potent inducers of DC maturation, could also stimulate or modulate DC cytotoxicity toward tumoral cells. We observed that dsRNA, but not CD40L, is a potent inducer of TRAIL expression in human monocyte-derived DCs. As revealed by cytotoxicity assays, DCs acquire the ability to kill tumoral cells via the TRAIL pathway when treated with dsRNA. More precisely, dsRNA is shown to induce IFN-beta synthesis that consecutively mediates TRAIL expression by the DCs. In contrast, we demonstrate that TRAIL expression in dsRNA- or IFN-alpha-treated DCs is potently inhibited after CD40L stimulation. Unexpectedly, CD40L-activated DCs still developed cytotoxicity toward tumoral cells. This latter appeared to be partly mediated by TNF-alpha induction and a yet unidentified pathway. Altogether, these results demonstrate that dsRNA and CD40L, that were originally characterized as maturation signals for DCs, also stimulate their cytotoxicity that is mediated through TRAIL-dependent or -independent mechanisms.     

Biophysical characterization of a soluble CD40 ligand (CD154) coiled-coil trimer: evidence of a reversible acid-denatured molten globule

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region.     

In vivo CD40-CD154 (CD40 ligand) interaction induces integrated HIV expression by APC in an HIV-1-transgenic mouse model

Because of their relative resistance to viral cytopathic effects, APC can provide an alternative reservoir for latently integrated HIV. We used an HIV-transgenic mouse model in which APC serve as the major source of inducible HIV expression to study mechanisms by which integrated virus can be activated in these cells. When admixed with transgenic APC, activated T lymphocytes provided a major contact-dependent stimulus for viral protein expression in vitro. Using blocking anti-CD154 mAb as well as CD154-deficient T cells, the HIV response induced by activated T lymphocytes was demonstrated to require CD40-CD154 interaction. The role of this pathway in the induction of HIV expression from APC in vivo was further studied in an experimental model involving infection of the HIV-transgenic mice with PLASMODIUM: chabaudi parasites. Enhanced viral production by dendritic cells and macrophages in infected mice was associated with up-regulated CD40 expression. More importantly, in vivo treatment with blocking anti-CD154 mAb markedly reduced viral expression in P. chabaudi-infected animals. Together, these findings indicate that immune activation of integrated HIV can be driven by the costimulatory interaction of activated T cells with APC. Because chronic T cell activation driven by coinfections as well as HIV-1 itself is a characteristic of HIV disease, this pathway may be important in sustaining viral expression from APC reservoirs.