A TNF family member LIGHT transduces costimulatory signals into human T cells

DcR3/TR6 is a secreted protein belonging to the TNFR family. It binds to Fas ligand, LIGHT, and TL1A, all of which are TNF family members. LIGHT is expressed on activated T cells. Its known receptors are TR2 and LTbetaR on the cell surface, and TR6 in solution. In the present study, we report soluble TR6-Fc or solid-phase TR6-Fc costimulated proliferation, lymphokine production, and cytotoxicity of human T cells in the presence of TCR ligation. These costimulating effects were blocked by soluble LIGHT but not by soluble Fas-Fc. TR6-Fc could also effectively costimulate gld/gld mouse T cells. We further demonstrated that TR6 bound to both Th1 and Th2 cells, according to flow cytometry, and that the association was inhibited by soluble LIGHT. Cross-linking Th1 and Th2 cells with solid-phase TR6-Fc along with a suboptimal concentration of anti-CD3 enhanced proliferation of both Th1 and Th2 cells, and augmented Th1 but not Th2 lymphokine production. These data suggest that TR6 delivers costimulation through its ligand(s) on the T cell surface, and at least the major part of such costimulation is via LIGHT.     

DcR3/TR6 modulates immune cell interactions

DcR3/TR6, a secreted protein, is a member of TNF receptor family. Its ligands include FasL, LIGHT, and TL1A, all TNF family members. TR6 can interfere with FasL- or LTbetaR-mediated apoptosis; it can also inhibit T-cell costimulation by blocking the two-way signaling between TR2 and LIGHT, and the one-way signaling from TL1A to DR3. In this study, we discovered that TR6 was secreted by peripheral blood mononuclear cells (PBMC) stimulated by T-cell mitogens. It inhibited actin polymerization of T cells upon mitogen stimulation, and repress T-cell pseudopodium formation, which is known to be important for cell-cell interaction. As a consequence, T-cell aggregation stimulated by alloantigens, anti-CD3 or PHA was suppressed by either soluble or solid phase TR6-Fc. This result suggests that TR6 might regulate T-cell interaction with other cells such as antigen-presenting cells (APC) or their fellow T cells by preventing them from forming inseparable cell clusters, which are undesirable for the progression of immune responses.     

Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.     

A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis.

TR6 (decoy receptor 3 (DcR3)) is a new member of the tumor necrosis factor receptor (TNFR) family. TR6 mRNA is expressed in lung tissues and colon adenocarcinoma, SW480. In addition, the expression of TR6 mRNA was shown in the endothelial cell line and induced by phorbol 12-myristate 13-acetate/ionomycin in Jurkat T leukemia cells. The open reading frame of TR6 encodes 300 amino acids with a 29-residue signal sequence but no transmembrane region. Using histidine-tagged recombinant TR6, we screened soluble forms of TNF-ligand proteins with immunoprecipitation. Here, we demonstrate that TR6 specifically binds two cellular ligands, LIGHT (herpes virus entry mediator (HVEM)-L) and Fas ligand (FasL/CD95L). These bindings were confirmed with HEK 293 EBNA cells transfected with LIGHT cDNA by flow cytometry. TR6 inhibited LIGHT-induced cytotoxicity in HT29 cells. It has been shown that LIGHT triggers apoptosis of various tumor cells including HT29 cells that express both lymphotoxin beta receptor (LTbetaR) and HVEM/TR2 receptors. Our data suggest that TR6 inhibits the interactions of LIGHT with HVEM/TR2 and LTbetaR, thereby suppressing LIGHT- mediated HT29 cell death. Thus, TR6 may play a regulatory role for suppressing in FasL- and LIGHT-mediated cell death.     

Tumor vaccine based on cell surface expression of DcR3/TR6

DcR3/TR6, a secreted protein belonging to the TNF receptor superfamily, interacts with lymphotoxin-like, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entrance mediator (LIGHT), Fas ligand (FasL), and TL1A, all members of the TNF superfamily. Solid-phase TR6 can trigger reverse signaling of LIGHT and FasL expressed on T cells, and lead to T cell costimulation. In this study, we engineered tumor cells to express cell surface TR6 and used these cells as a tumor vaccine. We demonstrated that mastocytoma P815 cells expressing surface TR6 (TR6-P815) effectively augmented the T cells response in vitro and ex vivo in terms of proliferation, as well as IL-2 and IFN-gamma secretion. TR6-P815 cells had reduced tumorigenicity compared with parental P815 cells. When inactivated TR6-P815 cells were employed as a vaccine, they protected the mice from challenge with live parental P815 cells, and eliminated established P815 tumors. The cell surface TR6-based tumor vaccine was also effective against low antigenicity tumors, such as B16 melanoma; co-administration of bacillus Calmette-Guérin further enhanced the vaccine's efficacy. Thus, cell surface TR6 expression is a useful addition to our tumor vaccine arsenal.     

Constitutive expression of LIGHT on T cells leads to lymphocyte activation, inflammation, and tissue destruction.

LIGHT, a member of the TNF family of cytokines (homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator, a receptor expressed on T cells), is induced on activated T cells and mediates costimulatory and antitumor activity in vitro. Relatively little information is available on the in vivo effects of LIGHT expression, particularly within the T cell compartment. In this work, we describe transgenic mice that express human LIGHT under the control of the CD2 promoter, resulting in constitutive transgene expression in cells of the T lymphocyte lineage. LIGHT-transgenic animals exhibit abnormalities in both lymphoid tissue architecture and the distribution of lymphocyte subsets. They also show signs of inflammation that are most severe in the intestine, along with tissue destruction of the reproductive organs. These LIGHT-mediated effects were recapitulated when immune-deficient mice were reconstituted with bone marrow from LIGHT-transgenic donor mice. T cells in the LIGHT-transgenic mice have an activated phenotype and mucosal T cells exhibit enhanced Th1 cytokine activity. The results indicate that LIGHT may function as an important regulator of T cell activation, and implicate LIGHT signaling pathways in inflammation focused on mucosal tissues.     

LIGHT expression by mucosal T cells may regulate IFN-gamma expression in the intestine

The TNF superfamily of cytokines play an important role in T cell activation and inflammation. Sustained expression of lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T cells (LIGHT) (TNFSF14) causes a pathological intestinal inflammation when constitutively expressed by mouse T cells. In this study, we characterized LIGHT expression on activated human T cell subsets in vitro and demonstrated a direct proinflammatory effect on regulation of IFN-gamma. LIGHT was induced in memory CD45RO CD4+ T cells and by IFN-gamma-producing CD4+ T cells. Kinetic analysis indicated rapid induction of LIGHT by human lamina propria T cells, reaching maximal levels by 2-6 h, whereas peripheral blood or lymph node-derived T cells required 24 h. Further analysis of intestinal specimens from a 41 patient cohort by flow cytometry indicated membrane LIGHT induction to higher peak levels in lamina propria T cells from the small bowel or rectum but not colon, when compared with lymph node or peripheral blood. Independent stimulation of the LIGHT receptor, herpesvirus entry mediator, induced IFN-gamma production in lamina propria T cells, while blocking LIGHT inhibited CD2-dependent induction of IFN-gamma synthesis, indicating a role for LIGHT in the regulation of IFN-gamma and as a putative mediator of proinflammatory T-T interactions in the intestinal mucosa. Taken together, these findings suggest LIGHT-herpesvirus entry mediator mediated signaling as an important immune regulatory mechanism in mucosal inflammatory responses.     

LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response

LIGHT is a recently identified member of the TNF superfamily and its receptors, herpesvirus entry mediator and lymphotoxin beta receptor, are found in T cells and stromal cells. In this study, we demonstrate that LIGHT is selectively expressed on immature dendritic cells (DCs) generated from human PBMCs. In contrast, LIGHT is not detectable in DCs either freshly isolated from PBMCs or rendered mature in vitro by LPS treatment. Blockade of LIGHT by its soluble receptors, lymphotoxin beta receptor-Ig or HVEM-Ig, inhibits the induction of DC-mediated primary allogeneic T cell response. Furthermore, engagement of LIGHT costimulates human T cell proliferation, amplifies the NF-kappaB signaling pathway, and preferentially induces the production of IFN-gamma, but not IL-4, in the presence of an antigenic signal. Our results suggest that LIGHT is a costimulatory molecule involved in DC-mediated cellular immune responses.     

The effect of LIGHT in inducing maturation of monocyte-derived dendritic cells from MDS patients

LIGHT is a recently cloned novel cytokine belonging to the TNF family that is selectively expressed on immature dendritic cells (iDCs) generated from monocytes isolated from human PBMCs. In these studies, we demonstrate that exogenous soluble LIGHT or soluble CD40 ligand (CD40L) can promote monocyte-derived dendritic cell maturation in vitro by the up-regulation of CD86, CD80, CD83, and HLA-DR antigen expression. Immature dendritic cells differentiated from monocytes of MDS patients displayed lower levels of costimulatory and HLA-DR molecules compared with iDCs differentiated from monocytes of normal subjects. However, upon induction of maturation by LIGHT or CD40L, the expression of costimulatory and HLA-DR molecules is comparable between DCs from MDS and normal subjects. Exogenous LIGHT- and CD40L-stimulated mature DCs (mDCs) also displayed increased antigen presentation to autologous T lymphocytes (tetanus toxin) or allogeneic T lymphocytes in mixed lymphocyte reactions. DCs matured by LIGHT showed increased secretion of IL-6, IL-12p75, and TNF-, but not IL-1. We conclude that both LIGHT and CD40L are immunoregulating factors that induce monocyte-derived iDCs from MDS patients to undergo maturation resulting in increased antigen presentation and T-cell activation. Monocyte-derived DCs can be stimulated to undergo phenotypic and functional changes with LIGHT that might be applied in the development of a DC-based vaccine for MDS treatment.     

Immunotherapeutic targeting of LIGHT/LTβR/HVEM pathway fully recapitulates the reduced cytotoxic phenotype of LIGHT-deficient T cells

Tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily members play essential roles in the development of the different phases of the immune response. Mouse LIGHT (TNFSF14) is a type II transmembrane protein with a C-terminus extracellular TNF homology domain (THD) that assembles in homotrimers and regulates the course of the immune responses by signaling through 2 receptors, the herpes virus entry mediator (HVEM, TNFSFR14) and the lymphotoxin β receptor (LTβR, TNFSFR3). LIGHT is a membrane-bound protein transiently expressed on activated T cells, natural killer (NK) cells and immature dendritic cells that can be proteolytically cleaved by a metalloprotease and released to the extracellular milieu. The immunotherapeutic potential of LIGHT blockade was evaluated in vivo. Administration of an antagonist of LIGHT interaction with its receptors attenuated the course of graft-versus-host reaction and recapitulated the reduced cytotoxic activity of LIGHT-deficient T cells adoptively transferred into non-irradiated semiallogeneic recipients. The lack of LIGHT expression on donor T cells or blockade of LIGHT interaction with its receptors slowed down the rate of T cell proliferation and decreased the frequency of precursor alloreactive T cells, retarding T cell differentiation toward effector T cells. The blockade of LIGHT/LTβR/HVEM pathway was associated with delayed downregulation of interleukin-7Rα and delayed upregulation of inducible costimulatory molecule expression on donor alloreactive CD8 T cells that are typical features of impaired T cell differentiation. These results expose the relevance of LIGHT/LTβR/HVEM interaction for the potential therapeutic control of the allogeneic immune responses mediated by alloreactive CD8 T cells that can contribute to prolong allograft survival.     

Modulation of Ia-like antigen expression and antigen-presenting activity of human monocytes by endotoxin and zymosan A

The environmental agents E. coli endotoxin and zymosan A modulated antigen-specific T cell proliferation in vitro, assessed by 3H-TdR uptake. In the continual presence of these agents, human mononuclear leukocyte responses to the antigens tuberculin PPD, Candida albicans, and mumps were significantly reduced. Treatment of adherent cell-depleted T cells with the agents did not affect their subsequent reactivity to soluble antigens in the presence of normal M phi. However, cultures consisting of pretreated M phi, normal T cells, and soluble antigen gave responses that were only 7 to 38% of control values, indicating that the function of the antigen-presenting cell, not the T cell, was inhibited. This effect was observed only when treatment with endotoxin or zymosan A preceded antigen stimulation by at least 24 hr, suggesting that a gradual inhibition of antigen presentation had occurred. When various ratios of normal antigen-pulsed and agent-treated M phi were cultured with normal T cells, antigen-specific responses were not significantly different from control cultures; this indicated that M phi-mediated suppression was not involved. It did not appear that the inhibition was due to enhanced antigen degradation by the treated M phi because responses were not reconstituted in the presence of excess antigen. After endotoxin or zymosan A treatment of the M phi population the proportion of Ia+ cells was reduced significantly, and surface expression of Ia antigen correlated with the ability of the cell population to present antigens to immune T cells. This suggested that endotoxin and zymosan A induce a loss of surface Ia antigen on antigen-presenting cells that inhibits immune T cell activation.     

LIGHT regulates CD86 expression on dendritic cells through NF-kappaB, but not JNK/AP-1 signal transduction pathway

The members of the tumor necrosis factor (TNF) family play pivotal roles in the regulation of the immune system. LIGHT is a type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. This cytokine has been extensively studied for its role in T cell regulation. Recently, we identified its role in inducing maturation of dendritic cells, such as LIGHT upregulated CD86 expression on dendritic cells in our previous report. However, the signal transduction pathway on this regulation remains unknown. In this study, we found that LIGHT activated NF-kappaB, p44/42 MAPK, but not JNK. LIGHT upregulates CD86 expression on DCs through activation of NF-kappaB, but not p44/42 signal pathway, because inhibition of NF-kappaB activity by its inhibitor could blunt the effect of LIGHT in up-regulation of CD86 expression, but neither inhibitor of p44/42 MAPK nor JNK inhibitor has this effect. Thus we demonstrate that LIGHT regulates CD86 expression through NF-kappaB signal transduction pathway but neither p44/42 MAPK nor JNK/AP-1 signaling pathway. We conclude that NF-kappaB signal plays a key role in LIGHT-mediated upregulation of CD86 expression.     

The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.     

Antigen-specific blockade of T cells in vivo using dimeric MHC peptide

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR zeta-chain and recruitment and phosphorylation of zeta-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.     

Trimerization of murine TNF ligand family member LIGHT increases the cytotoxic activity against the FM3A mammary carcinoma cell line

LIGHT is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. Recombinant soluble human LIGHT produced by mammalian cells or Escherichia coli is functional at nanomolar concentrations. However, there is little information about the biological activity of mouse LIGHT (mLIGHT) because of the difficulty in producing bioactive soluble mLIGHT. In this study, recombinant trimeric soluble mLIGHT, or Foldon-mLIGHT, was produced by fusing mLIGHT with the trimerization domain foldon from bacteriophage T4 fibritin. Foldon-mLIGHT was secreted from 293F cells as a 68-kDa trimeric protein. The recombinant protein potently inhibited the growth of the FM3A mouse mammary carcinoma cell line with an IC₅₀ of 77 pM; however, the monomer or dimer forms of mLIGHT produced by E. coli or mammalian cell systems showed weak or no inhibitory activity. These data clearly indicated that trimerization of soluble mLIGHT is essential for its biological activity.     

B lymphocyte regulation of the immune system. I. In vivo biologic activity of the novel lymphokine, B cell-derived enhancing factor (BEF)

B cell-derived enhancing factor (BEF) is a lymphokine of B cell origin which was originally identified and characterized by its ability to enhance in vitro antibody responses, an effect shown to be due to the ability of BEF to reduce the activation of suppressor T cells. The present study was undertaken to determine whether BEF could also be active in modulating antibody responses in vivo. The data presented here demonstrate that BEF is biologically active in vivo, as manifested by significantly enhanced primary IgM and IgG antibody responses in mice that were either injected with BEF prepared exogenously or implanted with growing BEF-secreting cells of a B cell line. Moreover, BEF was shown to enhance subsequent development of immunologic memory in mice pretreated with BEF at the time of primary immunization; these mice then displayed enhanced secondary responses when challenged with the same antigen some weeks later. The mechanism by which BEF exerts biologic activities to positively modulate in vivo antibody responses and immunologic memory reflects the ability of BEF to modulate one or more T cell functions, as evidenced by the following findings. 1) Transient in vitro exposure to BEF of T cells, but not of B cells, endowed such cells with the capacity to adoptively transfer enhanced primary antibody responses to irradiated recipients. 2) Utilizing adoptive in vivo antibody responses, in which fractionated B cell or T cell populations were obtained from BEF-pretreated mice, revealed that one effect of BEF which results in enhanced immunologic memory is related to its activity on T cells during the priming phase of the immune response. Finally, the existence of this B cell-derived lymphokine and the demonstration of its in vivo regulatory effects on the immune system provide yet another example of the emerging biologic importance of B lymphocytes in the overall regulation of the immune system.     

The critical role of LIGHT, a TNF family member, in T cell development.

Negative selection refers to the selective deletion of autoreactive thymocytes but its molecular events have not been well defined. In this study, we demonstrate that a cellular ligand for herpes virus entry mediator and lymphotoxin receptor (LIGHT), a newly identified member of the TNF superfamily, may play a critical role in negative selection. Using TCR transgenic mice, we find that the blockade of LIGHT signaling in vitro and in vivo prevents negative selection induced by peptide and intrathymically expressed Ags, resulting in the rescue of thymocytes from apoptosis. Furthermore, the thymi of LIGHT transgenic mice show severe atrophy with remarkably reduced CD4(+)CD8(+) double-positive cells caused by increased apoptosis, suggesting that LIGHT can delete immature T cells in vivo. Taken together, these results demonstrate a critical role of LIGHT in thymic negative selection of the T cell repertoire.     

LIGHT Is critical for IL-12 production by dendritic cells, optimal CD4+ Th1 cell response, and resistance to Leishmania major

Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4(+) Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT(-/-) mice were severely impaired in IL-12p40 production following IFN-gamma and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT beta receptor (LTbetaR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-gamma production in vivo. In an infection model, injection of HVEM-Ig or LTbetaR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-gamma production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTbetaR-Ig was also reproduced in LIGHT(-/-) --> RAG1(-/-) chimeric mice. In contrast, L. major-infected LTbeta(-/-) mice do not develop acute disease, suggesting that the effect of LTbetaR-Ig is not due to blockade of membrane LT (LTalpha1beta2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-gamma-producing CD4(+) Th1 cells and its blockade results in severe susceptibility to Leishmania major.