CD161 DEFINES EFFECTOR T CELLS THAT EXPRESS LIGHT AND RESPOND TO TL1A-DR3 SIGNALING.

Expression of NK cell markers identifies pro-inflammatory T cell subsets in the liver and intestinal immune compartments. Specifically, CD161 is expressed on Th17 cells which play an important role in the regulation of mucosal inflammation. In this study, we characterized human peripheral blood CD161+ T cells as an effector population partially resembling a gut T cell phenotype. CD161+ CD4+ T cells express the gut-associated TNF family member, LIGHT, and respond to crosslinking of DR3, a receptor to another gut-associated cytokine, TL1A. Robust IFN-γ production in response to DR3 signaling correlated with enhanced expression of surface DR3 on CD161+ T cells and co-stimulation with IL12 and IL18. CD161+ T cell effector function was directly demonstrated by activation of responder monocytes in co-culture leading to CD40 upregulation and CD14 downregulation. CD161+ T cells reciprocally responded to activated monocytes, inducing expression of activation marker, CD69, and production of IL2 and IFN-γ, further demonstrating effective CD161+ T cell cross-talk with monocytes. Finally, CD161 defined a subset of T cells that co-express CD56, a second NK marker. Our findings implicate human CD161+ T cells in gut-associated signaling mechanisms, and suggest a monocyte mediated effector function in mucosal inflammation.

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.

Genomic characterization of LIGHT reveals linkage to an immune response locus on chromosome 19p13.3 and distinct isoforms generated by alternate splicing or proteolysis.

LIGHT is a member of the TNF cytokine superfamily that signals through the lymphotoxin (LT)beta receptor and the herpesvirus entry mediator. LIGHT may function as a costimulatory factor for the activation of lymphoid cells and as a deterrent to infection by herpesvirus, which may provide significant selective pressure shaping the evolution of LIGHT. Here, we define the molecular genetics of the human LIGHT locus, revealing its close linkage to the TNF superfamily members CD27 ligand and 4-1BB ligand, and the third complement protein (C3), which positions LIGHT within the MHC paralog on chromosome 19p13.3. An alternately spliced isoform of LIGHT mRNA that encodes a transmembrane-deleted form is detected in activated T cells and gives rise to a nonglycosylated protein that resides in the cytosol. Furthermore, membrane LIGHT is shed from the cell surface of human 293 T cells. These studies reveal new mechanisms involved in regulating the physical forms and cellular compartmentalization of LIGHT that may contribute to the regulation and biological function of this cytokine.

Virus targeting of the tumor necrosis factor superfamily.

Herpesviruses appear to peacefully coexist with their natural hosts, with infection typically manifested as a benign, but lifelong process. However, coexistence depends on active resistance by innate and specific immune defenses as revealed in the striking virulence of herpesviruses when immunity fails. This pattern of infection is characteristic of a viral pathogen, such as cytomegalovirus, that has evolved efficient strategies targeted at host defense systems. Targeting members of the tumor necrosis factor (TNF)/lymphotoxin (LT) superfamily of cytokines is a strategy found in all herpesviruses, which suggests the existence of an intimate evolutionary link in their host-parasite relationship. Here we examine some of the strategies used by herpesvirus that target members of the TNF superfamily and discuss a recent study that revealed a novel mechanism that links LT-related ligands and interferons (IFN) to the establishment of coexistence between herpesvirus and its host cell.

Lymphotoxins and cytomegalovirus cooperatively induce interferon-beta, establishing host-virus détente.

Tumor necrosis factor (TNF)-related cytokines regulate cell death and survival and provide strong selective pressures for viruses, such as cytomegalovirus (CMV), to evolve counterstrategies in order to persist in immune-competent hosts. Signaling by the lymphotoxin (LT)-beta receptor or TNF receptor-1, but not Fas or TRAIL receptors, inhibits the cytopathicity and replication of human CMV by a nonapoptotic, reversible process that requires nuclear factor kappa B (NF-kappa B)-dependent induction of interferon-beta (IFN-beta). Efficient induction of IFN-beta requires virus infection and LT signaling, demonstrating the need for both host and viral factors in the curtailment of viral replication without cellular elimination. LT alpha-deficient mice and LT beta R-Fc transgenic mice were profoundly susceptible to murine CMV infection. Together, these results reveal an essential and conserved role for LTs in establishing host defense to CMV.

Suppression by apoptotic cells defines tumor necrosis factor-mediated induction of glomerular mesangial cell apoptosis by activated macrophages.

Activated macrophages (M(phi)) isolated from inflamed glomeruli or generated by interferon-gamma and lipopolysaccharide treatment in vitro induce glomerular mesangial cell apoptosis by hitherto incompletely understood mechanisms. In this report we demonstrate that nitric oxide-independent killing of co-cultured mesangial cells by interferon-gamma/lipopolysaccharide-activated M(phi) is suppressed by binding/ingestion of apoptotic cells and is mediated by tumor necrosis factor (TNF). Thus, soluble TNF receptor-1 significantly inhibited induction of mesangial cell apoptosis by 1) rodent M(phi) in the presence of nitric oxide synthase inhibitors or 2) human M(phi), both situations in which nitric oxide release was minimal. Furthermore, murine TNF knockout M(phi) were completely unable to induce mesangial cell apoptosis in the presence of nitric oxide synthase inhibitors. We conclude that TNF-restricted M(phi)-directed apoptosis of glomerular mesangial cells can be down-regulated by M(phi) binding/ingestion of apoptotic cells, suggesting a new mechanism for negative feedback regulation of M(phi) controls on resident cell number at inflamed sites.

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells.

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.

Identification of tumor necrosis factor (TNF) amino acids crucial for binding to the murine p75 TNF receptor and construction of receptor-selective mutants.

The bioactivity of tumor necrosis factor (TNF) is mediated by two TNF receptors (TNF-Rs), more particularly TNF-RI and TNF-RII. Although human TNF (hTNF) and murine TNF (mTNF) are very homologous, hTNF binds only to mTNF-RI. By measuring the binding of a panel of mTNF/hTNF chimeras to both mTNF-R, we pinpointed the TNF region that mediates the interaction with mTNF-RII. Using site-specific mutagenesis, we identified amino acids 71-73 and 89 as the main interacting residues. Mutein hTNF-S71D/T72Y/H73 Delta/T89E interacts with both types of mTNF-R and is active in CT6 cell proliferation assays mediated by mTNF-RII. Mutein mTNF-D71S/Y72T/Delta 73H/E89T binds to mTNF-RI only and is no longer active on CT6 cells. However, the L929s cytotoxicity of this mutein (an effect mediated by mTNF-RI triggering) was also 100-fold lower than that of wild-type mTNF due to enhanced dissociation during incubation at subnanomolar concentrations. The additional mutation of amino acid 102, resulting in the mutein mTNF-D71S/Y72T/Delta 73H/E89T/P102Q, restored the trimer stability, which led to an enhanced specific activity on L929s cells. Hence the specific activity of a TNF species is governed not only by its receptor binding characteristics but also by its trimer stability after incubation at subnanomolar concentrations. In conclusion, the mutation of TNF amino acids 71-73, 89, and 102 is sufficient to obtain a mTNF mutein selective for mTNF-RI and a hTNF mutein that, unlike wild-type hTNF, also acts on mTNF-RII.

Hepatitis C virus core protein enhances FADD-mediated apoptosis and suppresses TRADD signaling of tumor necrosis factor receptor.

Hepatitis C virus (HCV) core protein has been shown to interact with the death domain (DD) of tumor necrosis factor receptor-1 (TNFR1). In this study, we further examined the interaction of the core protein with the signaling molecules of TNFR1, including FADD, TRADD, and TRAF2, in a human embryonic kidney cell line, HEK-293, that overexpresses the HCV core protein. This core protein-expressing cell line exhibited enhanced sensitivity to TNF-induced apoptosis. By in vitro binding and in vivo coimmunoprecipitation assays, we showed that the HCV core protein interacted with the DD of FADD and enhanced apoptosis induced by FADD overexpression. This enhancement could be blocked by a dominant-negative mutant of FADD. In contrast, the core protein did not directly interact with the DD of TRADD, but could disrupt the binding of TRADD to TNFR1. TRAF2 recruitment to the TNFR1 signaling complex was also disrupted by the core protein. Correspondingly, TRAF2-dependent activation of the protein kinase JNK was suppressed in the core protein-expressing cells. However, NF kappa B activation by TNF was not significantly altered by the HCV core protein, suggesting the existence of TRAF2-independent pathways for NF kappa B activation. These results combined indicate that the HCV core protein sensitizes cells to TNF-induced apoptosis primarily by facilitating FADD recruitment to TNFR1. The inhibition of JNK activation by the HCV core protein may also contribute to the increased propensity of cells for apoptosis. These results, in comparison with other published studies, suggest that the effects of the HCV core protein and their underlying mechanisms vary significantly among cells of different origins.

p53-dependent radiation-induced crypt intestinal epithelial cells apoptosis is mediated in part through TNF-TNFR1 system.

Radiation induces apoptosis of crypt intestinal epithelial cells (IEC) through a pathway that is largely dependent on p53. However, exactly how p53 mediates IEC apoptosis is unclear. Studies in vitro suggest that one mechanism by which p53 mediates apoptosis is through its ability to transactivate members of the TNF receptor family of 'Death Receptors'. Here, we examined the role of one of its member, TNF receptor type 1 (TNFR1), in an in vivo model of p53-dependent radiation-induced IEC apoptosis. We demonstrate that mice genetically engineered to be deficient in TNF receptor type 1 (TNFR1(-/-)) and mice injected with TNFR1-fusion chimeric protein (TNFR1-Fc; a competitive inhibitor of TNFR1) were partially protected (30-40%) from p53-dependent radiation-induced IEC apoptosis. However, we found no evidence to support the possibility p53 transcriptionally regulates the expression of TNFR1 nor increases the susceptibility of IEC to TNF-mediated apoptosis. Interestingly, we found that injection of TNF readily induced IEC apoptosis and that radiation induced a p53-dependent increase in the intestinal level of TNF. Furthermore, injection of a neutralizing anti-TNF mAb reduced p53-dependent radiation-induced IEC apoptosis by approximately 60%. Overall, these results suggest that p53-dependent radiation-induced IEC apoptosis is mediated in part through ability of p53 to regulate TNF, which subsequently induces IEC apoptosis through TNFR1.

A diverse family of proteins containing tumor necrosis factor receptor-associated factor domains.

We have identified three new tumor necrosis factor-receptor associated factor (TRAF) domain-containing proteins in humans using bioinformatics approaches, including: MUL, the product of the causative gene in Mulibrey Nanism syndrome; USP7 (HAUSP), an ubiquitin protease; and SPOP, a POZ domain-containing protein. Unlike classical TRAF family proteins involved in TNF family receptor (TNFR) signaling, the TRAF domains (TDs) of MUL, USP7, and SPOP are located near the NH(2) termini or central region of these proteins, rather than carboxyl end. MUL and USP7 are capable of binding in vitro via their TDs to all of the previously identified TRAF family proteins (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6), whereas the TD of SPOP interacts weakly with TRAF1 and TRAF6 only. The TD of MUL also interacted with itself, whereas the TDs of USP7 and SPOP did not self-associate. Analysis of various MUL and USP7 mutants by transient transfection assays indicated that the TDs of these proteins are necessary and sufficient for suppressing NF-kappaB induction by TRAF2 and TRAF6 as well as certain TRAF-binding TNF family receptors. In contrast, the TD of SPOP did not inhibit NF-kappaB induction. Immunofluorescence confocal microscopy indicated that MUL localizes to cytosolic bodies, with targeting to these structures mediated by a RBCC tripartite domain within the MUL protein. USP7 localized predominantly to the nucleus, in a TD-dependent manner. Data base searches revealed multiple proteins containing TDs homologous to those found in MUL, USP7, and SPOP throughout eukaryotes, including yeast, protists, plants, invertebrates, and mammals, suggesting that this branch of the TD family arose from an ancient gene. We propose the moniker TEFs (TD-encompassing factors) for this large family of proteins.

Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2.

Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors.

Human immunodeficiency virus type 1 Nef mediates sustained membrane expression of tumor necrosis factor and the related cytokine LIGHT on activated T cells.

Human immunodeficiency virus (HIV) Nef downregulates the antigen recognition molecules major histocompatibility complex class I and CD4. Downregulation of surface CD4 by Nef relies on the ability of this viral protein to redirect the endocytic machinery to CD4. However, by redirecting the endocytic machinery, Nef may affect the internalization rates of other proteins. Here we show that Nef simultaneously enhances surface expression of the effector cytokines tumor necrosis factor (TNF) and LIGHT, leading to enhanced cytokine activity. A dileucine motif in Nef, which is essential for CD4 downregulation and is involved in the recruitment of adapter protein complexes by Nef, was required to increase surface levels of both cytokines. The physiological impact of the Nef-mediated interference with endocytosis was demonstrated by the fact that a TNF-responsive T-cell line chronically infected with HIV produced higher levels of p24 viral protein following expression of a Nef-green fluorescent protein (GFP) fusion protein. This enhancement was dependent on the levels of membrane-bound TNF, since it was abrogated by a recombinant soluble TNF receptor. Expression of Nef-GFP in human 293T cells reduced the endocytosis of LIGHT, whereas at the same time CD4 internalization was accelerated. Taken together, these results suggest that in infected cells Nef interferes with the internalization of these effector cytokines. By increasing TNF expression, Nef could accelerate disease progression in infected individuals. These findings may help explain the pleiotropic functions that Nef plays during infection and disease.

Expression of lymphotoxins and their receptor-Fc fusion proteins by baculovirus.

The tumor necrosis factor (TNF) cytokine and receptor superfamily plays critical roles in immune physiology. Several members of this family, such as the lymphotoxins (LT alpha and LT beta), Fas ligand, and TNF, induce cell death in some normal and transformed cells, but also induce cell growth and differentiation. The receptors for these ligands, when expressed as fusion proteins with the Fc region of IgG, function as potent antagonists of biological activity. The receptor-Fc fusion protein is a highly versatile reagent that can be utilized in virtually all the formats designed for antibodies. In this chapter we describe the expression, purification, and assays for lymphotoxins and their receptors, using a recombinant baculovirus system.

Membrane lymphotoxin is required for the development of different subpopulations of NK T cells.

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTalpha and LTbeta, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTalpha-/- and LTbeta-/- mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTalpha-/- and LTbeta-/- mice did not respond to the lipoglycan alpha-galactosylceramide, which is presented by mouse CD1 to Valpha14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and alpha-galactosylceramide reactive and those that are not, were affected by disruption of the LTalpha and LTbeta genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTbeta receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.

Varicella-zoster virus infection of a human CD4-positive T-cell line.

Varicella-zoster virus (VZV) is a human alpha-herpesvirus that causes varicella (chickenpox) at primary infection and may reactivate as herpes zoster. VZV is a T-lymphotropic virus in vivo. To investigate the T-cell tropism of VZV, we constructed a recombinant virus expressing green fluorescent protein (VZV-GFP) under the CMV IE promoter. Coculture of VZV-GFP-infected fibroblasts with II-23 cells, a CD4-positive human T-cell hybridoma, resulted in transfer of virus to II-23 cells. II-23 cells are susceptible to VZV-GFP infection as demonstrated by expression of immediate/early (IE62), early (ORF4), and late (gE) genes. Recovery of infectious virus was limited, with only 1 to 3 in 10(6) cells releasing infectious virus by plaque assay, indicating that transfer of virus results in a limited productive infection. In vitro infection of II-23 cells will be useful for further analysis of VZV tropism for T-lymphocytes.