Lymphotoxin and LIGHT signaling pathways and target genes

Summary: Lymphotoxins (LTα and LTβ), LIGHT [homologous to LT, inducible expression, competes with herpes simplex virus (HSV) glycoprotein D for HSV entry mediator (HVEM), a receptor expressed on T lymphocytes], tumor necrosis factor (TNF), and their specific receptors LTβR, HVEM, and TNF receptor 1 (TNFR1) and TNFR2, form the immediate family of the larger TNF superfamily. These cytokines establish a critical communication system required for the development of secondary lymphoid tissues; however, knowledge of the target genes activated by these signaling pathways is limited. Target genes regulated by the LTαβ‐LTβR pathway include the tissue‐organizing chemokines, CXCL13, CCL19, and CCL21, which establish cytokine circuits that regulate LT expression on lymphocytes, leading to organized lymphoid tissue. Infectious disease models have revealed that LTαβ pathways are also important for innate and adaptive immune responses involved in host defense. Here, regulation of interferon‐β by LTβR and TNFR signaling may play a crucial role in certain viral infections. Regulation of autoimmune regulator in the thymus via LTβR implicates LT/LIGHT involvement in central tolerance. Dysregulated expression of LIGHT overrides peripheral tolerance leading to T‐cell‐driven autoimmune disease. Blockade of TNF/LT/LIGHT pathways as an intervention in controlling autoimmune diseases is attractive, but such therapy may have risks. Thus, identifying and understanding the target genes may offer an opportunity to fine‐tune inhibitory interventions.     

Investigation of osteoclastogenic signalling of the RANKL substitute LIGHT

LIGHT (TNFSF14) is a member of the TNF superfamily and is known to substitute for RANKL to induce osteoclast differentiation. LIGHT binds HVEM and LTβR, but it is not known whether these receptors play a role in osteoclast formation or whether LIGHT acts via RANKL signalling pathways. We found that both RANKL and LIGHT strongly induced phosphorylation of Akt and NFκB but not JNK in mouse osteoclast precursor cells. The addition of an Akt inhibitor showed decreased osteoclast differentiation and resorption mediated by both RANKL and LIGHT. RT-PCR and FACS analysis showed that CD14⁺ human osteoclast precursors expressed HVEM and LTβR; expression levels of HVEM increased in the course of osteoclastogenesis and a decrease in LIGHT expression was associated with an increase in HVEM suggesting that there is a feedback loop related to this receptor. Our findings show that LIGHT is not inhibited by the soluble RANKL receptor OPG and that LIGHT is a potent osteoclastogenesis factor that activates the Akt, NFκB and JNK pathways.     

Herpesvirus entry mediator (HVEM) attenuates signals mediated by the lymphotoxin β receptor (LTβR) in human cells stimulated by the shared ligand LIGHT

Signals mediated by members of the tumor necrosis factor receptor superfamily modulate a network of diverse processes including initiation of inflammatory responses and altering cell fate between pathways favoring survival and death. Although such pathways have been well-described for the TNF-α receptor, less is known about signaling induced by the TNF superfamily member LIGHT and how it is differentially altered by expression of its two receptors LTβR and HVEM in the same cell. We used cell lines with different relative expression of HVEM and LTβR to show that LIGHT-induced signals mediated by these receptors were associated with altered TRAF2 stability and RelA nuclear translocation. Production of the inflammatory chemokine CXCL10 was primarily mediated by LTβR. Higher expression of HVEM was associated with cell survival, while unopposed LTβR signaling favored pathways leading to apoptosis. Importantly, restoring HVEM expression in cells with low endogenous expression recapitulated the phenotype of cells with higher endogenous expression. Together, our data provide evidence that relative expression of HVEM and LTβR modulates canonical NF-κB and pro-apoptotic signals stimulated by LIGHT.     

BST2/Tetherin is constitutively expressed on human thymocytes with the phenotype and function of Treg cells

In contrast to peripheral plasmacytoid DCs (pDCs), thymic pDCs constitutively express low levels of IFN‐α. This leads to induction of interferon secondary genes (ISGs) in medullary thymocytes, raising the question whether IFN‐α may play a role in T‐cell development. When characterizing further differences between peripheral and thymic pDCs, we found that thymic pDCs have a phenotype consistent with an “activated signature” including expression of TNF‐α and bone marrow stromal cell antigen 2 (BST2), but no expression of ILT7. Given that BST2 is induced by IFN‐α, and IFN‐α secretion is controlled by interaction between ILT7 and BST2, this regulatory pathway is apparently lost in thymic pDCs. Further, we also show that BST2 is constitutively expressed on a subset of medullary thymocytes at the mRNA and protein level reflecting a history of IFN‐α transduced signals. The majority of BST2⁺ thymocytes express CCR5 rendering them prevalent targets for R5‐tropic HIV infection. Moreover, BST2⁺ thymocytes express Foxp3 and CD25, consistent with the phenotype of natural Treg cells, and exert suppressive activity as they impair the proliferation of autologous CD3⁺ thymocytes. Collectively, our results suggest that low levels of IFN‐α secreted by thymic pDCs play an important role in the development of natural Treg cells.     

Expression of cytokines and their receptors by human thymocytes and thymic stromal cells.

The repertoire of cytokine and cytokine receptor mRNA expressed by unstimulated human thymocytes and thymic stromal cells was explored by a quantitative polymerase chain reaction (PCR) using sequence specific internal standards. Of the 18 cytokines tested we found a considerable overlap in the expression of cytokines by human thymocytes and by thymic stromal cells; both cell types express the mRNA for interleukin-1 beta(IL-1, IL-6, IL-7 and tumour necrosis factor-alpha (TNF-alpha). However, there are substantial differences in the levels of cytokine mRNA expressed in these two types of cells as revealed by the quantitative PCR assay. Stromal cells express considerably higher levels of IL-1 beta and IL-6 than thymocytes (14- and 27-fold respectively). In addition, a number of cytokines such as lymphotoxin and interferon-gamma (IFN-gamma), are expressed exclusively in thymocytes whereas others such as stem cell factor (SCF), IL-1 receptor antagonist-2 (IRAP-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are produced only in stromal cells. There is a complete overlap in the expression of a group of cytokine receptors tested in thymocytes and thymic stromal cells; these include IL-1R, IL-2R, IL-6R, IL-7R, TNFR and stem cell growth factor receptor (c-KIT). The expression of specific cytokines by thymic stromal cells and the parallel expression of their receptors on thymocytes under physiological conditions, support the hypothesis that these cytokines participate in paracrine interactions between these two cell populations during thymocyte differentiation.     

Increased thymic B cells but maintenance of thymic structure, T cell differentiation and negative selection in lymphotoxin-alpha and TNF gene-targeted mice

TNF, lymphotoxin (LT) and their receptors are expressed constitutively in the thymus. It remains unclear whether these cytokines play a role in normal thymic structure or function. We have investigated thymocyte differentiation, selection and thymic organogenesis in gene targeted mice lacking LTalpha, TNF, or both (TNF/LTalpha-/-). The thymus was normal in TNF/LTalpha-/- mice with regard to cell yields and stromal architecture. Detailed analysis of alphabeta and gammadelta T cell-lineage thymocyte subsets revealed no abnormalities, implying that neither TNF nor LT play an essential role in T cell differentiation or positive selection. The number and distribution of thymic CD11c+ dendritic cells was also normal in the absence of both TNF and LTalpha. A three-fold increase in B cell numbers was observed consistently in the TNF/LTalpha-/- thymus. This phenotype was due entirely to the LTalpha deficiency and associated with changes in the hemopoietic compartment, rather than the thymic stromal compartment of LTalpha-/- mice. Finally, specific Vbeta8+ T cell deletion within the thymus following intrathymic injection of staphylococcal enterotoxin B (SEB) was TNF/LT independent. Thus, despite the presence of these cytokines and their receptors in the normal thymus, there appears no essential role for either TNF or LT in development of organ structure or for those processes associated with T cell repertoire selection.     

MD-1 is a critical part of the mechanism causing Th1-cytokine-triggered murine fetal loss syndrome

PROBLEM: Fetal loss syndrome (abortion/resorption) occurring on or after gestation day (gd) 9.5 in CBA/J×DBA/2 matings is dependent upon presence of TNF‐α + IFN‐γ, which act by increasing expression of fgl2 prothrombinase at the feto‐maternal interface. The magnitude by which the abortion rate can be boosted by an injection of these cytokines on gd 7.5 depends on endogenous rate of loss, and appears to depend on microbial flora. Is cytokine‐triggered abortion dependent upon a third signaling pathway that senses ‘danger'? METHODS: Female CBA/J were mated to DBA/2 males and, C57Bl/6 and C57Bl/6 TNFαR1^?/?Mak were mated to C57Bl/6 control or TNFαR1^?/?Mak males. LPS from Escherichia coli and Salmonella enteritidis, or the combination of TNF‐α + IFN‐γ, was injected to stimulate abortions. The effect of anti‐MD‐1, which interferes with expression of CD14 and, hence, with signaling by LPS via the CD14‐tlr4 complex, on TNF‐α + IFN‐γ was tested. The presence of MD‐1 in the uterus was evaluated by in situ hybridization, and effect of lipopolysaccharide (LPS) on mice lacking TNF‐αR1 was tested. RESULTS: Anti‐MD‐1 completely abrogated TNF‐α + IFN‐γ ‐induced abortions. MD‐1 was expressed on trophoblast and in deciduas on gd 8.5 but LPS could not abort mice that lacked the type 1 receptor for TNF‐α. Pregnant CBA/J females had classical resorptions (abortions) countable on gd 13.5–14.5 in response to LPS from E. coli or S. enteritidis, but C57Bl/6 strain mice resorbed only in response to the latter, and E. coli LPS appeared to induce ‘occult’ losses. ‘Occult’ loss did not require TNF‐α R1. CONCLUSIONS: TNF‐α + IFN‐γ could not induce murine abortions without co‐presence of a ‘danger’ signal such as LPS acting via CD14 on toll receptors, and LPS could not act without co‐signaling by TNF‐α. Classical resorptions/abortions and ‘occult’ losses have a different mechanism in these models as reflected in type of endotoxin and requirement for TNF‐αR1 signaling.     

Overexpression of lymphotoxin in T cells induces fulminant thymic involution

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.     

Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.     

Increased Thymic B Cells but Maintenance of Thymic Structure,T Cell Differentiation and Negative Selection in Lymphotoxin-α and TNF Gene-Targeted Mice

TNF, lymphotoxin (LT) and their receptors are expressed constitutively in the thymus. It remains unclear whether these cytokines play a role in normal thymic structure or function. We have investigated thymocyte differentiation, selection and thymic organogenesis in gene targeted mice lacking LTα, TNF, or both (TNF/LTα^-/-). The thymus was normal in TNF/LTα^-/- mice with regard to cell yields and stromal architecture. Detailed analysis of αβ and γδ T cell-lineage thymocyte subsets revealed no abnormalities, implying that neither TNF nor LT play an essential role in T cell differentiation or positive selection. The number and distribution of thymic CD11c⁺ dendritic cells was also normal in the absence of both TNF and LTα. A three-fold increase in B cell numbers was observed consistently in the TNF/LTα^-/- thymus. This phenotype was due entirely to the LTα deficiency and associated with changes in the hemopoietic compartment, rather than the thymic stromal compartment of LTα^-/- mice. Finally, specific Vβ8⁺ T cell deletion within the thymus following intrathymic injection of staphylococcal enterotoxin B (SEB) was TNF/LT independent. Thus, despite the presence of these cytokines and their receptors in the normal thymus, there appears no essential role for either TNF or LT in development of organ structure or for those processes associated with T cell repertoire selection.     

Tumour necrosis factor receptor I blockade shows that TNF‐dependent and TNF‐independent mechanisms synergise in TNF receptor associated periodic syndrome

TNF receptor associated periodic syndrome (TRAPS) is an autoinflammatory disease involving recurrent episodes of fever and inflammation. It is associated with autosomal dominant mutations in TNF receptor superfamily 1A gene localised to exons encoding the ectodomain of the p55 TNF receptor, TNF receptor‐1 (TNFR1). The aim of this study was to investigate the role of cell surface TNFR1 in TRAPS, and the contribution of TNF‐dependent and TNF‐independent mechanisms to the production of cytokines. HEK‐293 and SK‐HEP‐1 cell lines were stably transfected with WT or TRAPS‐associated variants of human TNF receptor superfamily 1A gene. An anti‐TNFR1 single domain antibody (dAb), and an anti‐TNFR1 mAb, bound to cell surface WT and variant TNFR1s. In HEK‐293 cells transfected with death domain‐inactivated (R347A) TNFR1, and in SK‐HEP‐1 cells transfected with normal (full‐length) TNFR1, cytokine production stimulated in the absence of exogenous TNF by the presence of certain TNFR1 variants was not inhibited by the anti‐TNFR1 dAb. In SK‐Hep‐1 cells, specific TRAPS mutations increased the level of cytokine response to TNF, compared to WT, and this augmented cytokine production was suppressed by the anti‐TNFR1 dAb. Thus, TRAPS‐associated variants of TNFR1 enhance cytokine production by a TNF‐independent mechanism and by sensitising cells to a TNF‐dependent stimulation. The TNF‐dependent mechanism requires cell surface expression of TNFR1, as this is blocked by TNFR1‐specific dAb.     

The complementation of lymphotoxin deficiency with LIGHT,a newly discovered TNF family member,for the restoration of secondary lymphoid structure and function

Highly organized lymphoid structures provide the intricate microenvironment essential for the mediation of the effective immune responses. Compared with lymphotoxin beta knockout mice (LTbeta-/-), LTbeta receptor knockout (LTbetaR-/-) mice present with more severely disorganized splenic structures, suggesting the potential involvement of another ligand. LIGHT, a newly identified TNF family member, is a costimulatory molecule for T cells and binds to LTbetaR and herpes virus entry mediator (HVEM) in vitro. Here, we show that the complementation of LTalpha-/- mice with a LIGHT transgene (LIGHT Tg/LTalpha-/-) leads to the restoration of secondary lymphoid tissue chemokine and T/B cell zone segregation. LIGHT Tg/LTalpha-/- mice also preserve dendritic cells, follicular dendritic cell networks, and germinal centers, though not the marginal zone. Consequently, IgG responses to soluble, but not particulate, antigens are restored, confirming the role of primary follicle and marginal zone in the responses to soluble and particulate antigens. The failure of the LIGHT transgene to rescue the defective splenic structures in LTbetaR-/- mice demonstrates that LIGHT can interact with LTbetaR in vivo. More severely disorganized splenic structures developed after blockade of endogenous LIGHT in LTbeta-/- mice. These findings uncover the potential interaction between LIGHT and one of its receptors, LTbetaR, in supporting even in the absence of LT the development and maintenance of lymphoid microenvironment.     

Metalloproteinase-dependent control of thymocyte differentiation and proliferation

The development of T cells in the thymus is dependent on interactions between thymocytes and thymic stromal cells, on stimulation by growth factors, and on the binding to and migration along extracellular matrix (ECM) components. As metalloproteinases (MP) are involved in processes such as growth factor release and ECM modelling, we assessed the effect of MP inhibitors on T-cell development using fetal thymic organ culture systems. MP inhibitors significantly reduced the numbers of CD4/CD8 double-positive (DP) and mature single-positive thymocytes generated, correlated with a reduced number of cell cycles between the double-negative (DN)3 and DP stages. The progression of early thymocyte progenitors through the DN1-4 stages of development was also severely affected, including incomplete upregulation of CD25, decreased DN3 cell numbers, reduced rearrangement of the T-cell receptor (TCR)-beta locus and expression of intracellular TCR-beta by fewer DN3 cells. When purified DN1 cells were utilized as donor cells in reaggregate thymic organ cultures, essentially no DP thymocytes were produced in the presence of MP inhibitors. The results suggest that MP inhibitors affect the differentiation of developing thymocytes before, and reduce proliferation after, pre-TCR-mediated selection.     

Epstein–Barr virus‐specific CD8+ T lymphocytes from diffuse large B cell lymphoma patients are functionally impaired

Epstein–Barr virus (EBV) is a persistent virus with oncogenic capacity that has been implicated in the development of aggressive B cell lymphomas, primarily in immunosuppressed individuals, although it can be present in immunocompetent individuals. Changes in the function and clonal diversity of T lymphocytes might be implied by viral persistence and lymphoma development. The aim of the present study was to evaluate the frequency, phenotype, function and clonotypical distribution of EBV‐specific T cells after peripheral blood stimulation with a virus lysate in newly diagnosed patients with diffuse large B cell lymphoma (DLBCL) aged more than 50 years without prior histories of clinical immunosuppression compared with healthy controls. Our results showed impaired EBV‐specific immune responses among DLBCL patients that were associated primarily with decreased numbers of central and effector memory CD8⁺ T lymphocytes. In contrast to healthy controls, only a minority of the patients showed CD4⁺/tumour necrosis factor (TNF)‐α⁺ T cells expressing T cell receptor (TCR)‐Vβ17 and CD8⁺/TNF‐α⁺ T cells with TCR‐Vβ5·2, Vβ9 and Vβ18 in response to EBV. Notably, the production of TNF‐α was undetectable among TCR‐Vβ5·3⁺, Vβ11⁺, Vβ12⁺, Vβ16⁺ and Vβ23⁺ CD8⁺ T cells. In addition, we observed decreased numbers of CD4⁺/TNF‐α⁺ and CD8⁺/TNF‐α⁺, CD8⁺/interleukin (IL)‐2⁺ and CD8⁺/TNF‐α⁺/IL‐2⁺ T lymphocytes in the absence of T cells capable of producing TNF‐α, IL‐2 and IFN‐γ after EBV stimulation simultaneously. Moreover, DLBCL patients displayed higher IL‐10 levels both under baseline conditions and after EBV stimulation. These findings were also observed in patients with positive EBV viral loads. Prospective studies including a large number of patients are needed to confirm these findings.     

Functional discrepancies between tumor necrosis factor and lymphotoxin α explained by trimer stability and distinct receptor interactions

Tumor necrosis factor (TNF) and lymphotoxin α (LTα) are closely related cytokines which bind with nearly identical affinities to the same pair of cell surface receptors, p55 and p75TNFR. Therefore it is assumed that TNF and LTα are redundant cytokines. This study, however, demonstrates that TNF and LTα differ significantly with regard to their mitogenic and cytotoxic potentials. LTα's superior mitogenic effect could be explained by its formation of a more stable trimer. In contrast to the TNF trimer, which disintegrated under physiological conditions into biologically inactive monomers, the LTα trimer remained stable for several days. Accordingly, LTα more effectively induced fibroblast growth which demands long-term presence of the cytokine. TNF's superior cytotoxicity, which requires only short-term impact of the cytokine, could be attributed to a distinct interaction with the human p55TNFR. This was demonstrated in NIH 3T3 cells transfected with the human p55TNFR, where cytotoxicity is mediated exclusively by the transfected receptor. Although the p55TNFR had virtually identical affinities for TNF and LTα, as defined by Scatchard analysis, it nevertheless discriminated between binding of each cytokine and showed a 200-fold enhanced cytotoxicity mediated by TNF.