LIGHT Elevation Enhances Immune Eradication of Colon Cancer Metastases.

The majority of patients with colon cancer will develop advanced disease, with the liver being the most common site of metastatic disease. Patients with increased numbers of tumor-infiltrating lymphocytes in primary colon tumors and liver metastases have improved outcomes. However, the molecular factors that could empower antitumor immune responses in this setting remain to be elucidated. We reported that the immunostimulatory cytokine LIGHT (TNFSF14) in the microenvironment of colon cancer metastases associates with improved patient survival, and here we demonstrate in an immunocompetent murine model that colon tumors expressing LIGHT stimulate lymphocyte proliferation and tumor cell-specific antitumor immune responses. In this model, increasing LIGHT expression in the microenvironment of either primary tumors or liver metastases triggered regression of established tumors and slowed the growth of liver metastases, driven by cytotoxic T-lymphocyte-mediated antitumor immunity. These responses corresponded with significant increases in tumor-infiltrating lymphocytes and increased expression of lymphocyte-homing signals in the metastatic tumors. Furthermore, we demonstrated evidence of durable tumor-specific antitumor immunity. In conclusion, increasing LIGHT expression increased T-cell proliferation, activation, and infiltration, resulting in enhanced tumor-specific immune-mediated tumor regressions in primary tumors and colorectal liver metastases. Mechanisms to increase LIGHT in the colon cancer microenvironment warrant further investigation and hold promise as an immunotherapeutic strategy. Cancer Res; 77(8); 1880-91. ©2017 AACR.

Dextran sodium sulfate alters cytokine production in macrophages in vitro.

Macrophages have been assumed to have a crucial role in the development of inflammatory bowel disease (IBD). However, involvement of intestinal macrophages in IBD onset and functional alterations of macrophages during IBD development has not been clarified. We investigated the effect of exposure of compounds used in the induction of colitis in mice on the immune responses of peritoneal macrophages in mice. 2,4,6- trinitrobenzenesulfonic acid and oxazolone did not affect the production of interleukin (IL)-10 and IL-12 from lipopolysaccharide (LPS)-stimulated peritoneal macrophages from BALB/c mice. A significant increase in IL-10 secretion and decrease in IL-12 production from LPS-stimulated macrophages were observed upon exposure to dextran sodium sulfate (DSS). TNF-α production was enhanced significantly by exposure to DSS and LPS. The level of nitric-oxide production from macrophages was increased slightly by exposure to DSS and LPS. Expression of sphingosine kinase-1 and LIGHT (both of which are specific biomarkers of M2b macrophages) was observed in macrophages upon DSS exposure. Alteration of cytokine production in macrophages was observed upon DSS exposure in the absence of LPS stimulation. Peritoneal macrophages from C57BL/6 mice showed similar responses to peritoneal macrophages from BALB/c mice against DSS. These results suggest that DSS directs the immune response of macrophages towards the M2b phenotype.

LIGHT/TNFSF14 increases osteoclastogenesis and decreases osteoblastogenesis in multiple myeloma-bone disease.

LIGHT, a TNF superfamily member, is involved in T-cell homeostasis and erosive bone disease associated with rheumatoid arthritis. Herein, we investigated whether LIGHT has a role in Multiple Myeloma (MM)-bone disease. We found that LIGHT was overproduced by CD14+ monocytes, CD8+ T-cells and neutrophils of peripheral blood and bone marrow (BM) from MM-bone disease patients. We also found that LIGHT induced osteoclastogenesis and inhibited osteoblastogenesis. In cultures from healthy-donors, LIGHT induced osteoclastogenesis in RANKL-dependent and -independent manners. In the presence of a sub-optimal RANKL concentration, LIGHT and RANKL synergically stimulated osteoclast formation, through the phosphorylation of Akt, NFκB and JNK pathways. In cultures of BM samples from patients with bone disease, LIGHT inhibited the formation of CFU-F and CFU-OB as well as the expression of osteoblastic markers including collagen-I, osteocalcin and bone sialoprotein-II. LIGHT indirectly inhibited osteoblastogenesis in part through sclerostin expressed by monocytes. In conclusion, our findings for the first time provide evidence for a role of LIGHT in MM-bone disease development.

Somatic and axonal LIGHT signaling elicit degenerative and regenerative responses in motoneurons, respectively.

A receptor-ligand interaction can evoke a broad range of biological activities in different cell types depending on receptor identity and cell type-specific post-receptor signaling intermediates. Here, we show that the TNF family member LIGHT, known to act as a death-triggering factor in motoneurons through LT-ßR, can also promote axon outgrowth and branching in motoneurons through the same receptor. LIGHT-induced axonal elongation and branching require ERK and caspase-9 pathways. This distinct response involves a compartment-specific activation of LIGHT signals, with somatic activation-inducing death, while axonal stimulation promotes axon elongation and branching in motoneurons. Following peripheral nerve damage, LIGHT increases at the lesion site through expression by invading B lymphocytes, and genetic deletion of Light significantly delays functional recovery. We propose that a central and peripheral activation of the LIGHT pathway elicits different functional responses in motoneurons.

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus.

LIGHT/TNFSF14 is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. In this study, the cDNA of mefugu (Takifugu obscures) LIGHT (designated as fLIGHT) was amplified from spleen by reverse transcription polymerase chain reaction. The open reading frame of fLIGHT covers 765 bp and translates into a 254-aa protein. The predicted three-dimensional (3D) structure of the soluble LIGHT of mefugu (designated as fsLIGHT) monomer analyzed by comparative protein modeling revealed that it was very similar to its human counterpart. Real-time quantitative PCR analysis indicated that LIGHT is constitutively expressed in various tissues in mefugu. The soluble LIGHT binding of green fluorescent protein (GFP) (designated as GFP/fsLIGHT) had been cloned into the pET28a vector; SDS-PAGE and western blotting analysis confirmed that the recombinant protein pET28a-GFP/fsLIGHT was efficiently expressed in Escherichia coli BL21 (DE3). After purification, the GFP/fsLIGHT fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsLIGHT could bind to its receptors. In view of our basic research, LIGHT may be a potential immunologic factor for enhancing immunological efficacy in fish, and our results might provide a platform for further study into the effects of LIGHT.

LIGHT/TNFSR14 can regulate hepatic lipase expression by hepatocytes independent of T cells and Kupffer cells.

LIGHT/TNFSF14 is a costimulatory molecule expressed on activated T cells for activation and maintenance of T cell homeostasis. LIGHT over expressed in T cells also down regulates hepatic lipase levels in mice through lymphotoxin beta receptor (LTßR) signaling. It is unclear whether LIGHT regulates hepatic lipase directly by interacting with LTßR expressing cells in the liver or indirectly by activation of T cells, and whether Kupffer cells, a major cell populations in the liver that expresses the LTßR, are required. Here we report that LIGHT expression via an adenoviral vector (Ad-LIGHT) is sufficient to down regulate hepatic lipase expression in mice. Depletion of Kupffer cells using clodronate liposomes had no effect on LIGHT-mediated down regulation of hepatic lipase. LIGHT-mediated regulation of hepatic lipase is also independent of LIGHT expression by T cells or activation of T cells. This is demonstrated by the decreased hepatic lipase expression in the liver of Ad-LIGHT infected recombination activating gene deficient mice that lack mature T cells and by the Ad-LIGHT infection of primary hepatocytes. Hepatic lipase expression was not responsive to LIGHT when mice lacking LTßR globally or only on hepatocytes were infected with Ad-LIGHT. Therefore, our data argues that interaction of LIGHT with LTßR on hepatocytes, but not Kupffer cells, is sufficient to down regulate hepatic lipase expression and that this effect can be independent of LIGHT's costimulatory function.

In situ vaccination combined with androgen ablation and regulatory T-cell depletion reduces castration-resistant tumor burden in prostate-specific pten knockout mice.

There is no effective treatment for prostate cancer arising after androgen ablation. Previous studies have analyzed the short-term effects of androgen ablation on the immune system and suggest an abatement of immune suppression by hormone removal. Because castration-resistant disease can arise years after treatment, it is crucial to determine the duration of immune potentiation by castration. Because immunotherapeutic efficacy is determined by the balance of immune cell subsets and their location within the tumor, we assessed the acute and chronic effect of androgen ablation on the localization of T-cell subsets within castration-resistant murine prostate cancer. We observed a transient increase in CD4+ and CD8+ T-cell numbers at the residual tumor after androgen ablation. More than 2 months later, regulatory T cells (Treg) were increasingly found within prostate epithelium, whereas CTLs, which were evenly distributed before androgen ablation, became sequestered within stroma. Anti-CD25 antibody administration along with castration enhanced CTL access to cancerous glands but did not increase effector function. Intraprostatic injection of LIGHT-expressing tumor cells increased the proportion of CD8+ T cells with functional capacity within the cancerous gland. In addition, Treg depletion within the tumor was enhanced. Together, these manipulations significantly reduced castration-resistant tumor burden. Thus, our results indicate that immune modulations, which prevent Treg accumulation and augment effector cell infiltration of prostatic epithelium, may be effective in reducing tumor burden or preventing tumor recurrence after androgen ablation therapy.

CXCR3 binding chemokine and TNFSF14 over expression in bladder urothelium of patients with ulcerative interstitial cystitis.

PURPOSE: We investigated the genes responsible for ulcerative interstitial cystitis by DNA microarray analysis and quantitative real-time polymerase chain reaction. MATERIALS AND METHODS: Bladder urothelial tissues were taken from a site apart from the ulcerative lesion in 9 patients with ulcerative interstitial cystitis and from a normal-looking area in 9 controls, including 7 with bladder carcinoma and 2 with benign prostatic hyperplasia. Total RNA was extracted from bladder samples and gene expression was compared between these 2 groups using Whole Human Genome DNA microarray 44K (Agilent Technologies, Santa Clara, California). Microarray data were analyzed by GeneSpring GX software and Ingenuity Pathway Analysis. Chosen genes were confirmed for altered transcription by quantitative real-time polymerase chain reaction. RESULTS: We identified 564 probes that were significantly expressed in mRNA more than 4-fold vs those in controls using volcano plot analysis (p <0.001). Further network Ingenuity Pathway Analysis of these genes showed the top 3 functions, including 1) cell-to-cell signaling and interaction, and hematological system development and function, 2) inflammatory disease and 3) cellular development. Quantitative real-time polymerase chain reaction confirmed increased mRNA expression of several genes in the bladder samples of patients with ulcerative interstitial cystitis, including CXCR3 binding chemokines (CXCL9, 10 and 11) and TNFSF14 (LIGHT). CONCLUSIONS: Our study using DNA microarray analysis followed by quantitative real-time polymerase chain reaction reveals over expression of genes related to immune and inflammatory responses, including T-helper type 1 related chemokines, and cytokines such as CXCR3 binding chemokines and TNFSF14. These genes may be potential interstitial cystitis biomarkers.

Promoting immune responses by LIGHT in the face of abundant regulatory T cell inhibition.

CD4(+) regulatory T cell (Treg) populations are believed to play very important roles in the suppression of immune responses. Overriding Treg inhibition is necessary for initiating primary immune reaction upon inflammatory Ag stimulation. LIGHT, TNF superfamily member 14, has been shown to be a costimulatory molecule for effector T cells. Overexpression of lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) on T cells induces strong T cell-mediated experimental intestinal inflammation. How this process is initiated by LIGHT in suppressive intestinal environments remains incompletely understood. In this study, we assessed the effect of LIGHT on Tregs. Our results indicate that LIGHT can support the expansion and function of Tregs. However, when LIGHT was highly expressed, these abundant Tregs failed to suppress the development of T cell-mediated experimental colitis and antitumor immunity. We showed that this might be, in part, due to an ability of LIGHT to promote effector T cell proliferation and differentiation even in a Treg-abundant environment. Our data collectively suggest that LIGHT might be a critical cytokine involved in the development of autoimmune inflammatory diseases and that LIGHT-targeted immunotherapy might be useful in the treatment of these diseases.

LIGHT protein suppresses tumor growth by augmentation of immune response.

The tumor necrosis factor (TNF) superfamily member LIGHT has potent anti-tumor activities through activation of the immune response, and it is a promising candidate for use in cancer immunotherapy. However, there are no reports of the anti-tumor effects of LIGHT protein in vivo because of the lack of easy, efficient methods of manufacturing this protein. Here, we developed a method of manufacturing recombinant LIGHT protein using Escherichia coli through refolding of inclusion bodies; we then evaluated the anti-tumor activity of the protein. LIGHT protein expressed in E. coli showed the same biological activities and binding affinities to its receptors as did LIGHT expressed in mammalian cells. In addition, intratumoral injection of LIGHT significantly suppressed tumor growth, with augmentation of antigen-specific IFN-gamma-producing cells in the regional lymph nodes and spleen. These results indicate that LIGHT protein efficiently evokes the systemic tumor-specific immune response, and thus induces tumor suppression.

Developmental regulation of sensory neurite growth by the tumor necrosis factor superfamily member LIGHT.

In a PCR screen to identify novel cytokine candidates involved in neuronal development, we identified transcripts for the tumor necrosis factor superfamily member 14 (TNFSF14), generally known as LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells), together with its receptors, lymphotoxin-beta receptor (LTbetaR) and TNF family receptor herpesvirus entry mediator (HVEM), in the experimentally tractable sensory neurons of the mouse nodose ganglion. Immunocytochemistry revealed coexpression of LIGHT and its receptors in all nodose ganglion neurons in neonates. Enhancing LIGHT signaling in these neurons by overexpressing LIGHT inhibited BDNF-promoted neurite growth during a narrow window of development in the immediate perinatal period without affecting neuronal survival. Overexpressing a LIGHT mutant that selectively activates HVEM, but not one that selectively activates LTbetaR, also inhibited BDNF-promoted growth, suggesting that neurite growth inhibition is mediated via HVEM. Blocking HVEM signaling by a function-blocking anti-HVEM antibody significantly enhanced neurite growth from nodose neurons grown both with and without BDNF. Likewise, neurons from LIGHT-deficient neonates exhibited significantly greater neurite growth than neurons from wild-type littermates in both the presence and absence of BDNF. LIGHT overexpression significantly inhibited NF-kappaB activity, while preventing LIGHT-induced NF-kappaB inhibition by overexpressing the p65 and p50 NF-kappaB subunits prevented LIGHT-mediated growth inhibition. Together, these findings show that LIGHT/HVEM signaling negatively regulates neurite growth from developing sensory neurons via NF-kappaB inhibition.

Expression level of Bcl-XL critically affects sensitivity of hepatocellular carcinoma cells to LIGHT-enhanced and interferon-gamma-induced apoptosis.

The molecular mechanisms of apoptosis caused by IFN-gamma (interferon gamma)/LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells) have not been studied in detail. The present study was undertaken to gain insights into the signaling pathways involved in apoptosis induced by IFN-gamma/LIGHT in hepatocellular carcinoma (HCC) cell lines. Cell proliferation assay, flow cytometry, Western blotting, gene transfer and RNA interference were used in this study. LIGHT enhanced IFN-gamma-mediated apoptosis in Hep3B cells. IFN-gamma/LIGHT-induced apoptosis was inhibited by blocking peptides to the lymphotoxin beta receptor (LT-beta R), and not by the herpes virus entry mediator (HVEM). Expression of LT-beta R remained unchanged after cytokine treatments. IFN-gamma/LIGHT treatment resulted in the down-regulation of Bcl-XL and the activation of caspase-9 and caspase-3 as well as the decrease of phosphorylation of STAT3. HepG2 and SMMC-7721 cells, which showed high levels of endogenous Bcl-XL, displayed resistance to IFN-gamma/LIGHT-induced apoptosis. Overexpression of Bcl-XL in Hep3B cells increased the resistance to IFN-gamma/LIGHT induced apoptosis while the down-regulation of Bcl-XL in HepG2 and SMMC-7721 cells by RNA interference decreased the resistance. Our study provides important mechanistic insights into IFN-gamma/LIGHT- induced apoptosis in HCC cells and may help to select better therapeutic strategies for certain cancers with distinct Bcl-XL expression.

Biochemical and functional characterization of three activated macrophage populations.

We generated three populations of macrophages (Mphi) in vitro and characterized each. Classically activated Mphi (Ca-Mphi) were primed with IFN-gamma and stimulated with LPS. Type II-activated Mphi (Mphi-II) were similarly primed but stimulated with LPS plus immune complexes. Alternatively activated Mphi (AA-Mphi) were primed overnight with IL-4. Here, we present a side-by-side comparison of the three cell types. We focus primarily on differences between Mphi-II and AA-Mphi, as both have been classified as M2 Mphi, distinct from Ca-Mphi. We show that Mphi-II more closely resemble Ca-Mphi than they are to AA-Mphi. Mphi-II and Ca-Mphi, but not AA-Mphi, produce high levels of NO and have low arginase activity. AA-Mphi express FIZZ1, whereas neither Mphi-II nor Ca-Mphi do. Mphi-II and Ca-Mphi express relatively high levels of CD86, whereas AA-Mphi are virtually devoid of this costimulatory molecule. Ca-Mphi and Mphi-II are efficient APC, whereas AA-Mphi fail to stimulate efficient T cell proliferation. The differences between Ca-Mphi and Mphi-II are more subtle. Ca-Mphi produce IL-12 and give rise to Th1 cells, whereas Mphi-II produce high levels of IL-10 and thus, give rise to Th2 cells secreting IL-4 and IL-10. Mphi-II express two markers that may be used to identify them in tissue. These are sphingosine kinase-1 and LIGHT (TNF superfamily 14). Thus, Ca-Mphi, Mphi-II, and AA-Mphi represent three populations of cells with different biological functions.