Tumor Necrosis Factor Ligand Superfamily Research Articles

Design of vaccine adjuvants incorporating TNF superfamily ligands and TNF superfamily molecular mimics

TNF superfamily ligands play a critical role in the regulation of adaptive immune responses, including the costimulation of dendritic cells, T cells, and B cells. This costimulation could potentially be exploited for the development of prophylactic vaccines and immunotherapy. Despite this, there have been only a limited number of reports on the use of this family of molecules as gene-based adjuvants to enhance DNA and/or viral vector vaccines. In addition, the molecule latent membrane protein 1 (LMP1), a viral mimic of the TNF superfamily receptor CD40, provides an alternative approach for the design of novel molecular adjuvants. Here, we discuss advances in the development of recombinant TNF superfamily ligands as adjuvants for HIV vaccines and as cancer immunotherapy, including the use of LMP1 and LMP1-CD40 chimeric fusion proteins to mimic constitutive CD40 signaling.

Immune effects of dietary anethole on Eimeria acervulina infection

The effects of anethole on in vitro and in vivo parameters of chicken immunity during experimental avian coccidiosis were evaluated. Anethole reduced the viability of invasive Eimeria acervulina sporozoites after 2 or 4 h of treatment in vitro by 45 and 42%, respectively, and stimulated 6.0-fold greater chicken spleen cell proliferation compared with controls. Broiler chickens continuously fed from hatch with an anethole-supplemented diet and orally challenged with live E. acervulina oocysts showed enhanced BW gain, decreased fecal oocyst excretion, and greater E. acervulina profilin antibody responses compared with infected chickens given an unsupplemented standard diet. The levels of transcripts encoding the immune mediators IL6, IL8, IL10, and tumor necrosis factor ligand superfamily member 15 (TNFSF15) in intestinal lymphocytes were increased in E. acervulina-infected chickens fed the anethole-containing diet compared with untreated controls. Global gene expression analysis by microarray hybridization identified 1,810 transcripts (677 upregulated, 1,133 downregulated) whose levels were significantly altered in intestinal lymphocytes of anethole-fed birds compared with unsupplemented controls. From this transcriptome, 576 corresponding genes were identified. The most significant biological function associated with these genes was "Inflammatory Response" in the "Disease and Disorders" category. This new information documents the immunologic and genomic changes that occur in chickens following anethole dietary supplementation that may be relevant to host protective immune response to avian coccidiosis.

Identification of molecular phenotypic descriptors of breast capsular contracture formation using informatics analysis of the whole genome transcriptome

Breast capsular contracture formation following silicone implant augmentation/reconstruction is a common complication that remains poorly understood. The aim of this study was to identify potential biomarkers implicated in breast capsular contracture formation by using, for the first time, whole genome arrays. Biopsy samples were taken from 18 patients (23 breast capsules) with Baker Grade I-II (Control) and Baker Grade III-IV (Contracted). Whole genome microarrays were performed and six significantly dysregulated genes were selected for further validation with quantitative reverse transcriptase polymerase chain reaction and immunohistochemistry. Hematoxylin and eosin was also carried out to compare the histological characteristics of control and contracted samples. Microarray results showed that aggrecan, tissue inhibitor of metalloproteinase 4 (TIMP4), and tumor necrosis factor superfamily (ligand) member 11 were significantly down-regulated in contracted capsules; while matrix metallopeptidase 12, serum amyloid A 1, and interleukin 8 (IL8) were significantly up-regulated. The dysregulation of aggrecan, tumor necrosis factor superfamily (ligand) member 11, TIMP4, and IL8 was validated by quantitative reverse transcriptase polymerase chain reaction (p < 0.05). Immunohistochemistry confirmed an increased protein expression for IL8 and matrix metallopeptidase 12 in contracted capsules (p < 0.05), and decreased protein expression of TIMP4 (p < 0.05). This study has shown, for the first time, a number of unique biomarkers of significance in capsular contracture formation. IL8 and TIMP4 may serve as potential key diagnostic, therapeutic, and prognostic biomarkers in capsular contracture formation.

Lymphotoxin α induces apoptosis, necroptosis and inflammatory signals with the same potency as tumour necrosis factor

Both of the TNF superfamily ligands, TNF and LTα, can bind and signal through TNFR1 and TNFR2, yet mice mutant for each have different phenotypes. Part of this difference is because LTα but not TNF can activate Herpes Virus Entry Mediator and also heterotrimerise with LTβ to activate LTβR, which is consistent with the similar phenotypes of the LTα and LTβR deficient mice. However, it has also been reported that the LTα3 homotrimer signals differently than TNF through TNFR1, and has unique roles in initiation and exacerbation of some inflammatory diseases. Our modeling of the TNF/TNFR1 interface compared to the LTα3/TNFR1 structure revealed some differences that could affect signalling by the two ligands. To determine whether there were any functional differences in the ability of TNF and LTα3 to induce TNFR1-dependent apoptosis or necroptosis, and if there were different requirements for cIAPs and Sharpin to transmit the TNFR1 signal, we compared the ability of cells to respond to TNF and LTα3. Contrary to our hypothesis, we were unable to discover differences in signalling by TNFR1 in response to TNF and LTα3. Our results imply that the reasons for the conservation of LTα are most likely due either to differential regulation, the ability to signal through Herpes Virus Entry Mediator or the ability of LTα to form heterotrimers with LTβ.

MicroRNA regulate immune pathways in T-cells in multiple sclerosis (MS)

BackgroundMicroRNA are small noncoding RNA molecules that are involved in the control of gene expression. To investigate the role of microRNA in multiple sclerosis (MS), we performed genome-wide expression analyses of mRNA and microRNA in T-cells from MS patients and controls.MethodsHeparin-anticoagulated peripheral blood was collected from MS-patients and healthy controls followed by isolation of T-cells. MicroRNA and RNA from T-cells was prepared and hybridized to Affymetrix miR 2.0 array and Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA), respectively. Verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsWe identified 2,452 differentially expressed genes and 21 differentially expressed microRNA between MS patients and controls. By Kolmogorov-Smirnov test, 20 of 21 differentially expressed microRNA were shown to affect the expression of their target genes, many of which were involved in the immune system. Tumor necrosis factor ligand superfamily member 14 (TNFSF14) was a microRNA target gene significantly decreased in MS. The differential expression of mir-494, mir-197 and the predicted microRNA target gene TNFSF14 was verified by real-time PCR and ELISA.ConclusionThese findings indicate that microRNA may be important regulatory molecules in T-cells in MS.

Association of TNFSF4 polymorphisms with systemic lupus erythematosus: a meta-analysis

Introduction To more precisely estimate the association between the tumor necrosis factor ligand superfamily member 4 (TNFSF4) gene polymorphisms and systemic lupus erythematosus (SLE) risk, we surveyed studies on the association of the TNFSF4 rs2205960, rs1234315, rs844644, and rs844648 polymorphisms with SLE.Methods A literature-based search was conducted to identify all relevant studies. A total of eight independent studies were identified and subsequently reviewed in the meta-analysis.Results The meta-analysis showed an association between the TNFSF4 rs2205960 polymorphism and SLE in all subjects [ odds ratio (OR) 1.327, 95 % confidence interval (CI) 1.227–1.436, P < 0.001]. In a subgroup analysis by ethnicity, a significantly increased risk for SLE was associated with TNFSF4 rs2205960 T allele among patients of European (OR 1.254, 95 % CI 1.185–1.328, P < 0.001) and Asian ethnicity (OR 1.425, 95 % CI 1.352–1.501, P < 0.001). The meta-analysis of the rs1234315 polymorphism revealed no association between SLE and the rs1234315 T allele in all subjects (OR 1.167, 95 % CI 0.874–1.558, P = 0.296), but the results of the subgroup analysis revealed significant association in subjects of Asian ethnicity (OR 1.386, 95 % CI 1.318–1.458, P < 0.001). No association was found between the rs844644 and rs844648 polymorphisms and SLE.Conclusion The results of our meta-analysis suggest that the TNFSF4 rs2205960 polymorphism may confer susceptibility to SLE in different populations and that the TNFSF4 rs1234315 polymorphism is associated with susceptibility to SLE in Asians.

LIGHT (TNFSF14/CD258) Is a Decisive Factor for Recovery from Experimental Autoimmune Encephalomyelitis

The TNF superfamily ligand LIGHT (lymphotoxin-like, exhibits inducible expression and competes with HSV glycoprotein D for herpesvirus entry mediator [HVEM], a receptor expressed by T lymphocytes) has been shown to play a role in T cell costimulation and be involved in apoptosis of mononuclear cells. As both T cells and monocytes are key components in the development and progression of experimental autoimmune encephalomyelitis (EAE), we studied the role of LIGHT in EAE. Following immunization with myelin oligodendrocyte glycoprotein peptide (35-55), LIGHT-deficient mice developed severe EAE that resulted in an atypically high mortality rate. Histological examinations revealed intensive activation of microglia/macrophages in the CNS and higher numbers of apoptotic cells within the CNS parenchyma of LIGHT-deficient mice. However, myelin oligodendrocyte glycoprotein peptide-specific CD4(+) T cells from LIGHT-deficient mice showed reduced IFN-γ and IL-17 production and migration. Serum levels of reactive nitrogen intermediates and CNS transcripts of several proinflammatory cytokines and chemokines were also substantially decreased in the absence of LIGHT. EAE adoptive transfer experiments and bone marrow chimeras indicated that expression of LIGHT on donor cells is not required for disease induction. However, its expression on CNS host cells is a decisive factor to limit disease progression and tissue damage. Together, these data show that LIGHT expression is crucially involved in controlling activated macrophages/microglia during autoimmune CNS inflammation.

Molecular cloning and characterization of TNFSF14 (LIGHT) and its receptor TNFRSF14 (HVEM) in guinea pig (Cavia porcellus)

LIGHT (lymphotoxin-related inducible ligand that competes with herpes simplex virus (HSV) glycoprotein D for herpesvirus entry mediator on T cells) is a member of the tumor necrosis factor (TNF) ligand superfamily, which plays important roles in inflammatory and immune responses. In the present study, the cDNAs of guinea pig (Cavia porcellus) LIGHT (designated as gpLIGHT) and its receptor herpes virus entry mediator (designated as gpHVEM) were amplified from spleen by reverse transcription polymerase chain reaction (RT-PCR). The ORFs of gpLIGHT and gpHVEM cover 726 and 861bp, encoding predicted proteins with 241 and 286 aas, respectively. The three-dimensional (3D) structure, phylogenetic relationships, and characterization of both genes were also analyzed. We also generated a 3D model to verify interaction between the two proteins. Real-time quantitative PCR (qPCR) analysis revealed that both LIGHT and HVEM are constitutively expressed in guinea pig various tissues. A fusion protein SUMO (Small Ubiquitin-like Modifier)-gpsLIGHT (the soluble mature part of gpLIGHT) was efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy (LSCM) showed that gpsLIGHT can bind its receptors on T cells. The LIGHT-HVEM signaling pathway plays an important role in the immune system, and our results might provide a platform for further research into the effects of LIGHT and HVEM.

TWEAK/Fn14 pathway promotes chronic colitis and fibrosis mediated by IL-13-TSLP axis (P3244)

Abstract Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF ligand superfamily that is constitutively expressed by many immune cell types including monocytes/macrophages, DC, neutrophils, natural killer cells, and also T cells. The only known signaling receptor for TWEAK, FGF-inducible molecule 14 (Fn14), is a highly inducible molecule in epithelial cells (EC), endothelial cells, and other mesenchymal cell types wherein TWEAK signals through Fn14 to promote the canonical and noncanonical NF-κB, MAPK and potentially other pathways. We previously reported that TWEAK promoted colitis in mice by enhancing production of inflammatory mediators by EC. TWEAK also induced EC damage in the presence of IL-13, which is known as a pathogenic profibrotic T helper 2-type cytokine. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2-type immunity were significantly reduced in Fn14 knockout (KO) mice when compared with wild type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon EC than in WT EC. TWEAK/Fn14 was required for the induction by IL-13 of TSLP in colon explants. TWEAK and IL-13 synergistically promote embryonic fibroblast proliferation. Thus we propose an IL-13-TWEAK/ Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.

Cysteine-rich Domain 1 of CD40 Mediates Receptor Self-assembly

The activation of CD40 on B cells, macrophages, and dendritic cells by its ligand CD154 (CD40L) is essential for the development of humoral and cellular immune responses. CD40L and other TNF superfamily ligands are noncovalent homotrimers, but the form under which CD40 exists in the absence of ligand remains to be elucidated. Here, we show that both cell surface-expressed and soluble CD40 self-assemble, most probably as noncovalent dimers. The cysteine-rich domain 1 (CRD1) of CD40 participated to dimerization and was also required for efficient receptor expression. Modelization of a CD40 dimer allowed the identification of lysine 29 in CRD1, whose mutation decreased CD40 self-interaction without affecting expression or response to ligand. When expressed alone, recombinant CD40-CRD1 bound CD40 with a K(D) of 0.6 μM. This molecule triggered expression of maturation markers on human dendritic cells and potentiated CD40L activity. These results suggest that CD40 self-assembly modulates signaling, possibly by maintaining the receptor in a quiescent state.

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.

Tumor Necrosis Factor Receptor–Associated Factor 2 Signaling Provokes Adverse Cardiac Remodeling in the Adult Mammalian Heart

Tumor necrosis factor superfamily ligands provoke a dilated cardiac phenotype signal through a common scaffolding protein termed tumor necrosis factor receptor-associated factor 2 (TRAF2); however, virtually nothing is known about TRAF2 signaling in the adult mammalian heart. We generated multiple founder lines of mice with cardiac-restricted overexpression of TRAF2 and characterized the phenotype of mice with higher expression levels of TRAF2 (myosin heavy chain [MHC]-TRAF2(HC)). MHC-TRAF2(HC) transgenic mice developed a time-dependent increase in cardiac hypertrophy, left ventricular dilation, and adverse left ventricular remodeling, and a significant decrease in LV+dP/dt and LV-dP/dt when compared with littermate controls (P<0.05 compared with littermate). During the early phases of left ventricular remodeling, there was a significant increase in total matrix metalloproteinase activity that corresponded with a decrease in total myocardial fibrillar collagen content. As the MHC-TRAF2(HC) mice aged, there was a significant decrease in total matrix metalloproteinase activity accompanied by an increase in total fibrillar collagen content and an increase in myocardial tissue inhibitor of metalloproteinase-1 levels. There was a significant increase in nuclear factor-κB activation at 4 to 12 weeks and jun N-terminal kinases activation at 4 weeks in the MHC-TRAF2(HC) mice. Transciptional profiling revealed that >95% of the hypertrophic/dilated cardiomyopathy-related genes that were significantly upregulated genes in the MHC-TRAF2(HC) hearts contained κB elements in their promoters. These results show for the first time that targeted overexpression of TRAF2 is sufficient to mediate adverse cardiac remodeling in the heart.

ImmunoChip Study Implicates Antigen Presentation to T Cells in Narcolepsy

Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin) deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip). Three loci located outside the Human Leukocyte Antigen (HLA) region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.

Do γδ T cells predict osteonecrosis of the jaw?

A large body of evidence has established that T cells and cytokine mediators originating from the immune system potently impact bone turnover in both physiologic and pathologic conditions. Indeed, osteoclasts derive from precursors that circulate within the immune compartment; however, new revelations continue to emerge attesting to the depth of integration of the skeletal and immune systems, and the consequences of the immuno-skeletal interface for bone metabolism. Activated T cells have long been recognized to promote osteoclast formation as a consequence of their capacity to secrete osteoclastogenic cytokines including receptor activator of NF-kB ligand (RANKL)(1–5); the RANKL-independent osteoclastogenic cytokines, secreted osteoclastogenic factor of activated T cells (SOFAT)(6) and tumor necrosis factor (TNF) ligand superfamily member 14 (TNSF14, or LIGHT),(7) and the inflammatory mediator TNFα.(8) TNFα stimulates RANKL production in osteoblastic cells(9) and potently synergizes with RANKL to intensify osteoclastogenesis(8,10,11) and resorption.(12) Indeed, RANKL and/or TNFα, derived in part from activated T cells, have been implicated in bone loss and joint destruction in animal models of rheumatoid arthritis,(13) alveolar bone loss in periodontal infection,(14,15) and in humans and animal models of the archetypal osteoporotic bone disease, postmenopausal osteoporosis.(5,16,17) Interestingly, although lymphocytes are protagonists of bone loss in inflammatory states, under physiologic conditions both human(18) and rodent B cells(19,20) secrete the RANKL decoy receptor osteoprotegerin (OPG), an activity potentiated by T cell costimulation.(19) T cells are thus central to the regulation of basal bone turnover as well as protagonists of pathological bone loss.

The Role of Ubiquitination in TWEAK-Stimulated Signaling

Tumor necrosis factor superfamily ligands and receptors are responsible for development, immunity, and homeostasis of metazoan organisms. Thus, it is not surprising that signals emanating from these receptors are tightly regulated. Binding of TNF-related weak inducer of apoptosis (TWEAK) to its cognate receptor, FN14, triggers the assembly of receptor-associated signaling complex, which allows the activation of canonical and non-canonical nuclear factor kappa B (NF-κB) as well as mitogen-activated protein kinase signaling pathways. Ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1 and 2) and adaptor proteins TNFR-associated factors 2 and 3 (TRAF2 and TRAF3) are crucial for the regulation of TWEAK signaling as they facilitate the recruitment of distal signaling components including IKK and linear ubiquitin chain assembly complex complexes. At the same time c-IAP1/2, together with TRAF2 and TRAF3, promote constitutive ubiquitination and proteasomal degradation of NF-κB inducing kinase (NIK) – a kinase with critical role in the activation of non-canonical NF-κB signaling. While c-IAP1/2 mediated ubiquitination allows the activation of TWEAK-stimulated canonical NF-κB signaling, these E3 ligases are negative regulators of non-canonical signaling. TWEAK stimulation prompts the recruitment of c-IAP1/2 as well as TRAF2 and TRAF3 to the FN14 signaling complex leading to c-IAP1/2 autoubiquitination and degradation, which stabilizes NIK and allows subsequent phosphorylation of IKKα and partial proteasomal processing of p100 to activate gene expression. Recent studies have revealed that the spatio-temporal pattern of TWEAK-stimulated ubiquitination is a carefully orchestrated process involving several substrates that are modified by different ubiquitin linkages. Understanding the significance of ubiquitination for TWEAK signaling is important for the overall understanding of TWEAK biology and for the design of therapeutics that can be used in the treatment of human pathologies that are driven by TWEAK/FN14 expression and activity.

Ampicillin-Improved Glucose Tolerance in Diet-Induced Obese C57BL/6NTac Mice Is Age Dependent

Ampicillin has been shown to improve glucose tolerance in mice. We hypothesized that this effect is present only if treatment is initiated prior to weaning and that it disappears when treatment is terminated. High-fat fed C57BL/6NTac mice were divided into groups that received Ampicillin at different ages or not at all. We found that both diet and Ampicillin significantly changed the gut microbiota composition in the animals. Furthermore, there was a significant improvement in glucose tolerance in Ampicillin-treated, five-week-old mice compared to nontreated mice in the control group. At study termination, expressions of mRNA coding for tumor necrosis factor, serum amyloid A, and lactase were upregulated, while the expression of tumor necrosis factor (ligand) superfamily member 15 was downregulated in the ileum of Ampicillin-treated mice. Higher dendritic cell percentages were found systemically in high-fat diet mice, and a lower tolerogenic dendritic cell percentage was found both in relation to high-fat diet and late Ampicillin treatment. The results support our hypothesis that a “window” exists early in life in which an alteration of the gut microbiota affects glucose tolerance as well as development of gut immunity and that this window may disappear after weaning.

Characterizing Immunophenotypic and Functional Pseudo-Exhaustion in T Cells From CLL Patients: The Impact of Lenalidomide

AbstractAbstract 564The ability to evade immune destruction is increasingly being recognised as a crucial feature of cancer cells. Chronic lymphocytic leukemia (CLL) is associated with profound defects in T-cell function, resulting in failure of anti-tumor immunity and increased susceptibility to infections. T cells from CLL patients exhibit functional defects and alterations in gene expression, that show similarities to exhausted T cells in chronic viral infections. However, it is unclear whether CLL T cells are truly exhausted, or whether these defects are restricted to expanded populations of CMV specific T cells. We investigated the phenotype and function of CD8+ T cells from CLL patients and controls matched for age and CMV-serostatus. We demonstrate an increased proportion of CCR7- effector T cells in both CLL patients and CMV-seropositive individuals (p<0.05). CD8+ and CD4+ T cells from CLL patients had increased expression of exhaustion markers CD160 and CD244 irrespective of CMV-serostatus (p<0.01), whereas increased PD1 expression on CD8+ T cells was limited to CMV-seronegative patients (p=0.002). CLL CD8+ T cells also showed functional defects in proliferation and cytotoxicity irrespective of CMV-serostatus, with the cytolytic defect caused by a combination of impaired granzyme B packaging into secretory vesicles and non-polarized degranulation. In contrast to virally-induced exhaustion, CLL T cells showed increased production of interferon-γ with increased T-BET expression (p<0.01), normal IL-2 production, and no downregulation of IL-7R. Therefore, while CLL CD8+ T cells exhibit some features of T-cell exhaustion, they show important differences (Table 1). These findings also exclude CMV as the sole cause of T cell defects in CLL.Lenalidomide has recently been demonstrated to have significant clinical activity in CLL. Its mechanism of action in this disease is not well understood, but it thought to act primarily by a combination of CLL cell and immune cell activation. We therefore examined the ability of lenalidomide to repair the observed T cell defects by investigating the impact of this agent on the gene expression profiles and function of CLL T cells. Treatment of CLL CD8+ T cells with lenalidomide increased the expression of 137 genes, while 34 genes were downregulated. The most prominent changes in expression were of genes involved in cytoskeletal signaling including WASF1 (Wiskott-Aldrich syndrome protein, family member 1), and TPM2 (tropomyosin 2). There was also upregulation of genes involved in lymphocyte activation, including TNFSF4 (Tumor necrosis factor ligand superfamily, member 4: OX40L), LAG3 (Lymphocyte-activation gene 3), and TNF, and genes involved in cell proliferation such as IKZF1 (Ikaros) and GRN (Granulin). Although lenalidomide treatment or anti-CD3 stimulation alone had no impact on T-bet expression, co-treatment with both anti-CD3 stimulation and lenalidomide resulted in significantly enhanced T-bet expression and increased production of interferon-γ. In contrast, lenalidomide treatment alone was able to improve T cell cytotoxic function, associated with repair of trafficking of granzyme B into the immunological synapse.In conclusion, T cells from CLL patients exhibit features of T-cell pseudo-exhaustion that are present irrespective of CMV serostatus. Treatment of CLL T cells with lenalidomide results in upregulation of genes involved in proliferation, activation, and cytoskeletal pathways, resulting in repair of the functional T cell defects.Table 1Comparison of the phenotypic and functional defects of T cells from CLL patients with T-cell “exhaustion” in chronic viral infectionsExhausted T cells in chronic viral infectionsT cells from CLL patientsIncreased expression of inhibitory receptorsYesYesAbnormal transcription factor profileYesYesReduced proliferative potentialYesYesDecreased expression of IL-7R (CD127)YesNoDecreased cytokine production ↓IL-2, ↓IFN-γYesNoImpaired cytotoxicityYesYes Disclosures:Riches:Celgene: Research Funding. Gribben:Celgene: Honoraria; Roche: Honoraria; Pharmacyclics: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria.

Molecular Pathways That Determine the Activation State of Macrophages within the Myeloma Niche

AbstractAbstract 443Benefit from cytotoxic therapy in myeloma may be limited by persistence of residual tumor cells nested within favorable niches. However, the contribution of macrophages to the regulation of myeloma niches is still incompletely understood. We have previously shown that macrophages provide growth and anti-apoptotic signals to myeloma cells when grown in co-culture. These results prompted us to investigate the regulation of primary monocytes/macrophages that reside within myeloma niches in the bone marrow.Unmanipulated CD14+ monocytic cells, freshly explanted from myeloma bone marrows, displayed a pre-dominantly pro-inflammatory transcriptomic profile when compared to normal monocytes. We found enhanced transcription of genes encoding pro-inflammatory mediators, known to support growth and survival of myeloma cells, such as TNFalpha, IL-1beta, IL-6, IL-8 and TACI. Downregulation of TGFbeta was also consistent with a pro-inflammatory (M1) signature. Interestingly, we also found concurrent transcription of some genes characteristic of “alternative macrophage activation” (M2 phenotype) such as IL-10 and IL1-receptor antagonist (IL-1RN). These results suggest that myeloma-associated macrophages, while being predominantly pro-inflammatory, display significant plasticity between the M1-M2 phenotypic extremes.To obtain insights into the underlying mechanisms, we examined the role of TPL2 (Cot, MAP3K8), a serine/threonine kinase with central and non-redundant roles in regulating innate immune responses in macrophages following activation by Toll receptor (TLR) ligands and members of the TNF ligand superfamily. In myeloma-associated macrophages, we found constitutive activation of a TPL2 kinase-dependent, ERK-mediated pathway that promotes synthesis and processing of pro-inflammatory cytokines, including TNFalpha and IL-1beta. We also discovered constitutive activation of AKT at Ser473, a site dependent for its phosphorylation on TPL2 activity in macrophages responding to TLR signaling. Notably, the Akt/mTOR pathway limits the magnitude and duration of macrophage activation, in part through synthesis of the anti-inflammatory cytokine IL-10. These events involve signaling through STAT3. Accordingly, we discovered constitutive phosphorylation of STAT3 at a site regulated by TPL2 in activated macrophages.In addition to non-tumor cell autonomous roles in regulating myeloma through macrophages, we showed a tumor cell-autonomous, growth-regulatory role of TPL2 kinase. Treatment of myeloma cells with a TPL2 small molecule inhibitor resulted in apoptosis that was not rescued by the presence of patient-derived stromal cells. We postulate that TPL2 inhibition interferes with growth signaling in myeloma cells because TPL2 has been shown to substitute for RAF proteins in growth signal transduction. Interestingly, we found that TPL2 was activated by phosphorylation as cells entered G2/M. Treatment with nocodazole increased the proportion of cells that co-expressed phosphorylated TPL2 and phosphorylated histone H3. Moreover, we found that TPL2 activity was required for MAPK pathway signal transduction in response to TNF receptor stimulation in myeloma cells.Taken together, our results provide important novel insights into the regulation of macrophages within primary myeloma niches in the bone marrow. Plasticity between M1 and M2 phenotypes may correlate tightly with the actions of TPL2 kinase. In the myeloma niche, TPL2 activity helps to fine-tune macrophage activation by promoting synthesis and release of pro-inflammatory cytokines required by the myeloma tumor cell while engaging counter-regulatory mechanisms to prevent tissue destruction mediated by activated macrophages. Additionally, we described a growth regulatory role of TPL2 in the tumor cell itself. Thus, TPL2 blockade may disrupt crucial macrophage-tumor cell interactions within myeloma niches. Disclosures:No relevant conflicts of interest to declare.

APRIL Binding to BCMA Activates a JNK2–FOXO3–GADD45 Pathway and Induces a G2/M Cell Growth Arrest in Liver Cells

The TNF superfamily ligands APRIL and BAFF bind with different affinity to two receptors, BCMA and TACI, and induce cell survival and/or proliferation, whereas BAFF also binds specifically to BAFFR. These molecules were considered specific for the immune system. Recently, however, they were also found in epithelial and mesenchymal noncancerous and cancerous tissues and cell lines. In this article, we report that hepatocellular carcinoma (HCC) cell lines HepG2 and Hep3B and HCC specimens express APRIL and BAFF and their receptors BCMA and BAFFR, but not TACI; APRIL/BCMA is enhanced in HCC, compared with normal liver tissue. In contrast to previous reports, APRIL binding to BCMA decreases cell proliferation by inducing G(2)/M cell cycle arrest, whereas BAFF has no effect on cell growth. HCC cells therefore represent a rare system in which these two ligands (APRIL and BAFF) exert a differential effect and may serve as a model for specific APRIL/BCMA actions. We show that the effect of APRIL is mediated via BCMA, which does not activate the classical NF-κB pathway, whereas it induces a novel signaling pathway, which involves JNK2 phosphorylation, FOXO3A activation, and GADD45 transcription. In addition, JNK2 mediates the phosphorylation of Akt, which is activated but does not participate in the antiproliferative effect of APRIL. Furthermore, transcriptome analysis revealed that APRIL modifies genes specifically related to cell cycle modulation, including MCM2/4/5/6, CDC6, PCNA, and POLE2. Our data, therefore, identify a novel APRIL/BCMA signaling pathway in HCC and suggest that APRIL could have a pleiotropic role in tumor biology.

Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands

Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane-associate proteins, secreted proteins and exosomes. Exosomes are 30–100 nm endosome-derived vesicles consisting in cytosolic material surrounded by a lipid bilayer containing transmembrane proteins. We have previously shown that dendritic cells (DCs) express on their surface multiple TNF superfamily ligands (TNFSFLs), by which they can induce the apoptotic demise of tumor cells as well as the activation of natural killer (NK) cells. In the present study, we demonstrate that, similar to DCs, DC-derived exosomes (DCex) express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. We also show that DCex activate NK cells and stimulate them to secrete interferonγ (IFNγ) upon the interaction of DCex TNF with NK-cell TNF receptors. These data demonstrate that DCex can mediate essential innate immune functions that were previously ascribed to DCs.