Tumor Necrosis Factor Ligand Family Research Articles

Expression and functional characterization of bovine receptor activator of NF-κB ligand (RANKL)

Receptor activator of nuclear factor Kappa-B Ligand (RANKL) is a member of the tumor necrosis factor ligand (TNF) family involved in immune responses and immunomodulation. Expressed in various cells types around the body, RANKL plays a crucial role in bone remodeling and development of the thymus, lymph nodes and mammary glands. Research in other species demonstrates that RANKL is required for the development of microfold cells (M cells) in the gut, however limited information specific to cattle is available. Cloning and expression of bovine RANKL (BoRANKL) was carried out and bioactivity of the protein was demonstrated in the induction of osteoclast differentiation from both bovine and ovine bone marrow cells. The effects of BoRANKL on particle uptake in bovine enteroids was also assessed. The production of cross-reactive bovine RANKL protein will enable further investigations into cell differentiation using the available ruminant organoid systems, and their role in investigating host-pathogen interactions in cattle and sheep.

Fracture risks and their mechanisms in atopic dermatitis, focusing on receptor activator of nuclear factor kappa-Β ligand.

Recent multiple studies have shown that the long-term consequences of AD include an increased risk of osteoporosis and fracture, especially in hip, pelvic, spinal, and wrist fractures. AD is very common worldwide, and some kinds of fractures, such as hip fractures, are associated with increased mortality, which has a substantial socio-economic impact; however, the precise mechanisms for that remain unclear. Receptor activator of nuclear factor kappa-Β (RANK) ligand (RANKL) and osteoprotegerin (OPG) are members of the tumor necrosis factor ligand and receptor family, members of which also are known as bone biomarkers. Alterations in the RANKL/RANK/OPG system and the balance among these factors (represented by the RANKL/OPG ratio) are central to the pathogenesis of bone loss from osteoporosis, and it is postulated that the potential association between the serum levels of RANKL and OPG, and bone density or fracture. Recently, we demonstrated that serum RANKL/OPG ratio positively correlated with AD severity and suggests fracture risk in older women with AD. This review summarizes and discusses the risk and mechanisms of osteoporotic fracture in AD. RANKL may be involved in the pathogenesis of AD, regarding not only bone abnormality but also inflammation. Although further investigation will be needed to verify the hypotheses, recent findings may provide new insights into AD's pathogenesis and therapeutic targets.

Epithelial-to-mesenchymal transition promotes immune escape by inducing CD70 in non-small cell lung cancer

IntroductionEpithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. MethodsWe evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). ResultsWe uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. ConclusionOur results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

Receptor Specificity Engineering of TNF Superfamily Ligands.

The tumor necrosis factor (TNF) ligand family has nine ligands that show promiscuity in binding multiple receptors. As different receptors transduce into diverse pathways, the study on the functional role of natural ligands is very complex. In this review, we discuss the TNF ligands engineering for receptor specificity and summarize the performance of the ligand variants in vivo and in vitro. Those variants have an increased binding affinity to specific receptors to enhance the cell signal conduction and have reduced side effects due to a lowered binding to untargeted receptors. Refining receptor specificity is a promising research strategy for improving the application of multi-receptor ligands. Further, the settled variants also provide experimental guidance for engineering receptor specificity on other proteins with multiple receptors.

Assessment of serum TWEAK levels in patients with familial Mediterranean fever

Background/Aim: Mediterranean fever is an autoinflammatory disease characterized by recurrent attacks. Tumor necrosis factor (TNF) - like weak inducer of apoptosis (TWEAK) is a member of the TNF ligand family, and it has been reported to contribute significantly to the initiation of many inflammatory and immunological processes. In previous studies, an increasing amount of evidence has implicated the participation of TWEAK / Fn14 pathway in the pathogenesis of rheumatic inflammatory diseases that include rheumatoid arthritis, systemic lupus erythematosus and Behçet's disease. The aim of this study was to investigate the serum TWEAK levels of patients with Familial Mediterranean fever (FMF) and its possible relationship with inflammatory markers and disease activity. Methods: Our study included 20 patients with FMF and 19 healthy volunteers. Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) were measured, and PRAS disease severity score was determined in patients with FMF. Also, the FMF attack period was questioned. Serum TWEAK levels were measured with available commercial Enzyme Linked Immunosorbent Assay kits. Results: There was no significant difference in terms of age and gender between the FMF group and the healthy control group (P>0.05, for all). ESR, CRP and serum TWEAK levels were significantly higher in patients with FMF (P<0.001 for all). PRAS score in FMF patients was 3.4. Serum TWEAK level was not correlated with ESR (r=-0.042, P=0.0801), CRP (r=-0.017 P=0.921), or PRAS score (r=0.247, P=0.149). The ESR and CRP levels of patients in FMF attack period were significantly higher compared to attack free period (P<0.001 for both) whereas there was no significant difference in serum TWEAK levels (P=0.686). Conclusions: Serum TWEAK levels are increased in FMF disease with attacks. However, this increase is not associated with increased ESR and CRP during FMF attacks. These results indicate that the TWEAK / Fn14 pathway plays a role in earlier stages where the inflammatory pathways have not differentiated yet. Serum TWEAK levels appear to be more successful in reflecting a lower degree of inflammation compared to ESR and CRP.

Evaluation of Computationally Designed Peptides against TWEAK, a Cytokine of the Tumour Necrosis Factor Ligand Family.

The tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumour necrosis factor ligand family and has been shown to be overexpressed in tumoral cells together with the fibroblast growth factor–inducible 14 (Fn14) receptor. TWEAK-Fn14 interaction triggers a set of intracellular pathways responsible for tumour cell invasion and migration, as well as proliferation and angiogenesis. Hence, modulation of the TWEAK-Fn14 interaction is an important therapeutic goal. The targeting of protein-protein interactions by external agents, e.g., drugs, remains a substantial challenge. Given their intrinsic features, as well as recent advances that improve their pharmacological profiles, peptides have arisen as promising agents in this regard. Here, we report, by in silico structural design validated by cell-based and in vitro assays, the discovery of four peptides able to target TWEAK. Our results show that, when added to TWEAK-dependent cellular cultures, peptides cause a down-regulation of genes that are part of TWEAK-Fn14 signalling pathway. The direct, physical interaction between the peptides and TWEAK was further elucidated in an in vitro assay which confirmed that the bioactivity shown in cell-based assays was due to the targeting of TWEAK. The results presented here are framed within early pre-clinical drug development and therefore these peptide hits represent a starting point for the development of novel therapeutic agents. Our approach exemplifies the powerful combination of in silico and experimental efforts to quickly identify peptides with desirable traits.

Transcriptome Remodeling in Gradual Development of Inverse Resistance between Paclitaxel and Cisplatin in Ovarian Cancer Cells

Resistance to anti-cancer drugs is the main challenge in oncology. In pre-clinical studies, established cancer cell lines are primary tools in deciphering molecular mechanisms of this phenomenon. In this study, we proposed a new, transcriptome-focused approach, utilizing a model of isogenic cancer cell lines with gradually changing resistance. We analyzed trends in gene expression in the aim to find out a scaffold of resistance development process. The ovarian cancer cell line A2780 was treated with stepwise increased concentrations of paclitaxel (PTX) to generate a series of drug resistant sublines. To monitor transcriptome changes we submitted them to mRNA-sequencing, followed by the identification of differentially expressed genes (DEGs), principal component analysis (PCA), and hierarchical clustering. Functional interactions of proteins, encoded by DEGs, were analyzed by building protein-protein interaction (PPI) networks. We obtained human ovarian cancer cell lines with gradually developed resistance to PTX and collateral sensitivity to cisplatin (CDDP) (inverse resistance). In their transcriptomes, we identified two groups of DEGs: (1) With fluctuations in expression in the course of resistance acquiring; and (2) with a consistently changed expression at each stage of resistance development, constituting a scaffold of the process. In the scaffold PPI network, the cell cycle regulator—polo-like kinase 2 (PLK2); proteins belonging to the tumor necrosis factor (TNF) ligand and receptor family, as well as to the ephrin receptor family were found, and moreover, proteins linked to osteo- and chondrogenesis and the nervous system development. Our cellular model of drug resistance allowed for keeping track of trends in gene expression and studying this phenomenon as a process of evolution, reflected by global transcriptome remodeling. This approach enabled us to explore novel candidate genes and surmise that abrogation of the osteomimic phenotype in ovarian cancer cells might occur during the development of inverse resistance between PTX and CDDP.

Distinct dendritic cell responses

Dendritic Cells Dendritic cells (DCs) are critical for activating naive T cells by presenting antigens and providing costimulation, processes that are enhanced by cytokine signals from the surrounding environment. Girard et al. used single-cell proteogenomics and flow cytometry to examine cell type–specific responses of human peripheral blood monocyte and DC subsets to type I interferon. Interferon-β induced maturation of the recently identified CD1c+ CD5− DC3 subset, which was characterized by distinct expression of the costimulatory molecule GITRL, a tumor necrosis factor family ligand, and driven by nuclear factor κB signaling. These results identify conserved and cell type–specific features of the type I interferon response of human mononuclear phagocyte subsets, including the molecular signals that monocytes and DCs may leverage to instruct T cells. Sci. Immunol. 5 , eabe0347 (2020).

BAFF gets involved in response to bacterial infection and promotes IgM+ B cell survival in Nile tilapia (Oreochromis niloticus)

B-cell activating factor (BAFF), a member of the tumor necrosis factor (TNF) ligand family, is known to play a notable role in the survival and differentiation of B cells. In this study, we conducted functional analysis of BAFF in Nile tilapia (Oreochromis niloticus). Expression analysis revealed that OnBAFF was widely distributed in all tested tissues but expressed highly in the spleen. Analysis of sorted IgM positive (IgM+) and IgM negative (IgM−) lymphocyte populations from spleen and head kidney, the results indicated that OnBAFF had a high expression in the IgM+ B cells. In addition, in vivo, the OnBAFF expression was significantly up-regulated in spleen and head kidney after challenges of Streptococcus agalactiae (S. agalactiae) and lipopolysaccharide (LPS), and the similar significant up-regulation of OnBAFF was also observed in the spleen and head kidney leukocytes after stimulations with these two different immunogens in vitro. Furthermore, the recombinant OnBAFF ((r)OnBAFF) protein could improve the survival rate of IgM+ B cells in vitro. Moreover, the transcription levels of OnBAFF receptors were detected in IgM+ B cells, revealing that the OnBAFF-R and OnTACI were expressed much higher in the IgM+ B cells. Taken together, this study indicated that OnBAFF might get involved in host immune resistance against bacterial infection and promote IgM+ B cell survival in Nile tilapia.

Targeting CD70 with Cusatuzumab Eliminates Acute Myeloid Leukemia Stem Cells in Humans

Introduction Acute myeloid leukemia (AML) is a heterogenous hematological malignancy driven by leukemia stem cells (LSCs) (Lapidot et al, 1994). LSCs resistant against conventional chemotherapy represent the major cause of relapse. Elderly or unfit AML patients not eligible for intensive chemotherapy are treated in a palliative setting with hypomethylating agents (HMA) or low dose Ara-C, but responses are modest and not durable. The reason for the low efficacy of HMA treatment is their insufficient action on the disease initiating- and -maintaining LSC population (Craddock et al, 2013). We recently demonstrated that CD34+ AML cells (progenitors and LSC) consistently express the tumor necrosis factor family ligand CD70 as well as its receptor CD27 and that cell-autonomous CD70/CD27-signaling propagates the disease (Riether et al. 2017). The aim of the current study was to evaluate whether resistance to HMA treatment can be overcome by combining HMA with an anti-CD70 monoclonal antibody treatment. Experimental design The effect of HMA treatment on the expression of CD70 on primary human CD34+CD38- AML LSCs was determined in vitro cultures and in patients treated with HMA in vivo. The therapeutic potential of targeting CD70-expressing LSCs in presence and absence of HMA was assessed using the anti-CD70 ADCC-optimized monoclonal antibody (mAb), cusatuzumab, and an effector-dead anti-CD70 mAb in colony formation and re-plating assays as well as patient-derived xenograft models (Silence et al, 2014). The clinical relevance of the findings was determined in a clinical phase 1 trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with the HMA azacitidine (AZA, NCT03030612). Four different dose levels of cusatuzumab (1, 3, 10 and 20 mg/kg Q2W) were studied; AZA was administered at 75 mg/m² for 7 days every 28 days. Results We found that resistance of AML LSCs to HMA treatment is mediated by the up-regulation of the CD70. The up-regulation of CD70 triggered cell-autonomous CD70/CD27 signaling on AML LSCs. Based on these findings we hypothesized that the upregulation of CD70 by HMA may render LSCs more susceptible to CD70-targeting interventions. Targeting CD70-expressing LSCs by a blocking anti-CD70 mAb and the anti-CD70 mAb cusatuzumab, which blocks CD70/CD27-signaling and additionally mediates ADCC and CDC, eradicated LSCs in colony and re-plating assays in vitro and in xenotransplantation experiments in vivo. HMA in combination with blocking αCD70 mAb synergistically reduced LSC numbers in vivo and this was even more efficient when ADCC-enhanced αCD70 mAb cusatuzumab was added in the presence of NK cells. In order to test the hypothesis that targeting CD70 in combination with HMA eliminates LSCs in AML patients, we initiated a phase 1 dose-escalation trial in previously untreated elderly AML patients with a single dose of cusatuzumab monotherapy followed by a combination therapy with azacitidine. No dose-limiting toxicities (DLT) were observed in the dose-escalation phase 1 trial and responses were observed across the dose levels (1-20 mg/kg). A single dose of cusatuzumab reduced bone marrow blasts in just two weeks in all patients on average by 32%. Cusatuzumab monotherapy significantly reduced LSC numbers and frequencies in all patients analyzed in the bone marrow as assessed in limiting dilution colony assays. Single cell sequencing analysis revealed that cusatuzumab induced gene signatures related to myeloid differentiation and apoptosis in LSCs. In combination with azacitidine, cusatuzumab induced CR/CRi in 10 out of 12 patients. Responses were observed at all dose levels of cusatuzumab and median time to response was 3.3 months. Conclusions Blocking CD70/CD27-signaling and targeting CD70-expressing LSCs by the ADCC-optimized mAb, cusatuzumab, eliminated LSCs in vitro and in xenotransplantation experiments. In a phase 1 study promising activity of cusatuzumab in combination with HMA was observed in AML patients, in which translational data indicate that cusatuzumab selectively eliminates CD70-expressing LSCs. Disclosures Van Rompaey: argenx: Employment, Equity Ownership, Patents &amp; Royalties. Moshir:Argenx: Employment, Equity Ownership. Delahaye:argenx: Employment, Equity Ownership. Gandini:Argenx: Employment, Equity Ownership. Erzeel:argenx: Employment, Equity Ownership. Hultberg:Argenx: Employment. Fung:argenx: Consultancy, Equity Ownership. De Haard:Argenx: Employment, Equity Ownership, Patents &amp; Royalties. Leupin:Argenx: Employment, Equity Ownership, Patents &amp; Royalties.

The TNF Family of Ligands and Receptors: Communication Modules in the Immune System and Beyond.

The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies (TNFSF/TNFRSF) include 19 ligands and 29 receptors that play important roles in the modulation of cellular functions. The communication pathways mediated by TNFSF/TNFRSF are essential for numerous developmental, homeostatic, and stimulus-responsive processes in vivo. TNFSF/TNFRSF members regulate cellular differentiation, survival, and programmed death, but their most critical functions pertain to the immune system. Both innate and adaptive immune cells are controlled by TNFSF/TNFRSF members in a manner that is crucial for the coordination of various mechanisms driving either co-stimulation or co-inhibition of the immune response. Dysregulation of these same signaling pathways has been implicated in inflammatory and autoimmune diseases, highlighting the importance of their tight regulation. Investigation of the control of TNFSF/TNFRSF activities has led to the development of therapeutics with the potential to reduce chronic inflammation or promote anti-tumor immunity. The study of TNFSF/TNFRSF proteins has exploded over the last 30 yr, but there remains a need to better understand the fundamental mechanisms underlying the molecular pathways they mediate to design more effective anti-inflammatory and anti-cancer therapies.

Molecular mechanism of the selectivity between BAFF/APRIL and their receptors by molecular simulations

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) belonging to the tumour necrosis factor (TNF) ligand family can bind three unusual TNF receptors (BCMA, TACI and BR3) with various binding affinities. BAFF and APRIL are regarded as promising therapeutic targets for autoimmune diseases because of their pivotal roles in cell survival and immune regulation. In this work, we carried out molecular dynamics calculations to explore the structural and chemical features responsible for ligand recognition by extracellular functional segments of TNF receptors. We found that the conserved pocket Dcons of BAFF/APRIL contacted the DxL motif of TNF receptors, while the Dspe1–3 sub-domains were responsible for their different affinities, especially Dspe1 and Dspe2. The residues at position II–V of DxL motif were wrapped into the Dcons pocket via salt-bridge and hydrophobic interactions. The hydrophobic residues of strand3 and helix1 in TNF receptors provided remarkable contributions for the affinities to BAFF/APRIL. Additionally, ArgVI of DxL motif played a key role in the binding selectivity via salt-bridge interaction with residue D275B in BAFF. Arg27 in BCMA contributed to the high affinity for APRIL so that BCMA showed a preference for APRIL. Our studies indicated that Arg84 and Gln95 in TACI2 played an important role in the selectivity of two cysteine-rich domain segments in TACI, leading to the higher binding affinities of TACI2 than those of TACI1. The primary cause of the disability to bind APRIL was the space conflict with the rigid conformation of the C-terminus coil of BR3. These thorough understanding of the molecular mechanism for BAFF/APRIL recognition by their receptors provides new insights for guiding inhibitor design.

Identification and characteristic of three members of the C1q/TNF-related proteins (CTRPs) superfamily in Eudontomyzon morii

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and acquired immunity. C1q and the multifunctional tumor necrosis factor (TNF) ligand family is of similar crystal structures, are designated the C1q/TNF-related proteins (CTRPs) superfamily. They are involved in processes as diverse as host defense, inflammation, apoptosis, autoimmunity, cell differentiation, organogenesis, hibernation and insulinresistant obesity. In this study, three members of the CTRPs superfamily were isolated and characterized in Yalu River lampreys (Eudontomyzon morii), and are respectively named LaC1qC, LaCTRP1, LaCTRP9. The full-length cDNAs of C1qC-like (LaC1qAL), CTRP1-like (LaCTRP1), and CTRP9-like (LaCTRP9) consist of 723, 762 and 825 bp of nucleotide sequence encoding polypeptides of 241, 254 and 275 amino acids, respectively. All-three proteins share three common domains: a signal peptide at the N terminus, a collagenous domain (characteristic Gly-X-Y repeats), and a C-terminal globular domain. In addition, the higher expression level of the three proteins in heart by RT-PCR and real-time PCR tissue profiling implied that they might involve in immune response or injury repair of the heart in lamprey.

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Editorial: Biology-Driven Targeted Therapy of Pediatric Soft-Tissue and Bone Tumors: Current Opportunities and Future Challenges.

Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway.

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-κB in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-κB is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants.

Immunostimulation by OX40 Ligand Transgenic Ewing Sarcoma Cells.

Interleukin-2 (IL-2) transgenic Ewing sarcoma cells can induce tumor specific T and NK cell responses and reduce tumor growth in vivo and in vitro. Nevertheless, the efficiency of this stimulation is not high enough to inhibit tumor growth completely. In addition to recognition of the cognate antigen, optimal T-cell stimulation requires signals from so-called co-stimulatory molecules. Several members of the tumor necrosis factor superfamily have been identified as co-stimulatory molecules that can augment antitumor immune responses. OX40 (CD134) and OX40 ligand (OX40L = CD252; also known as tumor necrosis factor ligand family member 4) is one example of such receptor/ligand pair with co-stimulatory function. In the present investigation, we generated OX40L transgenic Ewing sarcoma cells and tested their immunostimulatory activity in vitro. OX40L transgenic Ewing sarcoma cells showed preserved expression of Ewing sarcoma-associated (anti)gens including lipase member I, cyclin D1 (CCND1), cytochrome P450 family member 26B1 (CYP26B1), and the Ewing sarcoma breakpoint region 1-friend leukemia virus integration 1 (EWSR1-FLI1) oncogene. OX40L-expressing tumor cells showed a trend for enhanced immune stimulation against Ewing sarcoma cells in combination with IL-2 and stimulation of CD137. Our data suggest that inclusion of the OX40/OX40L pathway of co-stimulation might improve immunotherapy strategies for the treatment of Ewing sarcoma.

Molecular characterization and expression analysis of B cell activating factor from rock bream (Oplegnathus fasciatus)

B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) ligand family. BAFF has been shown to induce survival and proliferation of lymphocytes. We characterized the gene encoding BAFF (RbBAFF) in rock bream (Oplegnathus fasciatus), and attempted to determine its biological functions upon immune responses. In silico analysis of RbBAFF demonstrated the presence of common TNF ligand family features, including a TNF domain, a D-E loop, and three cysteine residues that are crucial for trimer formation. Amino acid sequence alignment confirmed that RbBAFF and its homologs were conserved at secondary and tertiary levels. Transcriptional analysis indicated that RbBAFF mRNAs were ubiquitously expressed in wide array of tissues. The higher levels of constitutive expression were observed in the kidney, head kidney and spleen, suggesting an important physiological relationship with lymphocytes. Under pathological conditions, RbBAFF mRNA levels were significantly elevated. The role of RbBAFF in lymphocyte survival and proliferation was confirmed by MTT assays and flow cytometry. Recombinant RbBAFF protein (10 μg/mL) was able to prolong the survival and/or enhance the proliferation of rock bream lymphocytes by approximately 30%. Transcription of IL-10 and NFκB-1 was significantly stimulated by RbBAFF. Our findings provide further information regarding fish BAFF gene and its role in adaptive immunity.

The ever-expanding immunomodulatory role of calreticulin in cancer immunity.

Calreticulin is a pleiotropic molecule that normally resides in the lumen of the endoplasmic reticulum (ER). Here, it has various functions, ranging from regulation of calcium homeostasis to ensuring proper protein folding. More recently, calreticulin gained special interest for its extracellular functions, where it has direct immunomodulatory activity. In this respect, calreticulin activates dendritic cells and macrophages. In addition, certain anti-cancer therapies induce the translocation of calreticulin from the ER to the cell surface of dying cancer cells, where calreticulin dictates the immunogenicity of these cells. Interestingly, treatment with tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) also induces membrane calreticulin exposure on cancer cells. As shown here, calreticulin directly interacts with TRAIL and its receptor-signaling complex, as well as with other TNF family members. Of note, TRAIL is a well known immunomodulatory molecule, and is expressed on the surface of natural killer T-cells. Therefore, calreticulin may have an as yet unrecognized wide(r) impact on immunity, with the TNF-ligand family modulating virtually all aspects of the immune response.

Antibody-based targeting of TNF-ligands for cancer therapy

The tumor necrosis factor (TNF) ligand and cognate TNF receptor superfamily constitute an important immunoregulatory axis pivotal for the correct execution of immune responses. TNF ligand and receptor family members among others are involved in induction of cell death in malignant cells as well as in providing co-stimulatory signals that help mount effective anti-cancer immune responses. This diverse and important regulatory role in immunity has sparked great interest in the development of TNFL/TNFR-targeted cancer immunotherapeutics. Here, I will discuss our cancer immunotherapeutic drug discovery and development program using selected examples of the TNF-ligand superfamily.

RANKL/OPG; Critical role in bone physiology.

After it was proposed that the osteoblast lineage controlled the formation of osteoclasts, cell culture methods were developed that established this to be the case. Evidence was obtained that cytokines and hormones that promote osteoclast formation act first on osteoblast lineage cells to promote the production of a membrane-bound regulator of osteoclastogenesis. This proved to be receptor activator of NF-kB ligand (RANKL) a member of the tumor necrosis factor ligand family that acts upon its receptor RANK in the hematopoietic lineage, with interaction restricted by a decoy soluble receptor osteoprotegerin (OPG), also a product of the osteoblast lineage. The physiological roles of these factors were established through genetic and pharmacological studies, have led to a new physiology of bone, with complete revision of older ideas over the last 15 years, ultimately leading to the development of new pharmaceutical agents for bone disease.