Ovarian Cancer Ascites Inhibits Transcriptional Activation of NK Cells Partly through CA125.

Malignant ascites is a common clinical problem in ovarian cancer. NK cells are present in the ascites, but their antitumor activity is inhibited. The underlying mechanisms of the inhibition have yet to be fully elucidated. Using an Fcγ receptor-mediated NK cell activation assay, we show that ascites from ovarian cancer patients potently inhibits NK cell activation. Part of the inhibitory activity is mediated by CA125, a mucin 16 fragment shed from ovarian cancer tumors. Moreover, transcriptional analyses by RNA sequencing reveal upregulation of genes involved in multiple metabolic pathways but downregulation of genes involved in cytotoxicity and signaling pathways in NK cells purified from ovarian cancer patient ascites. Transcription of genes involved in cytotoxicity pathways are also downregulated in NK cells from healthy donors after in vitro treatment with ascites or with a CA125-enriched protein fraction. These results show that ascites and CA125 inhibit antitumor activity of NK cells at transcriptional levels by suppressing expression of genes involved in NK cell activation and cytotoxicity. Our findings shed light on the molecular mechanisms by which ascites inhibits the activity of NK cells and suggest possible approaches to reactivate NK cells for ovarian cancer immunotherapy.

Dengue Mosaic Vaccines Enhance Cellular Immunity and Expand the Breadth of Neutralizing Antibody Against All Four Serotypes of Dengue Viruses in Mice.

An estimated 400 million people in the world are infected with any of the four types of dengue virus (DENV) annually. The only licensed dengue vaccine cannot effectively prevent infection with all of the four DENVs, especially in those immunologically naïve at baseline. In this study, we explored a mosaic vaccine approach, which utilizes an artificial recombinant sequence for each serotype to achieve maximum coverage of variant epitopes in the four DENVs. We determined the immunogenicity and cross-reactivity of DNA plasmids encoding individual mosaic sequences or the natural sequences in mice. We show that the mosaic vaccines, particularly those targeting DENV serotype 1 and 2, improved vaccine immunogenicity by increasing the percentage of antigen-specific IFNγ- or TNFα-secreting CD4 and CD8 T cells, and titers of neutralizing antibodies. The mosaic vaccine diversified and broadened anti-dengue T cell responses and cross-reactive neutralizing antibodies against all four serotypes. The mosaic vaccines also induced higher level of antigen-specific B cell responses. These results suggest that mosaic vaccines comprising of DENV serotype 1 and 2 variant epitopes could stimulate strong and broad immune responses against all four serotypes.

Generation of a universal CD4 memory T cell recall peptide effective in humans, mice and non-human primates.

CD4T cells play a key role in humoral immunity by providing help to B cells, enabling effective antibody class switching and affinity maturation. Some vaccines may generate a poor response due to a lack of effective MHC class II epitopes, resulting in ineffective helper T cell activation and recall and consequently poor humoral immunity. It may be beneficial to provide a CD4T cell helper peptide with a vaccine particularly in the case of a poorly immunogenic antigen. Such a T cell helper peptide must be promiscuous in its ability to bind a broad range of MHC class II alleles due to broad allelic variation in the human population. We designed a chimeric MHC class II peptide (TpD) with epitopes from tetanus toxoid and diphtheria toxoid, separated by an internal cathepsin cleavage site. TpD was capable of inducing a memory recall response in peripheral blood mononuclear cells from 20/20 human donors. T cells responding to TpD showed a central memory phenotype. Immunization of mice with a synthetic nicotine nanoparticle vaccine containing TpD showed that the peptide was required for robust antibody production and resulted in a long term CD4 memory T cell recall response. As a pre-clinical model two non-human primate species, rhesus macaques and cynomolgus monkeys, were immunized with a nicotine nanoparticle vaccine and evaluated for an anti-nicotine antibody response and TpD specific memory T cells. We found that 4/4 rhesus monkeys had both sustained antibody production and TpD memory T cells for the duration of the experiment (119 days). In addition 30/30 cynomolgus monkeys dosed with nicotine vaccine nanoparticles showed dose-dependent antibody generation and T cell recall response compared to saline injected controls. In summary we have developed a potent universal memory T cell helper peptide (TpD) that is active in vitro in human PBMCs and in vivo in mice and non-human primates.

Selective amplification of glucocorticoid anti-inflammatory activity through synergistic multi-target action of a combination drug.

INTRODUCTION: Glucocorticoids are a mainstay of anti-inflammatory therapy, but significant adverse effects ultimately limit their utility. Previous efforts to design glucocorticoid structures with an increased therapeutic window have focused on dissociating anti-inflammatory transcriptional repression from adverse effects primarily driven by transcriptional activation. An alternative to this medicinal chemistry approach is a systems biology based strategy that seeks to amplify selectively the anti-inflammatory activity of very low dose glucocorticoid in immune cells without modulating alternative cellular networks that mediate glucocorticoid toxicity. METHODS: The combination of prednisolone and the antithrombotic drug dipyridamole was profiled using in vitro and in vivo models of anti-inflammatory activity and glucocorticoid-induced adverse effects to demonstrate a dissociated activity profile. RESULTS: The combination synergistically suppresses release of proinflammatory mediators, including tumour necrosis factor-alpha, IL-6, chemokine (C-C motif) ligand 5 (RANTES), matrix metalloproteinase-9, and others, from human peripheral blood mononuclear cells and mouse macrophages. In rat models of acute lipopolysaccharide-induced endotoxemia and delayed-type hypersensitivity, and in chronic models of collagen-induced and adjuvant-induced arthritis, the combination produced anti-inflammatory activity that required only a subtherapeutic dose of prednisolone. The immune-specific amplification of prednisolone anti-inflammatory activity by dipyridamole did not extend to glucocorticoid-mediated adverse effects, including corticosterone suppression or increased expression of tyrosine aminotransferase, in vivo after repeat dosing in rats. After 8 weeks of oral dosing in mice, treatment with the combination did not alter prednisolone-induced reduction in osteocalcin and mid-femur bone density, which are markers of steroid-induced osteoporosis. Additionally, amplification was not observed in the cellular network of corticotroph AtT-20/D16v-F2 cells in vitro, as measured by pro-opiomelanocortin expression and adrenocorticotropic hormone secretion. CONCLUSIONS: These data suggest that the multi-target mechanism of low-dose prednisolone and dipyridamole creates a dissociated activity profile with an increased therapeutic window through cellular network selective amplification of glucocorticoid-mediated anti-inflammatory signaling.

G protein-coupled receptor connectivity to NF-kappaB in inflammation and cancer.

Complex intracellular network interactions regulate gene expression and cellular behavior. Whether at the site of inflammation or within a tumor, individual cells are exposed to a plethora of signals. The transcription factor nuclear factor-kappaB (NF-kappaB) regulates genes that control key cellular activities involved in inflammatory diseases and cancer. NF-kappaB is regulated by several distinct signaling pathways that may be activated individually or simultaneously. Multiple ligands and heterologous cell-cell interactions have an impact on NF-kappaB activity. The G protein-coupled receptor (GPCR) superfamily makes up the largest class of transmembrane receptors in the human genome and has multiple molecularly distinct natural ligands. GPCRs regulate proliferation, differentiation, and chemotaxis and play a major role in inflammatory diseases and cancer. Both GPCRs and NF-kappaB have been, and continue to be, major targets for drug discovery. A clear understanding of network interactions between GPCR signaling pathways and those that control NF-kB may be valuable for the development of better drugs and drug combinations.

Regulatory T cell expression of herpesvirus entry mediator suppresses the function of B and T lymphocyte attenuator-positive effector T cells.

The binding of herpesvirus entry mediator (HVEM) to B and T lymphocyte attenuator (BTLA) is known to activate an inhibitory signaling cascade in effector T (Teff) cells, but we now report that the HVEM-BTLA pathway is also important to the suppressive function of regulatory T cells (Tregs). Although naive T cells up-regulated BTLA upon TCR activation, Treg expression of BTLA remained low, regardless of TCR activation. Moreover, BTLA(-/-) CD4(+)CD25(+) Tregs had normal suppressive activity, whereas BTLA(-/-) Teff cells were more resistant than wild-type Teff cells to suppression by Tregs, suggesting BTLA expression by Teff cells was required for their suppression by Tregs. In contrast to BTLA, HVEM expression was comparable in naive Tregs vs Teff cells, but after stimulation HVEM expression was quickly down-regulated by Teff cells, whereas HVEM was further up-regulated by Tregs. HVEM(-/-) Tregs had decreased suppressive activity as compared with wild-type Tregs, indicating that Treg expression of HVEM was required for optimal suppression. Consistent with this, T cells from Scurfy mice (FoxP3 mutant) lacked HVEM gene expression, and adoptively transferred wild-type but not HVEM(-/-) Tregs were able to control alloresponses in vivo by normal Teff cells. Our data demonstrate that Tregs can exert their effects via up-regulation of the negative costimulatory ligand HVEM, which upon binding to BTLA expressed by Teff cells helps mediate the suppressive functions of Tregs in vitro and in vivo.

Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Coordinated involvement of mast cells and T cells in allergic mucosal inflammation: critical role of the CC chemokine ligand 1:CCR8 axis.

CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by approximately 70% of CD4(+) T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4(+) T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4(+) effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.

Exploring the positive and negative consequences of NF-kappaB inhibition for the treatment of human disease.

The NF-kappaB (NF-kappaB) transcription factor-signaling pathway has become a major focus for research aimed at understanding its role in development, maintenance and progression of disease. A large body of recent research suggests inhibition of NF-kappaB blocks inflammation, cancer development and progression, diabetes, stroke, muscle wasting, and other diseases. The enormous potential for the treatment of disease by inhibiting NF-kappaB lead to the development of inhibitory drugs that specifically target this pathway. At the same time offering great clinical potential, inhibition of NF-kappaB in vivo can be detrimental. NF-kappaB controls multiple functions in homeostasis including a functional immune response, cell cycle, and cell death. Genetic studies in mice and analysis of naturally occurring mutations in humans point to specific developmental and immune consequences due to altering NF-kappaB activity. The balance between therapeutic benefit and potential changes in normal cellular function and response during drug induced NF-kappaB inhibition will be one of the challenges in future clinical studies.

Rapid TNFR1-dependent lymphocyte depletion in vivo with a selective chemical inhibitor of IKKbeta.

The transcription factor NF-kappaB plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKKbeta, an Ikappa-B kinase that regulates NF-kappaB. IKKbeta and NF-kappaB are required in vivo for prevention of TNFalpha-mediated apoptosis. ML120B sensitized mouse bone marrow progenitors and granulocytes, but not mature B cells to TNFalpha killing in vitro, and induced apoptosis in vivo in the bone marrow and spleen within 6 hours of a single oral dose. In vivo inhibition of IKKbeta with ML120B resulted in depletion of thymocytes and B cells in all stages of development in the bone marrow but did not deplete granulocytes. TNF receptor-deficient mouse thymocytes and B cells were resistant to ML120B-induced depletion in vivo. Surprisingly, surviving bone marrow granulocytes expressed TNFR1 and TNFR2 after dosing in vivo with ML120B. Our results show that inhibition of IKKbeta with a small molecule in vivo leads to rapid TNF-dependent depletion of T and B cells. This observation has several implications for potential use of IKKbeta inhibitors for the treatment of inflammatory disease and cancer.

Transgenic angiopoietin-like (angptl)4 overexpression and targeted disruption of angptl4 and angptl3: regulation of triglyceride metabolism.

Lipoprotein lipase (LPL) is a key regulator of triglyceride clearance. Its coordinated regulation during feeding and fasting is critical for maintaining lipid homeostasis and energy supply. Angiopoietin-like (Angptl)3 and Angptl4 are secreted proteins that have been demonstrated to regulate triglyceride metabolism by inhibiting LPL. We have taken a targeted genetic approach to generate Angptl4- and Angptl3-deficient mice as well as transgenic mice overexpressing human Angptl4 in the liver. The Angptl4 transgenic mice displayed elevated plasma triglycerides and reduced postheparin plasma (PHP) LPL activity. A purified recombinant Angptl4 protein inhibited mouse LPL and recombinant human LPL activity in vitro. In contrast to the transgenic mice, Angptl4-deficient mice displayed hypotriglyceridemia and increased PHP LPL activity, with greater effects in the fasted compared with the fed state. Angptl3-deficient mice also displayed hypotriglyceridemia with elevated PHP LPL activity, but these mice showed a greater effect in the fed state. Mice deficient in both Angptl proteins showed an additive effect on plasma triglycerides and did not survive past 2 months of age. Our results show that Angptl3 and Angptl4 function to regulate circulating triglyceride levels during different nutritional states and therefore play a role in lipid metabolism during feeding/fasting through differential inhibition of LPL.

Tumor necrosis factor-alpha triggers mucus production in airway epithelium through an IkappaB kinase beta-dependent mechanism.

Excessive mucus production by airway epithelium is a major characteristic of a number of respiratory diseases, including asthma, chronic bronchitis, and cystic fibrosis. However, the signal transduction pathways leading to mucus production are poorly understood. Here we examined the potential role of IkappaB kinase beta (IKKbeta) in mucus synthesis in vitro and in vivo. Tumor necrosis factor-alpha (TNF-alpha) or transforming growth factor-alpha stimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein. TNF-alpha stimulation induced IKKbeta-dependent p65 nuclear translocation, mucus synthesis, and production of cytokines from epithelial cells. TNF-alpha, but not transforming growth factor-alpha, induced mucus production dependent on IKKbeta-mediated NF-kappaB activation. In vivo, TNF-alpha induced NF-kappaB as determined by whole mouse body bioluminescence. This activation was localized to the epithelium as revealed by LacZ staining in NF-kappaB-LacZ transgenic mice. TNF-alpha-induced mucus production in vivo could also be inhibited by administration into the epithelium of an IKKbeta dominant negative adenovirus. Taken together, our results demonstrated the important role of IKKbeta in TNF-alpha-mediated mucus production in airway epithelium in vitro and in vivo.

PK1/EG-VEGF induces monocyte differentiation and activation.

Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up-regulating antigen-presenting and costimulatory molecules. We report a novel function for prokineticin-1 (PK1)/endocrine gland-derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down-regulation of B7-1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)-12 and tumor necrosis factor alpha and down-regulated IL-10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte-derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.

B7-H3 promotes acute and chronic allograft rejection.

The B7 homolog B7-H3 is important for the regulation of immune responses though its functions in vivo are controversial. We report the first clinical and experimental data concerning expression and function of B7-H3 in alloresponses. Immunohistological and molecular analyses showed B7-H3 expression by cells mediating rejection of human and mouse allografts. To analyze the significance of B7-H3 in rejecting allografts, we generated B7-H3-/- mice and showed that targeting of B7-H3 was synergistic with other forms of immune modulation; e.g. a regimen of rapamycin gave 12-14 days of survival in wild-type controls but led to permanent cardiac and islet allograft survival in B7-H3-/- mice. Cardiac allografts in treated B7-H3-/- mice showed markedly decreased production of key cytokine, chemokine and chemokine receptor mRNA transcripts as compared to wild-type controls. The incidence of chronic rejection in two different cardiac allograft models was also inhibited in B7-H3-/- as compared to wild-type recipients. Lastly, in addition to the expected antigen-presenting cell expression of B7-H3, CD4 and CD8 T cells showed B7-H3 induction upon cell activation, and both dendritic cell- and T cell-expressed B7-H3 each enhanced T cell proliferation in vitro and in vivo. We conclude that B7-H3 promotes T cell-mediated immune responses and the development of acute and chronic allograft rejection.

Transcriptional up-regulation of the signaling regulatory protein LNK in activated endothelial cells.

BACKGROUND: A better understanding of inflammatory processes in endothelial cells (ECs) might reveal new ways of controlling inflammation and graft rejection. This study investigates EC genes regulated in response to human tumor necrosis factor (TNF)-alpha and xenogeneic natural antibodies (XNAs) that contribute to endothelial activation during transplantation. METHODS: Gene expression between resting and activated ECs was investigated by RNA differential display reverse-transcriptase polymerase chain reaction and confirmed by reverse-Northern blot. RESULTS: Forty-five cDNA fragments corresponding to genes up-regulated in activated ECs were identified. Our findings show that TNF-alpha-mediated EC activation was associated with increased levels of mRNA for the adaptor protein Lnk, the nuclear protein RED, and the initiation factor eIF4G. We further show that Lnk and eIF4G were also up-regulated in response to XNA binding to ECs. CONCLUSION: Our data suggest that TNF-alpha and XNAs could share common signaling pathways involving Lnk and eIF4G but may also drive specific transcriptional events.

Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells.

BACKGROUND: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations. Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. MATERIALS AND METHODS: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. RESULTS: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. CONCLUSION: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.

Modulation of LIGHT-HVEM costimulation prolongs cardiac allograft survival.

LIGHT (TNFSF14), a tumor necrosis factor superfamily member expressed by activated T cells, binds to herpes virus entry mediator (HVEM) which is constitutively expressed by T cells and costimulates T cell activation in a CD28-independent manner. Given interest in regulating the effector functions of T cells in vivo, we examined the role of LIGHT-HVEM costimulation in a murine cardiac allograft rejection model. Normal hearts lacked LIGHT or HVEM mRNA expression, but allografts showed strong expression of both genes from day 3 after transplant, and in situ hybridization and immunohistology-localized LIGHT and HVEM to infiltrating leukocytes. To test the importance of LIGHT expression on allograft survival, we generated LIGHT-/- mice by homologous recombination. The mean survival of fully major histocompatibility complex-mismatched vascularized cardiac allografts in LIGHT-/- mice (10 days, P < 0.05) or cyclosporine A (CsA)-treated LIGHT+/+ mice (10 days, P < 0.05) was only slightly prolonged compared with LIGHT+/+ mice (7 days). However, mean allograft survival in CsA-treated LIGHT-/- allograft recipients (30 days) was considerably enhanced (P < 0.001) compared with the 10 days of mean survival in either untreated LIGHT-/- mice or CsA-treated LIGHT+/+ controls. Molecular analyzes showed that the beneficial effects of targeting of LIGHT in CsA-treated recipients were accompanied by decreased intragraft expression of interferon (IFN)-gamma, plus IFN-gamma-induced chemokine, inducible protein-10, and its receptor, CXCR3. Treatment of LIGHT+/+ allograft recipients with HVEM-Ig plus CsA also enhanced mean allograft survival (21 days) versus wild-type controls receiving HVEM-Ig (mean of 7 days) or CsA alone (P < 0.001). Our data suggest that T cell to T cell-mediated LIGHT/HVEM-dependent costimulation is a significant component of the host response leading to cardiac allograft rejection.

Identification and characterization of SF2000 and SF2001, two new members of the immune receptor SLAM/CD2 family.

The SLAM family of human genes currently consists of seven related members of the immunoglobulin superfamily, membrane-associated proteins, including CD150 (SLAM), CD244 (2B4), CD84, CD229 ( Ly-9), BLAME, CD48, and 19A. These genes are expressed to varying degrees in subsets of immune cells (T, B, natural killer, and myeloid cells) and may function as ligands or receptors. This set of genes, related to CD2 and CD58 on Chromosome (Chr) 1p98, are found clustered close together in the human genome on Chr 1q22. Four of these family members (CD150, CD244, CD84, CD229) contain conserved tyrosine motifs in their cytoplasmic tails that enable them to bind intracellular signaling molecules SAP and EAT-2. SAP is mutated in human X-linked lymphoproliferative disease (XLP), and studies in XLP patients have shown that improper signaling via molecules that bind SAP contributes to the disease. We have identified two new members of the SLAM family (SF), which we term SF2000 and SF2001, which are expressed in immune cells and map in the SLAM gene cluster. SF2001 does not contain SAP-binding motifs in its short cytoplasmic tail. SF2000, which is co-expressed with SAP in T cells, binds both SAP and EAT-2. The data suggest that signaling through SF2000, together with CD150, CD244, CD84, and CD229, is controlled by SAP and therefore contributes to the pathogenesis of XLP.