Simultaneous blockade of programmed death 1 and vascular endothelial growth factor receptor 2 (VEGFR2) induces synergistic anti‐tumour effect in vivo

Recent basic and clinical studies have shown that the programmed death ligand (PD‐L)/PD‐1 pathway has a significant role in tumour immunity, and its blockade has a therapeutic potential against several human cancers. We hypothesized that anti‐angiogeneic treatment might augment the efficacy of PD‐1 blockade. To this end, we evaluated combining the blockade of PD‐1 and vascular endothelial growth factor receptor 2 ( VEGFR2) in a murine cancer model using Colon‐26 adenocarcinoma. Interestingly, simultaneous treatment with anti‐PD‐1 and anti‐VEGFR2 monoclonal antibodies (mAbs) inhibited tumour growth synergistically in vivo without overt toxicity. Blocking VEGFR2 inhibited tumour neovascularization significantly, as demonstrated by the reduced number of microvessels, while PD‐1 blockade had no impact on tumour angiogenesis. PD‐1 blockade might promote T cell infiltration into tumours and significantly enhanced local immune activation, as shown by the up‐regulation of several proinflammatory cytokine expressions. Importantly, VEGFR2 blockade did not interfere with T cell infiltration and immunological activation induced by PD‐1 blockade. In conclusion, simultaneous blockade of PD‐1 and VEGFR2 induced a synergistic in‐vivo anti‐tumour effect, possibly through different mechanisms that might not be mutually exclusive. This unique therapeutic strategy may hold significant promise for future clinical application.     

NF‐κB‐inducing kinase is a key regulator of inflammation‐induced and tumour‐associated angiogenesis

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signalling pathways. In endothelial cells (ECs), expression of the pro‐angiogenic chemokine CXCL12 is regulated by non‐canonical nuclear factor (NF)‐κB signalling. Here, we report that NF‐κB‐inducing kinase (NIK) and subsequent non‐canonical NF‐κB signalling regulate both inflammation‐induced and tumour‐associated angiogenesis. NIK is highly expressed in endothelial cells (ECs) in tumour tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non‐canonical NF‐κB signalling in human microvascular ECs significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik^?/? mice exhibited normal angiogenesis during development and unaltered TNFα‐ or VEGF‐induced angiogenic responses, whereas angiogenesis induced by non‐canonical NF‐κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumour model was severely impaired in these mice. These studies provide evidence for a role of non‐canonical NF‐κB signalling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumour neoangiogenesis. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

pDCs efficiently process synthetic long peptides to induce functional virus‐ and tumour‐specific T‐cell responses

Robust cell‐mediated immunity is required for immune control of tumours and protection from viral infections, with both CD4⁺ and CD8⁺ T cells playing a pivotal role. Synthetic long peptides (SLPs) represent an attractive way to induce such combined responses, as they contain both class I and class II epitopes. The ability of plasmacytoid dendritic cells (pDCs) to cross‐present SLPs has not yet been investigated; yet, pDCs play a critical role in shaping immune responses and have emerged as novel vectors for immunotherapy. Using overlapping 15‐mer peptide pools covering the entire sequence of CMVpp65 and MelA, representing a viral disease (cytomegalovirus, CMV) and a tumour (melanoma), respectively, we showed that human pDCs can effectively process SLPs. Our results demonstrated that pDCs potently cross‐present virus‐ and tumour‐derived SLPs and cross‐prime broad‐ranging, effective and long‐lived CD4⁺ and CD8⁺ T‐cell responses, triggering more efficient immune responses than short peptide loaded pDCs. This ability required intracellular processing by the proteasome and was enhanced by co‐exposure to TLR7/9‐L. Combining SLPs with pDCs represents a powerful immunotherapeutic strategy to elicit potent immune responses, which are required for clinical success in cancers and viral infections.     

HOPX is methylated and exerts tumour‐suppressive function through Ras‐induced senescence in human lung cancer

HOPX acts as a tumour suppressor in various cancers. However, the regulation of HOPX in human lung cancer as well as the mechanism underlying its tumour‐suppressive function has not yet been well elucidated. Here we investigated the epigenetic regulation and molecular mechanism by which HOPX exerts growth inhibitory effects. We found that HOPX was down‐regulated in 12 out of 13 lung cancer cell lines and in 69 out of 120 primary lung tumours at mRNA and protein levels. Patients with lung adenocarcinoma (ADC) exhibited significantly more positive staining of HOPX protein compared with lung squamous cell carcinoma (SCC) (p =0.036). Again in ADC, patients with higher HOPX expression had a significantly longer disease‐free survival (p =0.001). Methylation analysis showed that down‐regulation of HOPX was associated with DNA methylation (p =0.011). To analyse the function of HOPX in lung cancer cells, stable transfection with an expression vector of HOPX was performed. It turned out that HOPX inhibited tumour cell proliferation rate, migration, and invasion, and, more interestingly, forced expression of HOPX enhanced cellular senescence via activation of oncogenic Ras and the downstream MAPK pathway, which in turn led to decreased MDM2 and increased p21. On the contrary, knockdown of HOPX by siRNA resulted in reduced Ras activity, inactivation of the MAPK pathway, and decreased p21 levels, accompanied by reduced cellular senescence. Additionally, the HOPX‐induced senescence pathway was also active in human bronchial epithelial cells. Taken together, our data suggest that down‐regulation of HOPX was related to DNA methylation and that HOPX exerts tumour‐suppressive activity by oncogenic Ras‐induced cellular senescence in lung cancer cells. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Anti‐PR3 immune responses induce segmental and necrotizing glomerulonephritis

Wegener's granulomatosis (WG) is a life‐threatening autoimmune vasculitis that affects lungs, kidneys and other organs. A hallmark of WG is the presence of classic anti‐neutrophil cytoplasmic antibodies (c‐ANCA) against self‐proteinase 3 (PR3). Little is known about the role of these antibodies and PR3‐specific immune responses in disease development. In this study, we demonstrate that PR3‐specific autoimmune responses are pathogenic in non‐obese diabetic (NOD) mice with an impaired regulatory arm of the immune response. Immunization of autoimmunity prone NOD mice with rmPR3 (recombinant mouse PR3) in complete Freund's adjuvant (CFA) resulted in high levels of c‐ANCA, without detectable disease development. However, when splenocytes from these immunized mice were transferred into immunodeficient NOD–severe combined immunodeficiency (SCID) mice, the recipient mice developed vasculitis and severe segmental and necrotizing glomerulonephritis. No disease developed in NOD–SCID mice that received splenocytes from the CFA‐alone‐immunized donors (controls), indicating that disease development depends upon PR3‐specific immune responses. In contrast to the pathology observed in NOD–SCID mice, no disease was observed when splenocytes from rmPR3‐immunized C57BL/6 mice were transferred into immunodeficient C57BL/6‐RAG‐1^–/– mice, suggesting that complex and probably multi‐genetic factors play a role in the regulation of disease development.     

Monocyte‐derived dendritic cells from patients with severe forms of chromoblastomycosis induce CD4+ T cell activation in vitro

Environmental and predisposing genetic factors are known to play a crucial role in the development of systemic autoimmune diseases. With respect to the role of environmental factors, it is not known how and to what extent they contribute to the initiation and exacerbation of systemic autoimmunity. In the present study, I considered this issue and asked if environmental factors can induce autoimmunity in the absence of specific susceptible genes. The development of genetically controlled mercury‐ and silver‐induced B cell activation and anti‐nucleolar autoantibodies (ANolA) production in genetically heterozygous outbred Institute of Cancer Research (ICR), Naval Medical Research Institute (NMRI) and Black Swiss mouse stocks were analysed. Four weeks of treatment with both mercury and silver induced a strong B cell activation characterized by increased numbers of splenic antibody‐secreting cells of at least one or more immunoglobulin (Ig) isotype(s) in all treated stocks. The three stocks also exhibited a marked increase in the serum IgE levels in response to mercury, but not silver. More importantly, in response to mercury a large numbers of ICR (88%), NMRI (96%) and Black Swiss (100%) mice produced different levels of IgG1 and IgG2a ANolA (a characteristic which is linked strictly to the H‐2 genes). Similarly, but at lower magnitudes, treatment with silver also induced the production of IgG1 and IgG2a ANolA in 60% of ICR, 75% of NMRI and 100% of Black Swiss mice. Thus, the findings of this study suggest that long‐term exposure to certain environmental factors can activate the immune system to produce autoimmunity per se, without requiring specific susceptible genes.     

Hypoxia augments cytokine (transforming growth factor-beta (TGF-β) and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts

Vascular endothelial growth factor (VEGF) is abundant in synovium and synovial fluids, where it probably contributes to vascular permeability and angiogenesis in arthritic joints. To investigate the probable sources of VEGF in synovium, we compared the ability of several cytokines (TGF-β, platelet-derived growth factor (PDGF), IL-1, tumour necrosis factor (TNF), basic fibroblast growth factor (bFGF) that are associated with arthritis and angiogenesis, to stimulate secretion of VEGF protein by human synovial fibroblasts. TGF-β was the strongest inducer of VEGF secretion; six times more VEGF was secreted when cells were stimulated by TGF-β than when stimulated by PDGF or IL-1 for 24 h. TNF-α and bFGF did not stimulate any secretion of VEGF. The stimulatory effects of TGF-β and IL-1 on VEGF secretion were additive. Hypoxic culture alone also stimulated VEGF secretion, but more importantly, hypoxic culture conditions doubled the rate of VEGF secretion stimulated by the cytokines TGF-β and IL-1. When dermal and synovial fibroblasts were stimulated identically by hypoxia and cytokines (TGF-β and IL-1), synovial fibroblasts secreted four times more VEGF than did dermal fibroblasts. Thus in rheumatoid arthritis, the capacity of synovial fibroblasts in the hypoxic environment to secrete large amounts of VEGF in response to cytokines such as TGF-β probably contributes significantly to angiogenesis in the synovium.     

Different cytokine production and toll‐like receptor expression induced by heat‐killed invasive and carrier strains of Neisseria meningitidis

Neisseria meningitidis may cause severe invasive disease. The carriage state of the pathogen is common, and the reasons underlying why the infection becomes invasive are not fully understood. The aim of this study was to compare the differences between invasive and carrier strains in the activation of innate immunity. The monocyte expression of TLR2, TLR4, CD14, and HLA‐DR, cytokine production, and the granulocyte oxidative burst were analyzed after in vitro stimulation by heat‐killed invasive (n = 14) and carrier (n = 9) strains of N. meningitidis. The expression of the cell surface markers in monocytes, the oxidative burst, and cytokine concentrations were measured using flow cytometry. Carrier strains stimulated a higher production of inflammatory cytokines and oxidative burst in granulocytes than invasive strains (all p < 0.001), whereas invasive strains significantly up‐regulated TLR2, TLR4 (p < 0.001), and CD14 (p < 0.01) expression on monocytes. Conversely, the monocyte expression of HLA‐DR was higher after the stimulation by carrier strains (p < 0.05) in comparison to invasive strains. The LPS inhibitor polymyxin B abolished the differences between the strains. Our findings indicate different immunostimulatory potencies of invasive strains of N. meningitidis compared with carrier strains.     

T‐helper 1 and T‐helper 2 adjuvants induce distinct differences in the magnitude,quality and kinetics of the early inflammatory response at the site of injection

Vaccine adjuvants activate the innate immune system and thus influence subsequent adaptive T‐cell responses. However, little is known about the initial immune mechanisms preceding the adjuvant‐induced differentiation of T‐helper (Th) cells. The effect of a T‐helper 1 (Th1) adjuvant, dimethyldioctadecylammonium liposomes with monophosphoryl lipid‐A (DDA/MPL), and a T‐helper 2 adjuvant, aluminium hydroxide [Al(OH)₃], on early, innate chemotactic signals and inflammatory cell influx at the site of injection was therefore investigated. Injection of the adjuvants into the peritoneal cavity of mice demonstrated distinct differences in the magnitude, quality and kinetics of the response. The inflammatory response to DDA/MPL was prominent, inducing high local levels of pro‐inflammatory cytokines, chemokines and a pronounced inflammatory exudate consisting of neutrophils, monocytes/macrophages and activated natural killer cells. This was in contrast to the response induced by Al(OH)₃, which, although sharing some of the early chemokine signals, was more moderate and consisted almost exclusively of neutrophils and eosinophils. Notably, Al(OH)₃ specifically induced the release of a significant amount of interleukin (IL)‐5, whereas DDA/MPL induced high amounts of tumour necrosis factor‐α (TNF‐α), IL‐1α and IL‐6. Finally, a microarray analysis confirmed that the effect of DDA/MPL was broader with more than five times as many genes being specifically up‐regulated after injection of DDA/MPL compared with Al(OH)₃. Thus, the adjuvants induced qualitatively distinct local inflammatory signals early after injection.     

Partial and transient modulation of the CD3–T‐cell receptor complex,elicited by low‐dose regimens of monoclonal anti‐CD3, is sufficient to induce disease remission in non‐obese diabetic mice

It has been established that a total of 250 μg of monoclonal anti‐mouse CD3 F(ab′)₂ fragments, administered daily (50 μg per dose), induces remission of diabetes in the non‐obese diabetic (NOD) mouse model of autoimmune diabetes by preventing β cells from undergoing further autoimmune attack. We evaluated lower‐dose regimens of monoclonal anti‐CD3 F(ab′)₂ in diabetic NOD mice for their efficacy and associated pharmacodynamic (PD) effects, including CD3–T‐cell receptor (TCR) complex modulation, complete blood counts and proportions of circulating CD4⁺, CD8⁺ and CD4⁺ FoxP3⁺ T cells. Four doses of 2 μg (total dose 8 μg) induced 53% remission of diabetes, similarly to the 250 μg dose regimen, whereas four doses of 1 μg induced only 16% remission. While the 250 μg dose regimen produced nearly complete and sustained modulation of the CD3 –TCR complex, lower doses, spaced 3 days apart, which induced similar remission rates, elicited patterns of transient and partial modulation. In treated mice, the proportions of circulating CD4⁺ and CD8⁺ T cells decreased, whereas the proportions of CD4⁺ FoxP3⁺ T cells increased; these effects were transient. Mice with greater residual β‐cell function, estimated using blood glucose and C‐peptide levels at the initiation of treatment, were more likely to enter remission than mice with more advanced disease. Thus, lower doses of monoclonal anti‐CD3 that produced only partial and transient modulation of the CD3–TCR complex induced remission rates comparable to higher doses of monoclonal anti‐CD3. Accordingly, in a clinical setting, lower‐dose regimens may be efficacious and may also improve the safety profile of therapy with monoclonal anti‐CD3, potentially including reductions in cytokine release‐related syndromes and maintenance of pathogen‐specific immunosurveillance during treatment.     

Heparin co‐factor II enhances cell motility and promotes metastasis in non‐small cell lung cancer

Using the Serial Analysis of Gene Expression (SAGE) database from the Cancer Genome Anatomy Project, we identified heparin co‐factor II (HCII), which is over‐expressed in non‐small cell lung cancer (NSCLC). Here, we investigated the clinical significance of HCII and provided molecular evidence to support the suggestion that HCII could enhance cancer metastasis in NSCLC. We found that high HCII expression in tumour tissue was associated with increased cancer recurrence and shorter overall survival times in 75 clinically operable NSCLC patients. High pretreatment plasma concentration of HCII was associated with reduced overall survival in 57 consecutive NSCLC patients. We over‐expressed and knocked down HCII expression in lung cancer cell lines and confirmed that HCII could promote cell motility, invasion ability and filopodium dynamics in NSCLC cells in vitro and increased metastatic colonization in an in vivo mouse model. Exogenous treatment of HCII promoted cancer cell migration, and this promigratory effect of HCII was independent of thrombin. We further showed that HCII could up‐regulate cancer cell migration through the activation of PI3K, which acts upstream of Rac1 and Cdc42, and this effect could be blocked by heparin. We suggest that HCII is a novel metastasis enhancer and may be used as a prognostic predictor for heparin treatment in NSCLC. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

A role for Myh1 in DNA repair after treatment with strand‐breaking and crosslinking chemotherapeutic agents

The highly conserved DNA glycosylase MutY is implicated in repair of oxidative DNA damage, in particular in removing adenines misincorporated opposite 7,8‐dihydro‐8‐oxoguanine (8‐oxo‐G). The MutY homologues (MutYH) physically associate with proteins implicated in replication, DNA repair, and checkpoint signaling, specifically with the DNA damage sensor complex 9‐1‐1 proteins. Here, we ask whether MutYH could have a broader function in sensing and repairing different types of DNA damage induced by conventional chemotherapeutics. Thus, we examined if deletion of the Schizosaccharomyces pombe MutY homologue, Myh1, alone or in combination with deletion of either component of the 9‐1‐1 sensor complex, influences survival after exposure to different classes of DNA damaging chemotherapeutics that do not act primarily by causing 8‐oxoG lesions. We show that Myh1 contributes to survival on genotoxic stresses induced by the oxidizing, DNA double strand break‐inducing, bleomycins, or the DNA crosslinking platinum compounds, particularly in a rad1 mutant background. Exposure of cells to cisplatin leads to a moderate overall accumulation of Myh1 protein. Interestingly, we found that DNA damage induced by phleomycin results in increased chromatin association of Myh1. Further, we demonstrate that Myh1 relocalizes to the nucleus after exposure to hydrogen peroxide or chemotherapeutics, most prominently seen after phleomycin treatment. These observations indicate a wider role of Myh1 in DNA repair and DNA damage‐induced checkpoint activation than previously thought. Environ. Mol. Mutagen. 54:327–337, 2013. © 2013 Wiley Periodicals, Inc.