Background Neuroblastoma is the commonest cancer in infants. Survival in high-risk groups is low (40-50%). Newer treatments are needed to improve survival and morbidity. Cytomegalovirus (CMV) is a ...common viral infection, which increases gamma delta T cells. We investigated the use of CMV-reactive gamma delta T cells as a potential new immunotherapy. Methods Peripheral blood mononuclear cells from 30 paediatric haemato-oncology patients with or without CMV infection were analysed by flow cytometry. gamma delta T cells were expanded, and then co-cultured with CMV targets or neuroblastoma cells. T-cell activation, cytokine secretion, killing activity, and mechanisms were determined by ELISA, MTT colorimetric assay, and blocking assays. Novel gamma delta T cell receptors (TCR) were identified by sequencing. Findings In paediatric haemato-oncology patients, acute CMV led to higher proportions of total gamma delta T cells (p=0.0002) with the dominant subtype V delta 1 (p=0.0035). CMV-reactive gamma delta T cells were of the effector memory phenotype and expanded to clinically significant numbers for adoptive transfer. When compared with gamma delta T cells from CMV-negative patients, CMV-reactive gamma delta T cells had statistically significantly higher interferon gamma release (p<0.001) in co-cultures with CMV-infected fibroblasts and showed cytolytic activity against CMV-infected fibroblasts and neuroblastoma cells which was mediated by gamma delta TCR and the NKG2D receptor. Sequencing showed that the dominant gamma delta T-cell chains in CMV-infected patients were V delta 1 V gamma 2. Within the TCRs, CDR3 sequences had minimal diversity, gamma chains had wide variations, and we identified a novel subpopulation in some patients. Interpretation We show that acute CMV infection in paediatric haemato-oncology patients leads to an increase in the V delta 1 V gamma 2 subtype of gamma delta T cells. They can be expanded for adoptive transfer and are likely to retain killing ability post expansion. They recognise and kill CMV-infected targets and neuroblastoma cells via the gamma delta TCR and the NKG2D receptor. We have identified a novel TCR in a subpopulation of CMV-positive patients. Funding Great Ormond Street Charity, National Institute for Health Research Biomedical Research Centre, Olivia Hodson Cancer Fund.
BackgroundSjogren's syndrome (SS) is an autoimmune disease with unknown aetiology. SS shares similarity with other autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid ...arthritis (RA). Our objective was to use bioinformatics tools to identify both unique and common genes involved in the pathogenesis of SS, SLE and RA.Materials and methodsLiterature mining tool PubTator was used to identify genes associated with each disease. Python scripts were used to extract information from PubTator and integrate it into a MySQL database management system. Three salivary glands (SGs) and one peripheral blood mononuclear cells (PBMCs) gene expression datasets (NCBI-GEO) of SS patients were analysed using GEO2R. MySQL tables were joined and organised to retrieve gene sets, which were utilised for molecular network analysis using STRINGdb. A web interface was designed and implemented to provide unique platform for search and retrieval of genes associated with SS.ResultsText mining yielded 847 genes in common in SS, SLE and RA. A total of 15 990 differentially expressed (DE) genes were represented in at least one dataset, including 683 in common with the 847 previously identified. Of these, 86 genes were differentially expressed in all SGs datasets. To identify DE genes unique to SGs of SS patients, PBMCs dataset was used to subtract DE genes common to SGs and PBMCs from the 86 genes. Remaining 71 genes were integrated into a molecular interaction network including 44 representing 22 SS-related biological processes. The biological processes included cytokine signalling, herpes virus infection, and toll-like receptor signalling. A web interface was established to aggregate and correlate gene expression and text mining data.ConclusionsBy combining text mining and gene expression analysis, we identified a molecular interaction network that contained DE genes representing candidate biomarkers for SS. Previous studies had reported no clear link between some of the identified genes and SS. These genes may be further investigated for their role in the progression of SS in experimental models. Further, the web interface provides dynamic platform to the investigator to correlate different data sources, which may provide additional insights on the development of SS.
Herpes simplex virus 1 (HSV-1) remodels nuclear membranes during virus egress. Although the UL31 and UL34 proteins control nucleocapsid transit in infected cells, the molecular interactions required ...for their function are unclear. Here we report that the gamma 134.5 gene product of HSV-1 facilitates nucleocapsid release to the cytoplasm through bridging the UL31/UL34 complex, cellular p32, and protein kinase C. Unlike wild-type virus, an HSV mutant devoid of gamma 134.5 or its amino terminus is crippled for viral growth and release. This is attributable to a defect in virus nuclear egress. In infected cells, wild-type virus recruits protein kinase C to the nuclear membrane and triggers its activation, whereas the gamma 134.5 mutants fail to exert such an effect. Accordingly, the gamma 134.5 mutants are unable to induce phosphorylation and reorganization of lamin A/C. When expressed in host cells gamma 134.5 targets p32 and protein kinase C. Meanwhile, it communicates with the UL31/UL34 complex through UL31. Deletion of the amino terminus from gamma 134.5 disrupts its activity. These results suggest that disintegration of the nuclear lamina mediated by gamma 134.5 promotes HSV replication. IMPORTANCE HSV nuclear egress is a key step that determines the outcome of viral infection. While the nuclear egress complex mediates capsid transit across the nuclear membrane, the regulatory components are not clearly defined in virus-infected cells. We report that the gamma 134.5 gene product, a virulence factor of HSV-1, facilitates nuclear egress cooperatively with cellular p32, protein kinase C, and the nuclear egress complex. This work highlights a viral mechanism that may contribute to the pathogenesis of HSV infection.
Dasatinib treatment markedly increases the number of large granular lymphocytes (LGLs) in a proportion of Ph super(+) leukemia patients, which associates with a better prognosis. The lymphocytosis is ...predominantly observed in cytomegalovirus (CMV)-seropositive patients, yet detectable CMV reactivation exists only in a small fraction of patients. Thus, etiology of the lymphocytosis still remains unclear. Here, we identified NK cells as the dominant LGLs expanding in dasatinib-treated patients, and applied principal component analysis (PCA) to an extensive panel of NK cell markers to explore underlying factors in NK cell activation. PCA displayed phenotypic divergence of NK cells that reflects CMV-associated differentiation and genetic differences, and the divergence was markedly augmented in CMV-seropositive dasatinib-treated patients. Notably, the CMV-associated highly differentiated status of NK cells was already observed at leukemia diagnosis, and was further enhanced after starting dasatinib in virtually all CMV-seropositive patients. Thus, the extensive characterization of NK cells by PCA strongly suggests that CMV is an essential factor in the NK cell activation, which progresses stepwise during leukemia and subsequent dasatinib treatment most likely by subclinical CMV reactivation. This study provides a rationale for the exploitation of CMV-associated NK cell activation for treatment of leukemias.
IntroductionCystic changes in the lung due to congenital/perinatal cytomegalovirus (CMV) infection have been reported in a few cases in the literature. In this case report, we presented a preterm ...infant with congenital CMV infection developed lung cysts during her clinical follow-up.CaseA female neonate was delivered by caesarean section at 35 gestational week due to fetal distress. Her birthweight was 1870 g (10-50th percentile) and Apgar scores 7 and 9, first and fifth minutes, respectively. She was transferred to our intensive care unit because of the prematurity and ventriculomegaly. It was reported that fetal ultrasonography revealed bowel hyperechogenicity at the 18th week of gestation. The serologic examination was negative for CMV IgM. Fetal magnetic resonance imaging was normal and fetal karyotype was 46, XX. The lateral ventricles were observed to be dilated in the repeated ultrasonography performed at 34th week. On physical examination she was well-appearing, there was no abnormality except for hepatosplenomegaly (3 cm below costal margin) and bilateral chorioretinitis. She failed auditory-brainstem-response test. Cranial ultrasonography performed after birth revealed bilateral ventriculomegaly and germinal matrix haemorrhage. Complete blood count showed thrombocytopenia. CMV IgM was reactive and CMV PCR was positive (7.60E+4 cp/mL) in serologic examination. Chest radiography was normal. Intravenous ganciclovir therapy was initiated to the patient at the dose of 12 mg/kg/day due to symptomatic congenital CMV infection. At the postnatal 20th day, a cystic lesion was detected in the basal part of the left lung on the abdomen radiography performed for nutrition intolerance and abdominal distension. Thoracic computed tomography revealed pulmonary cavitary lesions, the largest of them was located in the left lower lobe posterior basal segment. No signs of respiratory distress were detected during this period, infection parameters and blood culture were negative. Cystic lesions in the lung regressed during her clinical follow-up. She was discharged with oral valganciclovir treatment when she was 55 days old.ConclusionIt should be kept in mind that congenital CMV infection may cause lung cysts.
While it is established that cytomegalovirus (CMV) disease affects NK-cell profiles, the functional consequences of asymptomatic CMV replication are unclear. Here, we characterize NK cells in ...clinically stable renal transplant recipients (RTRs; n = 48) >2 years after transplantation. RTRs and age-matched controls (n = 32) were stratified by their CMV serostatus and the presence of measurable CMV DNA. CMV antibody or CMV DNA influenced expression of NKG2C, LIR-1, NKp30, NKp46, and FcR gamma , a signaling adaptor molecule, on CD56 super(dim) NK cells. Phenotypic changes ascribed to CMV were clearer in RTRs than in control subjects and affected NK-cell function as assessed by TNF- alpha and CD107a expression. The most active NK cells were FcR gamma super(-)LIR-1 super(+)NKG2C super(-) and displayed high antibody-dependent cell cytotoxicity responses in the presence of immobilized CMV glycoprotein B reactive antibody. However, perforin levels in supernatants from RTRs with active CMV replication were low. Overall we demonstrate that CMV can be reactivated in symptom-free renal transplant recipients, affecting the phenotypic, and functional profiles of NK cells. Continuous exposure to CMV may maintain and expand NK cells that lack FcR gamma but express LIR-1. In asymptomatic posttransplant renal transplant recipients, persistent, and active CMV expands a population of FcR gamma super(-)LIR-1 super(+)NKG2C super(-) NK cells, which on cross-linking with CMV antibody displays increased antibody dependent cell cytotoxicity, i.e. increased expression of CD107a and TNF- alpha .
Abstract
Triple-negative breast cancer (TNBC) accounts for 15-20% of the breast cancer cases.
These tumors are heterogeneous and aggressive, fail to respond to targeted therapy, have poor prognosis, ...and a high risk of relapse. The molecular basis of this breast cancer subtype is currently unknown. Viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human papillomavirus (HPV), are major suspects in the etiology of breast cancer. Since miRNAs have been implicated in the pathogenesis of breast cancer, including triple-negative breast cancer, we hypothesized that viral miRNAs may play a role in this aggressive disease. The objective of this project, therefore, was to determine the identity, prevalence, sequence variation, and differential expression of viral miRNAs in TNBC tumors as compared to control samples. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Five novel putative HCMV miRNAs were found to be differentially expressed in TNBC tumors as compared to controls. Two of the putative miRNAs were differentially expressed in 60-79% of the TNBC tumors as compared to 0% of normal controls. Two additional putative miRNAs were expressed in 67-88% of TNBC tumors as compared to 25-31% of normal controls. One putative miRNA was expressed in 67% of TNBC tumors as compared to 88% of normal samples, while 1 putative miRNA in addition to HCMV-US25-1-3p displayed no significant differences. These putative HCMV miRNAs localize within genomic regions of HCMV that encode UL56 DNA packaging terminase subunit 2, single-stranded DNA-binding protein, noncoding RNA4.9, and enveloped glycoprotein M. Ongoing work has so far computationally validated one of these miRNAs as a novel HCMV miRNA. This is the first report on the differential expression of HCMV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 60-80% of TNBC tumors specifically express the same HCMV miRNA. Our findings suggest that these differentially expressed HCMV miRNAs may potentially play a role in the pathogenesis of TNBC.
Citation Format: Tia Hudson, Scott Harrison, Dukka K C, Jan Prins, Perpetua M. Muganda. Differential expression of human cytomegalovirus microRNA in triple-negative breast cancer tumors. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1120.
Abstract
Talimogene laherparepvec, is a modified oncolytic herpes simplex virus type-1 (HSV-1) designed to selectively replicate in tumors and to initiate a systemic immune response to target cancer ...cells. Intralesional administration of talimogene laherparepvec is intended to result in oncolysis within injected tumors, with lytic cell destruction promoting the local release of progeny virus and tumor derived antigens. GM-CSF, a product of the viral transgene, is also produced locally and is designed to recruit and stimulate antigen presenting cells to further enhance systemic antitumor immune response.
While available evidence suggests that talimogene laherparepvec can induce effects in distant tumors via an immune-mediated effect, additional mechanistic evidence is being sought to help better understand the putative systemic effect. For preclinical mechanism of action studies we employed OncoVEXmGM-CSF, an HSV-1 virus modified in the same manner as talimogene laherparepvec except that murine GM-CSF is expressed. We utilized this virus to further dissect the underlying innate and adaptive immunity following viral administration in syngeneic tumor models.
Intratumoral administration of OncoVEXmGM-CSF into established murine tumors induced regressions in the injected lesion but also resulted in significant anti-tumor effects in contralateral (uninjected) lesions. We have previously demonstrated by immunohistochemistry that the systemic effects observed in the contralateral lesions are not driven by systemic spread of the virus, but instead correlate with the infiltration of CD3+ T cell populations. Here we report the full phenotypic characterization of immune infiltrates by flow cytometry in both the injected and contralateral lesions. In a time-course fashion, this characterization revealed the initial infiltration of the injected tumor by innate effector cells and a concomitant induction of a potent type I interferon response. This in turn drives the local release of proinflammatory cytokines and chemokines, resulting in the recruitment of adaptive immune subsets to the injected tumor and enhancing systemic anti-tumor reactivity. Additionally, both the primary oncolytic replication of the virus and the activation of type I interferon pathway are directly correlated with STING activity.
In conclusion, in vitro and in vivo observations suggest that intratumoral injection of OncoVEXmGM-CSF activates multiple immune-mediated mechanisms of action leading to T-cell activation and induction of anti-tumor immunity in mice.
Citation Format: Keegan Cooke, Karen Fitzgerald, Becky Yang, Juan Estrada, Brian Belmontes, Pedro Beltran, Achim K. Moesta. Intratumoral administration of OncoVEXmGM-CSF results in local innate immune alterations and induces systemic anti-tumor effects in syngeneic mouse tumor models. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2346.
Abstract
There has been a recent surge of interest in cancer immunotherapies due to newly FDA approved immunologic treatments based on targeting immune checkpoint inhibition pathways. Despite the ...dramatic responses in animal models and early clinical human trials, particularly in melanoma, durable clinical responses are observed only in approximately 30% of patients, therefore combinatorial immune therapeutic approaches may be needed to improve outcomes further. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body's natural checkpoint (ICI) response, the combination of these two approaches offers a potentially advantageous interaction. AdV-tk is an immunostimulatory virus-based approach that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (AdV-tk) followed by administration of an anti-herpetic prodrug (ganciclovir - GCV). This approach has shown benefit in clinical trials of glioblastoma among other tumor types. The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the thymidine kinase protein (TK). This approach has consistently demonstrated anti-tumor immune stimulation and an increased intra-tumoral CD8+ T-cell infiltrate. Not surprisingly, however, this immune stimulation also leads to increased expression of immune checkpoint inhibitory ligands on tumor cells, including PD-L1. Our initial in vitro studies showed that the immune checkpoint ligand PD-L1 is expressed across a panel of human and mouse conventional and stem-like glioblastoma-derived cells (GSCs). After AdV-tk infection and GCV treatment PD-L1 is consistently up-regulated both at protein and mRNA levels, while no changes were detected after AdV-tk alone. In vivo experiments in immunocompetent mouse models with GL261 glioma cell lines demonstrate a significant improvement in survival when we combine AdV-tk+GCV and anti-PD-1 antibodies. This data suggests that combination of immune checkpoint blockade with AdV-tk treatment should be explored in glioblastoma clinical trials.
Citation Format: Maria Carmela Speranza, Kazue Kasai, Johanna Kaufmann, Estuardo Aguilar-Cordova, Brian W. Guzik, E. Antonio Chiocca, Sean Lawler. Evaluation of the combination of the prodrug-mediated gene therapy vector AdV-tk and immune checkpoint inhibitor for glioblastoma treatment in a syngeneic mouse model. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2349.
Abstract
The major subsets of T cells that produce IL-17 include adaptive CD4+ Th17 cells and innate γδ T17 cells. IL-17 and both cellular sources are elevated in multiple human cancers and have been ...found to correlate with decreased patient survival. IL-17 produced by these cells promotes tumor growth by increasing the tumor infiltration and function of myeloid-derived suppressor cells (MDSCs), which in turn stimulate angiogenesis and suppress CD4+ and CD8+ T cell tumor homing and activation. Recently, two independent groups discovered that Th17 cell differentiation requires the transcription factor, hypoxia inducible factor 1α (HIF-1α), which promotes glycolytic enzymes and increases glucose metabolism. An established transcriptional target of HIF-1α and stimulator of glucose metabolism is 6-phosphofructo-2-kinase (PFKFB3) which synthesizes fructose 2,6-bisphosphate (F2,6BP), a potent allosteric activator of the rate-limiting enzyme 6-phosphofructo-1-kinase (PFK-1). In unpublished studies, we have found that human Th17 cells generated ex vivo produce increased PFKFB3 and F2,6BP relative to total T cells. We postulate that Th17 cells may selectively require the activity of PFKFB3 for their differentiation and tumor-promoting functions. We examined the immunomodulatory effects of a first-in-class PFKFB3 inhibitor, (E)-1-(pyridyn-4-yl)-3-(7-(trifluoromethyl)quinolin-2-yl)-prop-2-en-1-one (PFK-158) on Th17 cells in vitro, in B16 melanoma-bearing mice and in cancer patients participating in a phase 1 multi-center clinical trial (clinicaltrials.gov # NCT02044861) and found that PFK-158: (i) suppresses human Th17 cell differentiation in vitro (200 nM); (ii) decreases splenic and tumor-infiltrating Th17 cells, γδ T17 cells and MDSCs, and increases CD4+ and CD8+ T cells in the tumors of B16-F10 melanoma-bearing mice (0.06 mg/gm QD x 3 days); and (iii) decreases peripheral blood Th17 cells, γδ T cells and MDSCs and increases activated effector CD4+ and CD8+ T cells in cancer patients. Furthermore, we observed that homozygous genomic deletion of Pfkfb3 in C57Bl/6 mice (but not in implanted B16 melanoma cells) reduces B16 tumor growth, decreases splenic and tumor-infiltrating Th17 cells, γδ T17 cells and MDSCs, and increases tumor-filtrating CD4+ and CD8+ T cells. Based on these immunological effects, we predicted that PFK-158 would improve the anti-tumor activity of an immune checkpoint inhibitor, anti-CTLA4, in the B16-F10 model - we observed a marked increase in tumor growth inhibition by anti-CTLA4 when combined with PFK-158 in vivo. Taken together, these studies provide the first pre-clinical and clinical rationale for the conduct of phase 1/2 trials to examine the anti-cancer efficacy of PFKFB3 inhibitors in combination with FDA-approved immune checkpoint inhibitors and other immunostimulatory agents such as the GM-CSF-producing oncolytic herpes virus talimogene laherparepvec.
Citation Format: Sucheta Telang, Kavitha Yaddanadupi, Gilles Tapolsky, Rebecca Redman, Jason Chesney. Taking the sweet out of Th17 cells to potentiate immuno-oncology drugs. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 557.
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