Human Rhinovirus 16 Causes Golgi Apparatus Fragmentation without Blocking Protein Secretion

The replication of picornaviruses has been described to cause fragmentation of the Golgi apparatus that blocks the secretory pathway. The inhibition of major histocompatibility complex class I upregulation and cytokine, chemokine and interferon secretion may have important implications for host defense. Previous studies have shown that disruption of the secretory pathway can be replicated by expression of individual nonstructural proteins; however the situation with different serotypes of human rhinovirus (HRV) is unclear. The expression of 3A protein from HRV14 or HRV2 did not cause Golgi apparatus disruption or a block in secretion, whereas other studies showed that infection of cells with HRV1A did cause Golgi apparatus disruption which was replicated by the expression of 3A. HRV16 is the serotype most widely used in clinical HRV challenge studies; consequently, to address the issue of Golgi apparatus disruption for HRV16, we have systematically and quantitatively examined the effect of HRV16 on both Golgi apparatus fragmentation and protein secretion in HeLa cells. First, we expressed each individual nonstructural protein and examined their cellular localization and their disruption of endoplasmic reticulum and Golgi apparatus architecture. We quantified their effects on the secretory pathway by measuring secretion of the reporter protein Gaussia luciferase. Finally, we examined the same outcomes following infection of cells with live virus. We demonstrate that expression of HRV16 3A and 3AB and, to a lesser extent, 2B caused dispersal of the Golgi structure, and these three nonstructural proteins also inhibited protein secretion. The infection of cells with HRV16 also caused significant Golgi apparatus dispersal; however, this did not result in the inhibition of protein secretion. IMPORTANCE The ability of replicating picornaviruses to influence the function of the secretory pathway has important implications for host defense. However, there appear to be differences between different members of the family and inconsistent results when comparing infection with live virus to expression of individual nonstructural proteins. We demonstrate that individual nonstructural HRV16 proteins, when expressed in HeLa cells, can both fragment the Golgi apparatus and block secretion, whereas viral infection fragments the Golgi apparatus without blocking secretion. This has major implications for how we interpret mechanistic evidence derived from the expression of single viral proteins

Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer.

BackgroundT cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s.MethodsPatients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 1010 total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72.ResultsFourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had 111Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG-72 binding domain of humanized CC49, reflecting an anti-CAR immune response. No radiologic tumor responses were observed.ConclusionThese findings demonstrate the relative safety of CART72 cells. The limited persistence supports the incorporation of co-stimulatory domains in the CAR design and the use of fully human CAR constructs to mitigate immunogenicity

Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Background On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved chimeric antigen receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and costimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. Methods We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored on treatment with agonist anti-CD137 monoclonal antibodies (mAbs). Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 mAb (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISA and Luminex. Flow cytometry was used to monitor CD137 surface expression. Results CD137 costimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 costimulation inside subcutaneously implanted Matrigel plugs. The CD137 signaling domain-containing CAR T cells readily released sCD137 and acquired CD137 surface expression on antigen recognition. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. Conclusion sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents

Identification and selective expansion of functionally superior T cells expressing chimeric antigen receptors

Background: T cells expressing chimeric antigen receptors (CARs) have shown exciting promise in cancer therapy, particularly in the treatment of B-cell malignancies. However, optimization of CAR-T cell production remains a trial-and-error exercise due to a lack of phenotypic benchmarks that are clearly predictive of anti-tumor functionality. A close examination of the dynamic changes experienced by CAR-T cells upon stimulation can improve understanding of CAR–T-cell biology and identify potential points for optimization in the production of highly functional T cells. Methods: Primary human T cells expressing a second-generation, anti-CD19 CAR were systematically examined for changes in phenotypic and functional responses to antigen exposure over time. Multi-color flow cytometry was performed to quantify dynamic changes in CAR-T cell viability, proliferation, as well as expression of various activation and exhaustion markers in response to varied antigen stimulation conditions. Results: Stimulated CAR-T cells consistently bifurcate into two distinct subpopulations, only one of which (CARhi/CD25+) exhibit anti-tumor functions. The use of central memory T cells as the starting population and the resilience—but not antigen density—of antigen-presenting cells used to expand CAR-T cells were identified as critical parameters that augment the production of functionally superior T cells. We further demonstrate that the CARhi/CD25+ subpopulation upregulates PD-1 but is resistant to PD-L1-induced dysfunction. Conclusions: CAR-T cells expanded ex vivo for adoptive T-cell therapy undergo dynamic phenotypic changes during the expansion process and result in two distinct populations with dramatically different functional capacities. Significant and sustained CD25 and CAR expression upregulation is predictive of robust anti-tumor functionality in antigen-stimulated T cells, despite their correlation with persistent PD-1 upregulation. The functionally superior subpopulation can be selectively augmented by careful calibration of antigen stimulation and the enrichment of central memory T-cell type. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0519-8) contains supplementary material, which is available to authorized users

Identification of cell surface targets for CAR-T cell therapies and antibody-drug conjugates in breast cancer

Two promising therapeutic strategies in oncology are chimeric antigen receptor-T cell (CAR-T) therapies and antibody-drug conjugates (ADCs). To be effective and safe, these immunotherapies require surface antigens to be sufficiently expressed in tumors and less or not expressed in normal tissues. To identify new targets for ADCs and CAR-T specifically targeting breast cancer (BC) molecular and pathology-based subtypes, we propose a novel in silico strategy based on multiple publicly available datasets and provide a comprehensive explanation of the workflow for a further implementation

NETosis and associated host DNA orchestrate rhinovirus-induced type 2 allergic asthma exacerbation

Respiratory viral infections represent the most common cause of allergic asthma exacerbations. Amplification of type 2 immunity is strongly implicated in asthma exacerbation, but how virus infection boosts type 2 responses during exacerbation is poorly understood. We report a significant correlation between release of host double stranded DNA (dsDNA) following rhinovirus infection and exacerbation of type 2 allergic inflammation and disease severity in patients. In a mouse model, we show that rhinovirus infection triggers neutrophil extracellular traps (NETs) formation and host dsDNA release. We further demonstrate that inhibiting NETosis by blocking neutrophil elastase or degrading NETs with DNase protects mice from type 2 allergic asthma exacerbations. Furthermore, injection of host dsDNA alone is sufficient to recapitulate many features of rhinovirus-induced type 2 immune responses and asthma pathology. Thus, NETosis and host dsDNA contribute to exacerbation pathogenesis and may represent potential targets for novel treatments of rhinovirus-induced asthma exacerbations

First steps for the development of silk fibroin-based 3D biohybrid retina for age-related macular degeneration (AMD)

Age-related macular degeneration is an incurable chronic neurodegenerative disease, causing progressive loss of the central vision and even blindness. Up-to-date therapeutic approaches can only slow down he progression of the disease. Objective. Feasibility study for a multilayered, silk fibroin-based, 3D biohybrid retina. Approach. Fabrication of silk fibroin-based biofilms; culture of different types of cells: retinal pigment epithelium, retinal neurons, Müller and mesenchymal stem cells ; creation of a layered structure glued with silk fibroin hydrogel. Main results. In vitro evidence for the feasibility of layered 3D biohybrid retinas; primary culture neurons grow and develop neurites on silk fibroin biofilms, either alone or in presence of other cells cultivated on the same biomaterial; cell organization and cellular phenotypes are maintained in vitro for the seven days of the experiment. Significance. 3D biohybrid retina can be built using silk silkworm fibroin films and hydrogels to be used in cell replacement therapy for AMD and similar retinal neurodegenerative diseases