Christopher Silvin

Early-onset stroke and vasculopathy associated with mutations in ADA2

BACKGROUND We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. METHODS We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. RESULTS All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. CONCLUSIONS Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Measurement of Proliferative Responses of Cultured Lymphocytes

Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to induce proliferation in lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying protocol, which contains methods to determine cell proliferation by incorporation of [3H]thymidine into DNA by nonradioactive methods, e.g., reduction of tetrazolium salts (MTT or WST‐1). These protocols provide an estimate of cell proliferation indirectly by measuring DNA synthesis, and cell metabolic activity in an entire cell population, but no data on individual cells is obtained. A protocol for CFSE labeling allows direct detection of single proliferating cells and facilitates the quantification of cell divisions by flow cytometry according to the respective CFSE‐dilution, and following costaining with fluorescent labeled antibodies, the characterization of subpopulations in the cell culture. Curr. Protoc. Immunol. 94:7.10.1‐7.10.26.

Loss-of-Function Mutations in Rab Escort Protein 1 (REP-1) Affect Intracellular Transport in Fibroblasts and Monocytes of Choroideremia Patients

Background Choroideremia (CHM) is a progressive X-linked retinopathy caused by mutations in the CHM gene, which encodes Rab escort protein-1 (REP-1), an escort protein involved in the prenylation of Rabs. Under-prenylation of certain Rabs, as a result of loss of function mutations in REP-1, could affect vesicular trafficking, exocytosis and secretion in peripheral cells of CHM patients. Methodology/Principal Findings To evaluate this hypothesis, intracellular vesicle transport, lysosomal acidification and rates of proteolytic degradation were studied in monocytes (CD14+ fraction) and primary skin fibroblasts from the nine age-matched controls and thirteen CHM patients carrying 10 different loss-of-function mutations. With the use of pHrodo™ BioParticles® conjugated with E. coli, collagen I coated FluoSpheres beads and fluorescent DQ™ ovalbumin with BODYPY FL dye, we demonstrated for the first time that lysosomal pH was increased in monocytes of CHM patients and, as a consequence, the rates of proteolytic degradation were slowed. Microarray analysis of gene expression revealed that some genes involved in the immune response, small GTPase regulation, transcription, cell adhesion and the regulation of exocytosis were significantly up and down regulated in cells from CHM patients compared to controls. Finally, CHM fibroblasts secreted significantly lower levels of cytokine/growth factors such as macrophage chemoattractant protein-1 (MCP-1), pigment epithelial derived factor (PEDF), tumor necrosis factor (TNF) alpha, fibroblast growth factor (FGF) beta and interleukin (lL)-8. Conclusions/Significance We demonstrated for the first time that peripheral cells of CHM patients had increased pH levels in lysosomes, reduced rates of proteolytic degradation and altered secretion of cytokines. Peripheral cells from CHM patients expose characteristics that were not previously recognized and could used as an alternative models to study the effects of different mutations in the REP-1 gene on mechanism of CHM development in human population.

Anti-PD-L1 efficacy can be enhanced by inhibition of myeloid derived suppressor cells with a selective inhibitor of PI3Kδ/γ

Checkpoint inhibitors are relatively inefficacious in head and neck cancers, despite an abundance of genetic alterations and a T-cell-inflamed phenotype. One significant barrier to efficacy may be the recruitment of myeloid-derived suppressor cells (MDSC) into the tumor microenvironment. Here we demonstrate functional inhibition of MDSC with IPI-145, an inhibitor of PI3Kδ and PI3Kγ isoforms, which enhances responses to PD-L1 blockade. Combination therapy induced CD8+ T lymphocyte-dependent primary tumor growth delay and prolonged survival only in T-cell-inflamed tumor models of head and neck cancers. However, higher doses of IPI-145 reversed the observed enhancement of anti-PD-L1 efficacy due to off-target suppression of the activity of tumor-infiltrating T lymphocytes. Together, our results offer a preclinical proof of concept for the low-dose use of isoform-specific PI3Kδ/γ inhibitors to suppress MDSC to enhance responses to immune checkpoint blockade. Cancer Res; 77(10); 2607-19. ©2017 AACR.

Clinical efficacy of gene-modified stem cells in adenosine deaminase–deficient immunodeficiency

BACKGROUND. Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase–deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) &ggr;-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1–2.6) and granulocytes (VCN = 0.01–0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION. ClinicalTrials.gov NCT00794508. FUNDING. Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124.

Dynamic Effect of Bortezomib on Nuclear Factor-κB Activity and Gene Expression in Tumor Cells

Nuclear factor-κB (NF-κB) influences the initiation, progression, and maintenance of diverse cancer types. Despite current therapeutic efforts to block hyperactive NF-κB in cancer cells, the in vivo effects of a drug upon this complex pathway are unclear. We monitored NF-κB activity and a fast-expressing reporter level simultaneously in head and neck squamous carcinoma cells by quantitative live microscopy. The real-time single cell assay revealed the tumor necrosis factor-α-induced oscillation of NF-κB was echoed by equally dynamic reporter expression rate. Bortezomib is a proteasome inhibitor whose anticancer action is partly mediated through inhibition of NF-κB. When administered to preactivated cells, the drug gave rise to distinct inhibition dynamics, with discrete pulses of reporter induction remaining for hours. These findings suggest that, contrary to a simplistic presumption for a pathway “blockade,” the network dynamics and the intracellular pharmacokinetics of the inhibitor must be critically evaluated in developing strategies for optimal intervention of oncogenic pathways.

Avoiding phagocytosis-related artifact in myeloid derived suppressor cell T-lymphocyte suppression assays

Myeloid-derived suppressor cells (MDSCs) have garnered much attention in recent years as a potential target for altering the immunosuppressive tumor microenvironment in a variety of solid tumor types. The ability to accurately assess the immunosuppressive capacity of MDSCs is fundamental to the development of therapeutic approaches aimed at disabling these immunosuppressive functions. In this article we provide evidence that the use of CD3/28 coated microbeads leads to artefactual T-lymphocyte suppression due to sequestration of beads by MDSCs isolated from the spleens of wild-type mice bearing subcutaneous syngeneic, carcinogen-induced oral cavity carcinomas. Mechanisms of this finding may include early MDSC death and acquisition of phagocytic capacity. These artefactual findings were avoided by eliminating the use of microbeads and instead using plate bound CD3/28 antibody as the T-lymphocyte stimulus. We propose model-specific validation of microbead-based MDSC assays, or use of an alternative stimulation approach such as plate bound CD3/28 antibodies.

Attenuated TRAF3 Fosters Activation of Alternative NF-κB and Reduced Expression of Antiviral Interferon, TP53, and RB to Promote HPV-Positive Head and Neck Cancers

Human papilloma viruses (HPV) are linked to an epidemic increase in oropharyngeal head and neck squamous cell carcinomas (HNSCC), which display viral inactivation of tumor suppressors TP53 and RB1 and rapid regional spread. However, the role of genomic alterations in enabling the modulation of pathways that promote the aggressive phenotype of these cancers is unclear. Recently, a subset of HPV+ HNSCC has been shown to harbor novel genetic defects or decreased expression of TNF receptor-associated factor 3 (TRAF3). TRAF3 has been implicated as a negative regulator of alternative NF-κB pathway activation and activator of antiviral type I IFN response to other DNA viruses. How TRAF3 alterations affect pathogenesis of HPV+ HNSCC has not been extensively investigated. Here, we report that TRAF3-deficient HPV+ tumors and cell lines exhibit increased expression of alternative NF-κB pathway components and transcription factors NF-κB2/RELB. Overexpression of TRAF3 in HPV+ cell lines with decreased endogenous TRAF3 inhibited NF-κB2/RELB expression, nuclear localization, and NF-κB reporter activity, while increasing the expression of IFNA1 mRNA and protein and sensitizing cells to its growth inhibition. Overexpression of TRAF3 also enhanced TP53 and RB tumor suppressor proteins and decreased HPV E6 oncoprotein in HPV+ cells. Correspondingly, TRAF3 inhibited cell growth, colony formation, migration, and resistance to TNFα and cisplatin-induced cell death. Conversely, TRAF3 knockout enhanced colony formation and proliferation of an HPV+ HNSCC line expressing higher TRAF3 levels. Together, these findings support a functional role of TRAF3 as a tumor suppressor modulating established cancer hallmarks in HPV+ HNSCC.Significance: These findings report the functional role of TRAF3 as a tumor suppressor that modulates the malignant phenotype of HPV+ head and neck cancers. Cancer Res; 78(16); 4613-26. ©2018 AACR.

30. Phase II Clinical Trial of Gene Therapy for Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA-SCID) Using a γ-Retroviral Vector

We report follow-up of subjects treated in a Phase II study of gene therapy for ADA-SCID. Between 2009 and 2012, ten ADA-deficient SCID patients were treated by γ-retroviral-mediated gene transfer (MND-ADA) to their bone marrow CD34+ cells. The subjects were given non-myeloablative chemotherapy (busulfan @ 90 mg/m2) and were withdrawn from PEG-ADA enzyme replacement therapy (ERT) prior to infusion of autologous gene-modified cells. Subject age at the time of treatment ranged from 3 months to 15 years (median = 11.5 months). Follow-up times range from 2 to 5 years. All but one subject, who was 15-years old at the time of treatment, remain off PEG-ADA ERT with immune reconstitution that reached maximal level between 6 and 12 months after transplant and was maintained thereafter. Vector marking in peripheral blood cells remained consistently detectable (> 0.1 copy/PBMC and ≥ 0.003 copy/granulocyte) at 2 years and later after transplant in subjects who discontinued ERT. These subjects also had PBMC ADA enzymatic activity in the normal range and red blood cell deoxynucleotide levels below 10%. Three subjects have discontinued intravenous immunoglobulin; five subjects have discontinued prophylactic antibiotics. All subjects have polyclonal gene marking with no sign of lymphoproliferative disease. The subjects remain in good health without infections or other complications.

UNIT 7.10 Measurement of Proliferative Responses of Cultured Lymphocytes

Measurement of proliferative responses of human lymphocytes is a fundamental technique for the assessment of their biological responses to various stimuli. Most simply, this involves measurement of the number of cells present in a culture before and after the addition of a stimulating agent. This unit contains several different prototype protocols to measure the proliferation response of lymphocytes following exposure to mitogens, antigens, allogeneic or autologous cells, or soluble factors. Each of these protocols can be used in conjunction with an accompanying support protocol which contains methods for pulsing cultures with [3H]thymidine and determining incorporation of [3H]thymidine into DNA. The protocols described here provide an estimate of DNA synthesis and cell proliferation in an entire cell population, but do not provide information on the proliferation of individual cells.