Matching Items (9)
Filtering by
- All Subjects: Immunology
- Creators: Blattman, Joseph N
Description
Vaccination remains one of the most effective means for preventing infectious diseases. During viral infection, activated CD8 T cells differentiate into cytotoxic effector cells that directly kill infected cells and produce anti-viral cytokines. Further T cell differentiation results in a population of memory CD8 T cells that have the ability to self-renew and rapidly proliferate into effector cells during secondary infections. However during persistent viral infection, T cell differentiation is disrupted due to sustained antigen stimulation resulting in a loss of T cell effector function. Despite the development of vaccines for a wide range of viral diseases, efficacious vaccines for persistent viral infections have been challenging to design. Immunization against virus T cell epitopes has been proposed as an alternative vaccination strategy for persistent viral infections, such as HIV. However, vaccines that selectively engage T cell responses can result in inappropriate immune responses that increase, rather than prevent, disease. Quantitative models of virus infection and immune response were used to investigate how virus and immune system variables influence pathogenic versus protective T cell responses generated during persistent viral infection. It was determined that an intermediate precursor frequency of virus-specific memory CD8 T cells prior to LCMV infection resulted in maximum T cell mediated pathology. Increased pathology was independent of antigen sensitivity or the diversity of TCR in the CD8 T cell response, but was dependent on CD8 T cell production of TNF and the magnitude of initial virus exposure. The threshold for exhaustion of responding CD8 T cells ultimately influences the precursor frequency that causes enhanced disease.In addition, viral infection can occur in the context of co-infection by heterologous pathogens that modulate immune responses and/or disease. Co-infection of two unrelated viruses in their natural host, Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV) infection in mice, were studied. ECTV infection can be a lethal infection in mice due in part to the blockade of antiviral cytokines, including Type I Interferons (IFN-I). It was determined that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, presumably due to IFN-I induction by LCMV. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and biased toward effector-memory cell generation. Thus, providing evidence for bi-directional effects of viral co-infection that modulate disease and immunity. Together the results suggest heterogeneity in T cell responses during vaccination with viral vectors may be in part due to heterologous virus infection or vaccine usage and that TNF-blockade may be useful for minimizing pathology while maintaining protection during virus infection. Lastly, quantitative mathematical models of virus and T cell immunity can be useful to generate predictions regarding which molecular and cellular pathways mediate T cell protection versus pathology.
ContributorsMcAfee, Megan (Author) / Blattman, Joseph N (Thesis advisor) / Anderson, Karen (Committee member) / Jacobs, Bertram (Committee member) / Hogue, Brenda (Committee member) / Arizona State University (Publisher)
Created2015
Description
The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is essential for the innate immune response to danger signals. Importantly, the NLRP3 inflammasome responds to structurally and functionally dissimilar stimuli. It is currently unknown how the NLRP3 inflammasome responds to such diverse triggers. This dissertation investigates the role of ion flux in regulating the NLRP3 inflammasome. Project 1 explores the relationship between potassium efflux and Syk tyrosine kinase. The results reveal that Syk activity is upstream of mitochondrial oxidative signaling and is crucial for inflammasome assembly, pro-inflammatory cytokine processing, and caspase-1-dependent pyroptotic cell death. Dynamic potassium imaging and molecular analysis revealed that Syk is downstream of, and regulated by, potassium efflux. Project 1 reveals the first identified intermediate regulator of inflammasome activity regulated by potassium efflux. Project 2 focuses on P2X7 purinergic receptor-dependent ion flux in regulating the inflammasome. Dynamic potassium imaging revealed an ATP dose-dependent efflux of potassium driven by P2X7. Surprisingly, ATP induced mitochondrial potassium mobilization, suggesting a mitochondrial detection of purinergic ion flux. ATP-induced potassium and calcium flux was found to regulate mitochondrial oxidative signaling upstream of inflammasome assembly. First-ever multiplexed imaging of potassium and calcium dynamics revealed that potassium efflux is necessary for calcium influx. These results suggest that ATP-induced potassium efflux regulates the inflammasome by calcium influx-dependent mitochondrial oxidative signaling. Project 2 defines a coordinated cation flux dependent on the efflux of potassium and upstream of mitochondrial oxidative signaling in inflammasome regulation. Lastly, this dissertation contributes two methods that will be useful for investigating inflammasome biology: an optimized pipeline for single cell transcriptional analysis, and a mouse macrophage cell line expressing a genetically encoded intracellular ATP sensor. This dissertation contributes to understanding the fundamental role of ion flux in regulation of the NLRP3 inflammasome and identifies potassium flux and Syk as potential targets to modulate inflammation.
ContributorsYaron, Jordan Robin (Author) / Meldrum, Deirdre R (Thesis advisor) / Blattman, Joseph N (Committee member) / Glenn, Honor L (Committee member) / Arizona State University (Publisher)
Created2015
Description
There are many biological questions that require single-cell analysis of gene sequences, including analysis of clonally distributed dimeric immunoreceptors on lymphocytes (T cells and B cells) and/or the accumulation of driver/accessory mutations in polyclonal tumors. Lysis of bulk cell populations results in mixing of gene sequences, making it impossible to know which pairs of gene sequences originated from any particular cell and obfuscating analysis of rare sequences within large populations. Although current single-cell sorting technologies can be used to address some of these questions, such approaches are expensive, require specialized equipment, and lack the necessary high-throughput capacity for comprehensive analysis. Water-in-oil emulsion approaches for single cell sorting have been developed but droplet-based single-cell lysis and analysis have proven inefficient and yield high rates of false pairings. Ideally, molecular approaches for linking gene sequences from individual cells could be coupled with next-generation high-throughput sequencing to overcome these obstacles, but conventional approaches for linking gene sequences, such as by transfection with bridging oligonucleotides, result in activation of cellular nucleases that destroy the template, precluding this strategy. Recent advances in the synthesis and fabrication of modular deoxyribonucleic acid (DNA) origami nanostructures have resulted in new possibilities for addressing many current and long-standing scientific and technical challenges in biology and medicine. One exciting application of DNA nanotechnology is the intracellular capture, barcode linkage, and subsequent sequence analysis of multiple messenger RNA (mRNA) targets from individual cells within heterogeneous cell populations. DNA nanostructures can be transfected into individual cells to capture and protect mRNA for specific expressed genes, and incorporation of origami-specific bowtie-barcodes into the origami nanostructure facilitates pairing and analysis of mRNA from individual cells by high-throughput next-generation sequencing. This approach is highly modular and can be adapted to virtually any two (and possibly more) gene target sequences, and therefore has a wide range of potential applications for analysis of diverse cell populations such as understanding the relationship between different immune cell populations, development of novel immunotherapeutic antibodies, or improving the diagnosis or treatment for a wide variety of cancers.
ContributorsSchoettle, Louis (Author) / Blattman, Joseph N (Thesis advisor) / Yan, Hao (Committee member) / Chang, Yung (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2017
Description
The HIV-1 pandemic continues to cause millions of new infections and AIDS-related deaths each year, and a majority of these occur in regions of the world with limited access to antiretroviral therapy. Therefore, an HIV-1 vaccine is still desperately needed. The most successful HIV-1 clinical trial to date used a non-replicating canarypox viral vector and protein boosting, yet its modest efficacy left room for improvement. Efforts to derive novel vectors which can be both safe and immunogenic, have spawned a new era of live, viral vectors. One such vaccinia virus vector, NYVAC-KC, was specifically designed to replicate in humans and had several immune modulators deleted to improve immunogenicity and reduce pathogenicity. Two NYVAC-KC vectors were generated: one expressing the Gag capsid, and one with deconstructed-gp41 (dgp41), which contains an important neutralizing antibody target, the membrane proximal external region (MPER). These vectors were combined with HIV-1 Gag/dgp41 virus-like particles (VLPs) produced in the tobacco-relative Nicotiana benthamiana. Different plant expression vectors were compared in an effort to improve yield. A Geminivirus-based vector was shown to increase the amount of MPER present in VLPs, thus potentially enhancing immunogenicity. Furthermore, these VLPs were shown to interact with the innate immune system through Toll-like receptor (TLR) signaling, which activated antigen presenting cells to induce a Th2-biased response in a TLR-dependent manner. Furthermore, expression of Gag and dgp41 in NYVAC-KC vectors resulted in activation of antiviral signaling pathways reliant on TBK1/IRF3, which necessitated the use of higher doses in mice to match the immunogenicity of wild-type viral vectors. VLPs and NYVAC-KC vectors were tested in mice, ultimately showing that the best antibody and Gag-specific T cell responses were generated when both components were administered simultaneously. Thus, plant-produced VLPs and poxvirus vectors represent a highly immunogenic HIV-1 vaccine candidate that warrants further study.
ContributorsMeador, Lydia Rebecca (Author) / Mor, Tsafrir S (Thesis advisor) / Jacobs, Bertram L (Thesis advisor) / Blattman, Joseph N (Committee member) / Mason, Hugh S (Committee member) / Arizona State University (Publisher)
Created2016
Description
Agassiz’s desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.
Data Availability: All genomic and transcriptomic sequence files are available from the NIH-NCBI BioProject database (accession numbers PRJNA352725, PRJNA352726, and PRJNA281763). All genome assembly, transcriptome assembly, predicted protein, transcript, genome annotation, repeatmasker, phylogenetic trees, .vcf and GO enrichment files are available on Harvard Dataverse (doi:10.7910/DVN/EH2S9K).
ContributorsTollis, Marc (Author) / DeNardo, Dale F (Author) / Cornelius, John A (Author) / Dolby, Greer A (Author) / Edwards, Taylor (Author) / Henen, Brian T. (Author) / Karl, Alice E. (Author) / Murphy, Robert W. (Author) / Kusumi, Kenro (Author)
Created2017-05-31
Description
The innate immune system serves as an immediate response to pathogenic infection and an informant to the adaptive immune system. The 2′,5′-oligoadenylate (2-5A) synthetase (OAS)–RNase-L system is a component of the innate immune system induced by interferons (IFNs) and serves to eliminate viral infections. In humans, three enzymatically active OAS proteins exist, OAS1, OAS2, and OAS3. Recent evidence suggests variations in cellular localization of OAS proteins may influence the impact and influence of those proteins on viral replication. However, viral suppression mechanisms involving specific OAS proteins are still unclear for most viruses. Here, I overexpress different isoforms of OAS and determined that though viruses within the same family have similar replication strategies, the extent to which each OAS protein impacts viral replication for Flaviviruses, and Alphaviruses varies. In contrast to the innate immune system, the adaptive immune system provides specific and long-lived immune responses. In the context of cancer, T cells have been shown to play a prominent role in tumor regression. It has previously been demonstrated that administration α-CTLA-4/α-PD-L1 immune checkpoint blockade (ICB) to mice inoculated with a K7M2 metastatic osteosarcoma (mOS) cell line resulted in ~50% survival. Here, I sought to determine biological differences among murine responders and non-responders to ICB for mOS to understand better what factors could increase ICB efficacy. A prospective culprit is a variance in circulating antibodies (Abs). I have shown that sera from mice, before inoculation with mOS or ICB, display distinct differences in Ab repertoire between responders and non-responders, suggesting the presence or absence of particular Abs may influence the outcome of ICB. Recent studies have also shown that malleable environmental factors, such as differences in microbiome composition, can yield subsequent changes in circulating Abs.
Strong associations have been made between host-microbiome interactions and their effects on health. Here, I study potential associations of microbiome-mediated impacts on ICB efficacy for mOS. Additionally, I sought to determine potential changes in T-cellular response to mOS due to modulations in microbiome composition and showed that ICB efficacy can change in conjunction with microbiome composition changes in a murine model.
ContributorsDi Palma, Michelle Pina (Author) / Blattman, Joseph N (Thesis advisor) / Li, Yize (Thesis advisor) / Anderson, Karen S (Committee member) / McFadden, Grant (Committee member) / Arizona State University (Publisher)
Created2023
Description
Skeletal muscle can intrinsically repair itself in response to injury. This repair process has been shown to be mediated through signaling of the innate immune system. The immune response caused during repair helps to clear away debris in damage and promotes the activation and proliferation of muscle stem cells (MuSCs) that will repair the damage muscle. Dysregulation of this inflammation leads to fibrosis and decreased efficacy of the repair process. Despite the requirement of inflammatory signaling during muscle repair, muscle’s contribution during inflammation as only recently started to be explored. The objective of this dissertation is to assess the contribution of muscle in the early inflammatory response during repair as well attempting to modulate this inflammation during disease to ameliorate disease pathology in a model of Duchenne’s muscular dystrophy. I tested the hypotheses that 1) muscle is an active participant in the early inflammatory response, 2) the transcription factor Mohawk (Mkx) is a regulator of the early inflammatory response and, 3) If this inflammation can be modulated with a virally derived serine protease inhibitor in a model of muscle disrepair and chronic inflammation. I found that muscle is actively participating in the establishment early inflammation in repair through the production of chemokines used to promote infiltration of immune cells. As well as the identification of a new muscle subtype that produces more chemokines compared to the average MuSC and upregulated genes in the Interferon signaling pathway. I also discovered that presence of this muscle subtype is linked to the expression of Mkx. In Mkx null mice this population is not present, and these cells are deficient in chemokine expression compared to WT mice. I subsequently found that, using the myxomavirus derived serine protease inhibitor, Serp-1 I was able to modulate the chronic inflammation that is common in those affected with Duchenne’s muscular dystrophy (DMD) utilizing a high-fidelity mouse model of the disease. The result of this dissertation provides an expanded role for muscle in inflammation and gives a potential new class of therapeutics to be used in disease associated with chronic inflammation.
ContributorsAndre, Alex (Author) / Rawls, Alan (Thesis advisor) / Wilson-Rawls, Jeanne (Committee member) / Kusumi, Kenro (Committee member) / Lake, Doug (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2022
Description
Vitellogenin (Vg) is an ancient and highly conserved multifunctional protein. It is primarily known for its role in egg-yolk formation but also serves functions pertaining to immunity, longevity, nutrient storage, and oxidative stress relief. In the honey bee (Apis mellifera), Vg has evolved still further to include important social functions that are critical to the maintenance and proliferation of colonies. Here, Vg is used to synthesize royal jelly, a glandular secretion produced by a subset of the worker caste that is fed to the queen and young larvae and which is essential for caste development and social immunity. Moreover, Vg in the worker caste sets the pace of their behavioral development as they transition between different tasks throughout their life. In this dissertation, I make several new discoveries about Vg functionality. First, I uncover a colony-level immune pathway in bees that uses royal jelly as a vehicle to transfer pathogen fragments between nestmates. Second, I show that Vg is localized and expressed in the honey bee digestive tract and suggest possible immunological functions it may be performing there. Finally, I show that Vg enters to nucleus and binds to deoxyribonucleic acid (DNA), acting as a potential transcription factor to regulate expression of many genes pertaining to behavior, metabolism, and signal transduction pathways. These findings represent a significant advance in the understanding of Vg functionality and honey bee biology, and set the stage for many future avenues of research.
ContributorsHarwood, Gyan (Author) / Amdam, Gro V (Thesis advisor) / Kusumi, Kenro (Committee member) / Rabeling, Christian (Committee member) / Chang, Yung (Committee member) / Arizona State University (Publisher)
Created2019
Description
Glioblastoma (GBM) is a highly invasive and deadly late stage tumor that develops from abnormal astrocytes in the brain. With few improvements in treatment over many decades, median patient survival is only 15 months and the 5-year survival rate hovers at 6%. Numerous challenges are encountered in the development of treatments for GBM. The blood-brain barrier (BBB) serves as a primary obstacle due to its innate ability to prevent unwanted molecules, such as most chemotherapeutics, from entering the brain tissue and reaching malignant cells. The GBM cells themselves serve as a second obstacle, having a high level of genetic and phenotypic heterogeneity. This characteristic improves the probability of a population of cells to have resistance to treatment, which ensures the survival of the tumor. Here, the development and testing of two different modes of therapy for treating GBM is described. These therapeutics were enhanced by pathogenic peptides known to improve entry into brain tissue or to bind GBM cells to overcome the BBB and/or tumor cell heterogeneity. The first therapeutic utilizes a small peptide, RVG-29, derived from the rabies virus glycoprotein to improve brain-specific delivery of nanoparticles encapsulated with a small molecule payload. RVG-29-targeted nanoparticles were observed to reach the brain of healthy mice in higher concentrations 2 hours following intravenous injection compared to control particles. However, targeted camptothecin-loaded nanoparticles were not capable of producing significant treatment benefits compared to non-targeted particles in an orthotopic mouse model of GBM. Peptide degradation following injection was shown to be a likely cause for reduced treatment benefit. The second therapeutic utilizes chlorotoxin, a non-toxic 36-amino acid peptide found in the venom of the deathstalker scorpion, expressed as a fusion to antibody fragments to enhance T cell recognition and killing of GBM. This candidate biologic, known as anti-CD3/chlorotoxin (ACDClx) is expressed as an insoluble protein in Nicotiana benthamiana and Escherichia coli and must be purified in denaturing and reducing conditions prior to being refolded. ACDClx was shown to selectively activate T cells only in the presence of GBM cells, providing evidence that further preclinical development of ACDClx as a GBM immunotherapy is warranted.
ContributorsCook, Rebecca Leanne (Author) / Blattman, Joseph N (Thesis advisor) / Sirianni, Rachael W. (Thesis advisor) / Mor, Tsafrir (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2019