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- All Subjects: Immunology
- Creators: School of Life Sciences
Description
The amphibian pathogen Ambystoma Tigrinum Virus (ATV) has been an important topic of study within the amphibian community since its discovery. ATV threatens many salamander populations across the US, including those in east-central and southeast Arizona. These populations remain at risk since there are no treatments available. In this thesis, a novel method of inactivation is tested to produce a vaccine with the aim of safely eliciting an immune response within the salamander host. This novel form of inactivation has been tested on several human pathogens but has yet to be used on amphibian pathogens. It has the potential to revolutionize our traditional approach to inactivating viruses. After laser treatment, viral plaque assays suggested that inactivated ATV ceased to grow completely, pointing to the possibility of creating a vaccine. Animal challenge trials were conducted with 60 juvenile Ambystoma tigrinum, but surprisingly there was no protective effect from viral inactivation. Further study is needed to clarify why in vitro and in vivo tests of viral inactivation produced contradictory results.
ContributorsVazquez, Luis Ernesto (Author) / Collins, James (Thesis director) / Tsen, Kong-Thon (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer, are being developed and refined to treat such cancers. T cell immunotherapies in particular, where a patient’s T cell lymphocytes are isolated and amplified to be re-infused into the patient or where human cell lines are engineered to express T cell receptors for the recognition of common cancer antigens, are being expanded on because for some cancers, they could be the only option. Constructing an optimal pipeline for cloning and expression of antigen-specific TCRs has significant bearing on the efficacy of engineered cell lines for ACT. Adoptive T cell transfer, while making great strides, has to overcome a diverse T cell repertoire – cloning and expressing antigen-specific TCRs can mediate this understanding. Having identified the high frequency FluM1-specific TCR sequences in stimulated donor PBMCs, it was hypothesized that the antigen-specific TCR could be reconstructed via Gateway cloning methods and tested for expression and functionality. Establishing this pipeline would confirm an ability to properly pair and express the heterodimeric chains. In the context of downstream applications, neoantigens would be used to stimulate T cells, the α and β chains would be paired via single-cell or bulk methods, and instead of Gateway cloning, the CDR3 hypervariable regions α and β chains alone would be co-expressed using Golden Gate assembly methods.
ContributorsHirneise, Gabrielle Rachel (Author) / Anderson, Karen (Thesis director) / Mason, Hugh (Committee member) / Hariadi, Hugh (Committee member) / School of Life Sciences (Contributor, Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
T cells, a component of the adaptive immune system, play an instrumental role in directing immune responses and direct cell killing in response to pathogens and cancers. T cells recognize and signal through the T cell receptor, a protein heterodimer on the surface of T cells. The T cell receptor is a highly variable structure formed via somatic recombination; the structure recognizes peptides presented on the surface of nucleated cells by major histocompatibility complex proteins in a specific receptor-restricted, peptide-restricted manner. This balance between T cell diversity and T cell specificity stands as a barrier to efficacious development of articificial T cell receptors capable of clearing disease. T cell receptors may be tailored to produce pathogen- or cancer-specific immune responses from autologous T cell populations. This necessitates a pipeline for amplification, cloning, and expression of antigen-specific T cell receptors. This study aims to utilize influenza-specific T cell receptor chains from healthy donor T cells to test a model for T cell receptor cloning and expression. This study utilizes Gateway recombination for high-throughput cloning into mammalian expression vectors. This study has successfully amplified and cloned T cell receptor chains from a population of influenza-specific T cells from donor cell transcripts into mammalian cell expression vectors. Additionally, CD8, a coreceptor for the T cell receptor complex, was successfully cloned and inserted into a vector for expression in mammalian cells. Sanger sequencing has confirmed sequences for influenza-specific T cell receptor chains and the CD8 chain. Future application of this project includes expression in mammalian non-T cells to test for efficacy of expression and, ultimately, expression in cytotoxic cells to create lymphocytes capable of antigen-specific recognition and cytolytic killing of cells of interest.
ContributorsVale, Nolan Richard (Author) / Anderson, Karen (Thesis director) / Blattman, Joseph (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Immunology, the study of the immune system and its ability to distinguish self from non-self, is a rapidly advancing sector of molecular biology. Cancer, being host derived, provides a difficult challenge for immune cells to distinguish it from normal tissue. The historic treatment of cancer has had three main methods: radiation, chemotherapy, and surgery (1). Due to recent advancements in understanding the regulatory role of adaptive immunity against cancer, researchers have been attempting to engineer therapies to enhance patients’ immunities against their cancer. Immunotherapies, both passive and active, demonstrate potential for combating many diseases. Passive immunization provides temporary protection against a pathogen, whereas active immunization teaches the patient’s system to respond to the antigen independently, giving life-long immunity. Passive immunization, generally, is a much more expensive method of providing immunity and is commonly used in emergency situations. Anti-venom, for example, uses antibodies grown in lab to neutralize venom. Examples of active immunization are vaccines, which mimic the wild-type pathogen in a way that elicits an immune response, specifically naïve lymphocyte activation and maturation into memory lymphocytes. In terms of cancer therapy, both passive and active immunization are being tested for efficacy (2).
ContributorsMarquardt, Charles Andrew (Author) / Anderson, Karen S. (Thesis director) / Mason, Hugh S. (Committee member) / Lake, Douglas F. (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Cytokines induced by inflammasome has been used for blood cancer treatments, yet these treatments have been less successful in the solid tumor microenvironment. Here precise-morphology DNA origami structures were implemented to accurately test the effect and mechanism of activation in the NLRP3 inflammasome. THP1 WT cells, a macrophage cell line, were treated with eleven different DNA origami structures. The inflammasome activation of two cytokines, Interleukin 1 beta (IL-1β) and Interferon beta (IFN-β), was measured using HEK Blue IL-1β cells, HEK Blue IFN-β cells, and enzyme linked immunosorbent assay (ELISA). Differences in activation signaling have the potential to provide the characterization required to address the intrinsic complexity of modulating an immune response. It is hoped that DNA origami will help induce more inflammation for solid tumors. The DNA origami was tested in three different volumes: 1 μL, 5 μL, and 10 μL. Overall, the origami that showed promising results were Mg Square. Tetrahedral and P53 block also showed potential but not as well as Mg square. Further testing of more DNA origami structures and testing them in mice are key to the success of targeted cancer immunotherapies in the neoadjuvant setting.
ContributorsGreenwald, Elinor Vera (Co-author) / Ariola, Amanda (Co-author) / Ning, Bo (Thesis director) / Zhang, Fei (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Inhibitor of growth factor 4 (ING4) is a tumor suppressor of which low expression has been associated with poor patient survival and aggressive tumor progression in breast cancer. ING4 is characterized as a transcription regulator of inflammatory genes. Among the ING4-regulated genes is CXCL10, a chemokine secreted by endothelial cells during normal inflammation response, which induces chemotactic migration of immune cells to the site. High expression of CXCL10 has been implicated in aggressive breast cancer, but the mechanism is not well understood. A potential signaling molecule downstream of Cxcl10 is Janus Kinase 2 (Jak2), a kinase activated in normal immune response. Deregulation of Jak2 is associated with metastasis, immune evasion, and tumor progression in breast cancer. Thus, we hypothesized that the Ing4/Cxcl10/Jak2 axis plays a key role in breast cancer progression. We first investigated whether Cxcl10 affected breast cancer cell migration. We also investigated whether Cxcl10-mediated migration is dependent on ING4 expression levels. We utilized genetically engineered MDAmb231 breast cancer cells with a CRISPR/Cas9 ING4-knockout construct or a viral ING4 overexpression construct. We performed Western blot analysis to confirm Ing4 expression. Cell migration was assessed using Boyden Chamber assay with or without exogenous Cxcl10 treatment. The results showed that in the presence of Cxcl10, ING4-deficient cells had a two-fold increase in migration as compared to the vector controls, suggesting Ing4 inhibits Cxcl10-induced migration. These findings support our hypothesis that ING4-deficient tumor cells have increased migration when Cxcl10 signaling is present in breast cancer. These results implicate Ing4 is a key regulator of a chemokine-induced tumor migration. Our future plan includes evaluation of Jak2 as an intermediate signaling molecule in Cxcl10/Ing4 pathway. Therapeutic implications of these findings are targeting Cxcl10 and/or Jak2 may be effective in treating ING4-deficient aggressive breast cancer.
ContributorsArnold, Emily (Author) / Kim, Suwon (Thesis director) / Blattman, Joseph (Thesis director) / Mason, Hugh (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
An aim of fundamental immunology is quantifying the diversity of the T cell receptor (TCR) repertoire to elucidate the vast recognition by T cells for protection against pathogen and cancer. The utilization of DNA origami nanostructures engineered to capture single cell paired TCR mRNA sequences has transformed the financial and time requirements of repertoire establishment. To further support this protocol, confocal laser scanning microscopy was implemented following transfection to visualize the stability of the DNA origami within primary immune lymphocytes.
ContributorsReed, Abigail Elizabeth (Author) / Blattman, Joseph (Thesis director) / Glenn, Honor (Committee member) / Schoettle, Louis (Committee member) / School of Life Sciences (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Vaccinia virus is a cytoplasmic, double-stranded DNA orthopoxvirus. Unlike mammalian cells, vaccinia virus produces double-stranded RNA (dsRNA) during its viral life cycle. The protein kinase R, PKR, is one of the principal host defense mechanisms against orthopoxvirus infection. PKR can bind double-stranded RNA and phosphorylate eukaryotic translation initiation factor, eIF2α, shutting down protein synthesis and halting the viral life cycle. To combat host defenses, vaccinia virus encodes E3, a potent inhibitor of the cellular anti-viral eIF2α kinase, PKR. The E3 protein contains a C-terminal dsRNA-binding motif that sequesters dsRNA and inhibits PKR activation. We demonstrate that E3 also interacts with PKR by co-immunoprecipitation. This interaction is independent of the presence of dsRNA and dsRNA-binding by E3, indicating that the interaction is not due to dsRNA-bridging.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
PKR interaction mapped to a region within the dsRNA-binding domain of E3 and overlapped with sequences in the C-terminus of this domain that are necessary for binding to dsRNA. Point mutants of E3 were generated and screened for PKR inhibition and direct interaction. Analysis of these mutants demonstrates that dsRNA-binding but not PKR interaction plays a critical role in the broad host range of VACV. Nonetheless, full inhibition of PKR in cells in culture requires both dsRNA-binding and PKR interaction. Because E3 is highly conserved among orthopoxviruses, understanding the mechanisms that E3 uses to inhibit PKR can give insight into host range pathogenesis of dsRNA producing viruses.
ContributorsFoster, Clayton (Co-author) / Alattar, Hamed (Co-author) / Jacobs, Bertram (Thesis director) / Blattman, Joseph (Committee member) / McFadden, Grant (Committee member) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Purinergic receptors sense extracellular nucleotide DAMPs such as ATP and adenosine, which are expressed in high concentrations in the tumor microenvironment (TME). A2AR, an adenosine receptor that is expressed on both T cells and tumor cells, promotes immunosuppression. However, the impact of the TME on changes in purinergic receptor expression on CD8 T cells, as well as the overall dynamic between A2AR expression and tumor control, have not been clearly elucidated. Using in vitro co-culture experiments and in vivo murine tumor models, we found that A2AR is significantly upregulated on effector, tumor-infiltrating CD8 T cells. This upregulation was independent of the hypoxia, which we identified via inhibition of HIF1A. We found that this upregulation was partially dependent on CD8 T cell-tumor contact, but independent of cognate antigen recognition, by using transwell co-cultures, as well as combinations of different transgenic lines of CD8 T cells and tumor cells. We confirmed this observation in vivo using transfer of activated OTI cells into B16.OVA-bearing mice. Ultimately, we observed that the upregulation depended on inhibitory receptors such as Tim3 via the antibody blockade of Tim3. Using CRISPR/Cas9-mediated knockout of A2AR on activated CD8 T cells, we found that tumor-bearing mice receiving A2AR knockout CD8 T cells had increased tumor control. Taken together, these results suggest that inhibitory receptor-dependent, TCR-independent signals in the TME promotes upregulation of A2AR on CD8 T cells, leading to impairment of CD8 T cell-mediated tumor control.
ContributorsZhou, Maggie (Author) / Borges da Silva, Henrique (Thesis director) / Borges Florsheim, Esther (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Economics Program in CLAS (Contributor)
Created2022-12
Description
For my thesis, Professor Florsheim and I decided to focus on building lab experience in preparation for my master’s thesis. This included reading various research papers, starting a mast cell culture, and learning techniques essential for lab work. Additionally, I would conduct presentations and work with my peers to learn about various testing methods and components of the lab. One of the most crucial components of this experience included learning about how to collect bone marrow mast cells including how to properly sacrifice a mouse. My final product is the grant proposal which is what my focus will be for my master’s year.
ContributorsGalarza, Mayka (Author) / Florsheim, Esther (Thesis director) / Mana, Miyeko (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2022-05