Matching Items (34)
Filtering by
- All Subjects: Immunology
- Creators: School of Life Sciences
- Creators: Lake, Douglas
- Resource Type: Text
- Status: Published
Description
The majority of non-small cell lung cancer (NSCLC) patients (70%) are diagnosed with adenocarcinoma versus other histological subtypes. These patients often present with advanced, metastatic disease and frequently relapse after treatment. The tumor suppressor, Liver Kinase B1, is frequently inactivated in adenocarcinomas and loss of function is associated with a highly aggressive, metastatic tumor (1). Identification of the mechanisms deregulated with LKB1 inactivation could yield targeted therapeutic options for adenocarcinoma patients. Re-purposing the immune system to support tumor growth and aid in metastasis has been shown to be a feature in cancer progression (2). Tumor associated macrophages (TAMs) differentiate from monocytes, which are recruited to the tumor microenvironment via secretion of chemotaxic factors by cancer cells. We find that NSCLC cells deficient in LKB1 display increased secretion of C-C motif ligand 2 (CCL2), a chemokine involved in monocyte recruitment. To elucidate the molecular pathway regulating CCL2 up-regulation, we investigated inhibitors of substrates downstream of LKB1 signaling in A549, H23, H2030 and H838 cell lines. Noticeably, BAY-11-7082 (NF-κB inhibitor) reduced CCL2 secretion by an average 92%. We further demonstrate that a CCR2 antagonist and neutralizing CCL2 antibody substantially reduce monocyte migration to NSCLC (H23) cell line conditioned media. Using an in vivo model of NSCLC, we find that LKB1 deleted tumors demonstrate a discernible increase in CCL2 levels compared to normal lung. Moreover, tumors display an increase in the M2:M1 macrophage ratio and increase in tumor associated neutrophil (TAN) infiltrate compared to normal lung. This M2 shift was significantly reduced in mice treated with anti-CCL2 or a CCR2 antagonist and the TAN infiltrate was significantly reduced with the CCR2 antagonist. These data suggest that deregulation of the CCL2/CCR2 signaling axis could play a role in cancer progression in LKB1 deficient tumors.
ContributorsFriel, Jacqueline (Author) / Inge, Landon (Thesis advisor) / Lake, Douglas (Thesis advisor) / Blattman, Joseph (Committee member) / Arizona State University (Publisher)
Created2015
Description
Osteosarcoma is the most common bone cancer in children and adolescents. Patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T-lymphocytes (CTL) limit the development of metastatic osteosarcoma. I have investigated the role of Programmed Death Receptor-1 (PD-1) in limiting the efficacy of immune mediated control of metastatic osteosarcoma. I show that human metastatic, but not primary, osteosarcoma tumors express the ligand for PD-1 (PD-L1) and that tumor infiltrating CTL express PD-1, suggesting this pathway may limit CTL control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor infiltrating CTL during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTL in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. My results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy. However, PD-1/PD-L1 blockade treated mice still succumb to disease due to selection of PD-L1 mAb resistant tumor cells via up-regulation of other co-inhibitory T cell receptors. Combinational α-CTLA-4 and α-PD-L1 blockade treated mice were able to completely eradicate metastatic osteosarcoma, and generate immunity to disease. These results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma, although improves survival, may lead to tumor resistance, requiring combinational immunotherapies to combat and eradicate disease.
ContributorsLussier, Danielle (Author) / Blattman, Joseph N. (Thesis advisor) / Anderson, Karen (Committee member) / Goldstein, Elliott (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2015
Description
Antibodies are naturally occurring proteins that protect a host during infection through direct neutralization and/or recruitment of the innate immune system. Unfortunately, in some infections, antibodies present unique hurdles that must be overcome for a safer and more efficacious antibody-based therapeutic (e.g., antibody dependent viral enhancement (ADE) and inflammatory pathology). This dissertation describes the utilization of plant expression systems to produce N-glycan specific antibody-based therapeutics for Dengue Virus (DENV) and Chikungunya Virus (CHIKV). The Fc region of an antibody interacts with Fcγ Receptors (FcγRs) on immune cells and components of the innate immune system. Each class of immune cells has a distinct action of neutralization (e.g., antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP)). Therefore, structural alteration of the Fc region results in novel immune pathways of protection. One approach is to modulate the N-glycosylation in the Fc region of the antibody. Of scientific significance, is the plant’s capacity to express human antibodies with homogenous plant and humanized N-glycosylation (WT and GnGn, respectively). This allows to study how specific glycovariants interact with other components of the immune system to clear an infection, producing a tailor-made antibody for distinct diseases. In the first section, plant-produced glycovariants were explored for reduced interactions with specific FcγRs for the overall reduction in ADE for DENV infections. The results demonstrate a reduction in ADE of our plant-produced monoclonal antibodies in in vitro experiments, which led to a greater survival in vivo of immunodeficient mice challenged with lethal doses of DENV and a sub-lethal dose of DENV in ADE conditions. In the second section, plant-produced glycovariants were explored for increased interaction with specific FcγRs to improve ADCC in the treatment of the highly inflammatory CHIKV. The results demonstrate an increase ADCC activity in in vitro experiments and a reduction in CHIKV-associated inflammation in in vivo mouse models. Overall, the significance of this dissertation is that it can provide a treatment for DENV and CHIKV; but equally importantly, give insight to the role of N-glycosylation in antibody effector functions, which has a broader implication for therapeutic development for other viral infections.
ContributorsHurtado, Jonathan (Author) / Chen, Qiang (Thesis advisor) / Arntzen, Charles (Committee member) / Borges, Chad (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2019
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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
Viral infections are a significant cause of disease in humans. While some viral diseases have been eliminated, many more continue to infect millions. Viral infections are challenging to treat because viruses use host cell machinery to replicate, so it is difficult to develop drugs that can target viruses. Normally, the host’s immune system is capable of destroying the virus, but during chronic infections it becomes exhausted and T cells lose their effector functions necessary for the clearance of the virus. IL-2 can help relieve this exhaustion, but causes toxicity to the body. In mice infected with chronic LCMV, IL-2 administration causes death due to pulmonary hemorrhage. CD4 deficient mice were infected with chronic LCMV and then dosed with IL-2 and survived, but mice that were deficient for CD8 T cells died, indicating that toxicity was mediated by CD8 T cells. CD8 T cells can kill infected host cells directly by producing perforin, or can produce cytokines like IFN-γ and TNF to further activate the immune system and mediate killing. Mice that were deficient in perforin died after IL-2 administration, as well as mice that were deficient in IFN-γ. Mice deficient in TNF, however, survived, indicating that TNF was mediating the toxicity in response to IL-2. There are two different receptors for TNF, p55 and p75. p55 is known as TNFR1 and has been implicated in apoptosis of virally infected cells. P75 is known as TNFR2 and is associated more with inflammation in response to infection. My hypothesis was that if TNFR2 was knocked out, infected mice would survive IL-2 dosing. When single knockouts of TNFR1 and 2 were used in an experiment however, it was found that either receptor is capable of mediating toxicity, as both experimental groups failed to survive. This is relevant to current IL-2 therapies because there is no way to eliminate a single receptor in order to reduce toxicity. Further studies exploring the anti-viral capabilities of IFN-γ are suggested.
ContributorsJarvis, Jordan Alisa (Author) / Blattman, Joseph (Thesis director) / Denzler, Karen (Committee member) / McAfee, Megan (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Background: Coccidioidomycosis (Valley Fever) is a respiratory disease that is caused by the soil-dwelling fungi Coccidioides immitis and Coccidioides posadasii. Because fungal glycosylation patterns are distinct from mammalian glycosylation patterns, we hypothesized that certain lectins (carbohydrate-binding proteins) might have differential binding properties to coccidioidal glycoproteins, and therefore serve as a tool for the purification and characterization of these glycoproteins from patient specimens. Materials and Methods: To identify potential Coccidioides-binding lectins, lectin-based immunohistochemistry was performed using a panel of 21 lectins on lung tissue from human patients infected with Coccidioides. Enzyme-Linked Immunosorbent Assays (ELISAs) were used to confirm and test candidate Coccidioides-binding lectins for their ability to bind to proteins from antigen preparations of laboratory-grown Coccidioides. Inhibition IHC and ELISAs were used to confirm binding properties of these lectins. SDS-PAGE and mass spectrometry were performed on eluates from coccidioidal antigen preparations run through lectin-affinity chromatography columns to characterize and identify lectin-binding coccidioidal glycoproteins. Results: Two GlcNAc-binding lectins, GSLII and sWGA, bound specifically to spherules and endospores in infected human lung tissue, and not to adjacent lung tissue. The binding of these lectins to both Coccidioides proteins in lung tissue and to coccidioidal antigen preparations was confirmed to have lectin-like characteristics. SDS-PAGE analysis of eluates from lectin-affinity chromatography demonstrated that GSLII and sWGA bind to coccidioidal glycoproteins. Mass spectrometric identification of the top ten lectin affinity-purified glycoproteins demonstrated that GSLII and sWGA share affinity to a common set of coccidioidal glycoproteins. Conclusion: This is the first report of lectins that bind specifically to Coccidioides spherules and endospores in infected humans. These lectins may have the potential to serve as tools for a better method of detection and diagnosis of Valley Fever.
ContributorsChowdhury, Yasmynn (Author) / Lake, Douglas (Thesis director) / Grys, Thomas (Committee member) / Magee, Mitchell (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Human Evolution and Social Change (Contributor)
Created2015-05
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
The purpose of this study was to examine the effects of two positive discrete emotions, awe and nurturant love, on implicit prejudices. After completing an emotion induction task, participants completed Implicit Association Test blocks where they paired photos of Arab and White individuals with "good" and "bad" evaluations. We hypothesized that nurturant love would increase the strength of negative evaluations of Arab individuals and positive evaluations of White individuals, whereas awe would decrease the strength of these negative evaluations when compared to a neutral condition. However, we found that both awe and nurturant love increased negative implicit prejudices toward Arab individuals when compared to the neutral condition.
ContributorsCarrasco, Mia Annette (Author) / Shiota, Michelle (Thesis director) / O'Neil, Makenzie (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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