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- All Subjects: Immunology
- All Subjects: Virology
- Creators: Lake, Douglas
- Creators: Anderson, Karen S
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
CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
onself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy.
ContributorsKrishna, Sri (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Jacobs, Bertram L (Committee member) / Lake, Douglas F (Committee member) / Arizona State University (Publisher)
Created2017
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
Biomarkers find a wide variety of applications in oncology from risk assessment to diagnosis and predicting and monitoring recurrence and response to therapy. Developing clinically useful biomarkers for cancer is faced with several challenges, including cancer heterogeneity and factors related to assay development and biomarker performance. Circulating biomarkers offer a rapid, cost-effective, and minimally-invasive window to disease and are ideal for population-based screening. Circulating immune biomarkers are stable, measurable, and can betray the underlying antigen when present below detection levels or even no longer present. This dissertation aims to investigate potential circulating immune biomarkers with applications in cancer detection and novel therapies. Over 600,000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers. A key challenge in understanding HPV immunobiology and developing immune biomarkers is the diversity of HPV types and the need for multiplexed display of HPV antigens. In Project 1, nucleic acid programmable protein arrays displaying the proteomes of 12 HPV types were developed and used for serum immunoprofiling of women with cervical lesions or invasive cervical cancer. These arrays provide a valuable high-throughput tool for measuring the breadth, specificity, heterogeneity, and cross-reactivity of the serologic response to HPV. Project 2 investigates potential biomarkers of immunity to the bacterial CRISPR/Cas9 system that is currently in clinical trials for cancer. Pre-existing B cell and T cell immune responses to Cas9 were detected in humans and Cas9 was modified to eliminate immunodominant epitopes while preserving its function and specificity. This dissertation broadens our understanding of the immunobiology of cervical cancer and provides insights into the immune profiles that could serve as biomarkers of various applications in cancer.
ContributorsEwaisha, Radwa Mohamed Emadeldin Mahmoud (Author) / Anderson, Karen S (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas F (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2018
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
Vaccines against the arthropod-borne dengue virus (DENV) are still commercially nonexistent. A subunit immunization strategy may be of value, especially if a safe viral vector acts as a biologically active adjuvant. The DENV envelope protein (E), the main target for neutralizing immune responses, has three conformational domains. The immunoglobulin-like and independently folding domain III (DIII) contains epitopes that elicit highly specific neutralizing antibodies. The hepatitis B small surface antigen (HBsAg, S) was used as a scaffold to display DENV 2 DIII on a virus-like particle (VLP). A measles virus (MV) was engineered to vector HBsAg and the hybrid glycoprotein DIII-HBsAg in two different loci (DIII-S). Despite the relatively deleterious effect on replication caused by the insertion of two transcription cassettes, the recombinant virus MVvac2(DIII-S,S)P induced the secretion of DIII-S hybrid VLP with a similar sucrose density as HBsAg particles (1.10-1.12g/ml) and peaked at 48 h post-infection producing 1.3x106 TCID50/ml infectious MV units in vitro. A second recombinant virus, MVvac2(DIII-S)N, was engineered to vector only the hybrid DIII-S. However, it did not induce the secretion of hybrid HBsAg particles in the supernatant of infected cells. The immunogenicity of the recombinant viruses was tested in a MV-susceptible small animal model, the experimental group which received two 105 TCID50 I.P. doses of MVvac2(DIII-S,S)P in a 28 day interval developed a robust immune response against MV (1:1280), HBsAg (787 mIU/ml) and DENV2 (Log10 neutralization index of 1.2) on average. In summary, it is possible to display DENV E DIII on hybrid HBsAg particles vectored by MV that elicit an immune response. This forms the basis for a potential vaccine platform against DENV.
ContributorsHarahap, Indira (Author) / Reyes del Valle, Jorge (Thesis advisor) / Hogue, Brenda G (Thesis advisor) / Lake, Douglas (Committee member) / Mason, Hugh (Committee member) / Arizona State University (Publisher)
Created2015
Description
Despite the approval of a Dengue virus (DV) vaccine in five endemic countries, dengue prevention would benefit from an immunization strategy highly immunogenic in young infants and not curtailed by viral interference. Problematically, infants younger than 9 year of age, whom are particularly prone to Dengue severe infection and death, cannot be immunized using current approved DV vaccine. The most important issues documented so far are the lack of efficiency and enhancement of the disease in young seronegative recipients, as well as uneven protection against the four DV serotypes. Based on data from clinical trials that showed enhanced performance of dengue vaccines when the host has previous anti-flaviviral immunity, I proposed here an attractive solution to complement the current vaccine: a recombinant measles vaccine vectoring dengue protective antigens to be administered to young infants. I hypothesized that recombinant measles virus expressing Dengue 2 and 4 antigens would successfully induce neutralizing responses against DV2 and 4 and the vaccine cocktail of this recombinant measles can prime anti-flaviviral neutralizing immunity. For this dissertation, I generated and performed preclinical immune assessment for four novel Measles-Dengue (MV-DV) vaccine candidates. I generated four MVs expressing the pre membrane (prM) and full length or truncated (90%) forms of the major envelope (E) from DV2 and DV4. Two virus, MVvac2-DV2(prME)N and MVvac2-DV4(prME), expressed high levels of membrane associated full-length E, while the other two viruses, MVvac2-DV2(prMEsol)N and MVvac2-DV4(prMEsol)N, expressed and secreted truncated, soluble E protein to its extracellular environment. The last two vectored vaccines proved superior anti-dengue neutralizing responses comparing to its corresponding full length vectors. Remarkably, when MVvac2-DV2/4(prMEsol)N recombinant vaccines were combined, the vaccine cocktail was able to prime cross-neutralizing responses against DV 1 and the relatively distant 17D yellow fever virus attenuated strain. Thus, I identify a promising DV vaccination strategy, MVvac2-DV2/4(prMEsol)N, which can prime broad neutralizing immune responses by using only two of the four available DV serotypes. The current MV immunization scheme can be advantageus to prime broad anti-flaviviral neutralizing immunity status, which will be majorly boosted by subsequent chimeric Dengue vaccine approaches.
ContributorsAbdelgalel, Rowida (Author) / Reyes del Valle, Jorge (Thesis advisor) / Mason, Hugh (Thesis advisor) / Lake, Douglas (Committee member) / Stout, Valerie (Committee member) / Frasch, Wayne (Committee member) / Arizona State University (Publisher)
Created2016
Description
Host organisms have evolved multiple mechanisms to defend against a viral infection and likewise viruses have evolved multiple methods to subvert the host's anti-viral immune response. Vaccinia virus (VACV) is known to contain numerous proteins involved in blocking the cellular anti-viral immune response. The VACV E3L protein is important for inhibiting the anti-viral immune response and deletions within this gene lead to a severe attenuation. In particular, VACV containing N-terminal truncations in E3L are attenuated in animal models and fail to replicate in murine JC cells. Monkeypox virus (MPXV) F3L protein is a homologue of the VACV E3L protein, however it is predicted to contain a 37 amino acid N-terminal truncation. Despite containing an N-terminal truncation in the E3L homologue, MPXV is able to inhibit the anti-viral immune response similar to wild-type VACV and able to replicate in JC cells. This suggests that MPXV has evolved another mechanism(s) to counteract host defenses and promote replication in JC cells. MPXV produces less dsRNA than VACV during the course of an infection, which may explain why MPXV posses a phenotype similar to VACV, despite containing a truncated E3L homologue. The development of oncolytic viruses as a therapy for cancer has gained interest in recent years. Oncolytic viruses selectively replicate in and destroy cancerous cells and leave normal cells unharmed. Many tumors possess dysregulated anti-viral signaling pathways, since these pathways can also regulate cell growth. Creating a mutation in the N-terminus of the VACV-E3L protein generates an oncolytic VACV that depends on dysregulated anti-viral signaling pathways for replication allowing for direct targeting of the cancerous cells. VACV-E3Ldel54N selectively replicates in numerous cancer cells lines and not in the normal cell lines. Additionally, VACV-E3Ldel54N is safe and effective in causing tumor regression in a xenograph mouse model. Lastly, VACV-E3Ldel54N was capable of spreading from the treated tumors to the untreated tumors in both a xenograph and syngeneic mouse model. These data suggest that VACV-E3Ldel54N could be an effective oncolytic virus for the treatment of cancer.
ContributorsArndt, William D (Author) / Jacobs, Bertram (Thesis advisor) / Curtiss Iii, Roy (Committee member) / Chang, Yung (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2010
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