Browsing by Author "Henao Tamayo, Marcela, committee member"

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Development of a canine model to evaluate CAR-T cell subsets
(Colorado State University. Libraries, 2024) Brill, Samuel Austin, author; Thamm, Douglas H., advisor; Avery, Anne, committee member; Dow, Steven, committee member; Henao Tamayo, Marcela, committee member; Fry, Terry, committee member
Immunotherapy is a rapidly expanding therapeutic modality in the oncology clinic. Immunotherapy with chimeric antigen receptor (CAR) T cells has demonstrated success in human blood cancers, although relapse occurs in ~50% of patients. In the context of solid tumors, CAR-T cells show only transient effects. As a "living therapy", development of the next generation CAR-T cells requires a deeper understanding of how CAR biology, T cell biology, and tumor biology interact over the course of disease. CAR constructs are made up of an extracellular antigen binding domain fused with one or more intracellular signaling domains. The extracellular domain is frequently built from the fused heavy and light chains of an antibody, called a single chain fragmented variable (scFv) domain, and confers the CAR its tumor specificity. Intracellular signaling domains typically contain the zeta chain of CD3 along with one or more costimulatory domains, often CD28 and/or 4-1BB. Mechanisms of relapse and treatment failure for CAR-T cell therapy are related to loss of CAR-T targeting (e.g. target antigen loss) and dysfunction of CAR-T cells (e.g. immunosuppressive microenvironment). These mechanisms of resistance to CAR-T cell therapy have largely been resolved using in vitro and mouse models, where methods to overcome resistance have also been developed. While our mechanistic understanding of tumor and immune biology has been propelled to a place where beneficial therapies are being employed, there is a need for more clinically relevant models to help streamline clinical trials of promising next-generation therapies. Dogs provide an immune-intact animal model that can help address some fundamental questions of CAR-T cell biology in a clinically relevant animal model. Cancers in dogs share many pathobiological features to their human counterparts and receive similar treatments including chemotherapy, radiation, and surgery. Dogs' intact immune system, large size, and relatively outbred genetics provide an ideal environment to evaluate novel cellular therapies such as CAR T cell immunotherapies. This work sought to develop a model of CAR-T cell therapy in dogs to better support pre-clinical evaluation of novel CAR constructs and to better understand how CAR and T cell biology interact in the context of cancer. The first aim of this dissertation was to develop a canine CAR-T model for functional assessments of canine CAR-T cells. The second aim of this dissertation was to explore cell subsets using single cell sequencing. We first sought to establish a canine CAR-T cell in vitro model system. We hypothesized that the disialoganglioside GD2 would be a viable CAR tumor target in canine osteosarcoma (OS). The primary CAR used in these studies was a GD2 directed CAR which is currently undergoing human clinical trials. Using the same antibody clone from which the GD2 CAR is derived, we demonstrated that GD2 is expressed on a subset of canine OS and melanoma cell lines. We demonstrated that canine primary T cells are refractory towards lentiviral (HIV-1) based transduction, although they are susceptible to gammaretroviral (MSCV) transduction. Canine GD2+ cell lines were susceptible to GD2 CAR-T cells as assessed by IFN-g and IL-2 cytokine release, Incucyte live image microscopy, and luciferase killing assays. GD2 CARs with either CD28 or 4-1BB costimulatory domains were effective in killing GD2+ OS. The second aim of this proposal sought to elucidate and define canine immune cell subsets using single cell RNA sequencing (scRNASeq). We hypothesized that a phenotypically distinct population of CAR-T would preferentially expand and persist throughout CAR-T cell manufacturing and tumor engagement. To the aim of identifying specific immune subsets, we first sought to create an atlas of cells present in the canine lymph node (LN). In addition to peripheral blood and primary tumor lymphocytes, LNs are an additional key site for modulating cancer immunity. While the immune subsets in the peripheral blood and tumor have been described in dogs with OS, an atlas of canine LNs has not been made. We generated an immune atlas of cells with the LNs from healthy dogs and the regional LNs of OS bearing dogs. In addition to annotations of cell clusters present in l LNs, comparisons between dog populations revealed fewer B cells recovered in the OS group along with higher HLA-DQB2 gene expression across a variety of cell types. Taken together, these aims provide a basis for investigation of CAR-T cell therapy at single cell resolution. In the first aim, we developed canine GD2 directed CAR-T cells which could effectively kill GD2+ tumor in vitro. The second aim developed a workflow using scSeq to identify canine immune cell populations and provides a LN cell atlas to supplement those for the peripheral blood and tumor tissue. This platform will be used for translational oncology studies to assess novel CAR constructs, investigate CAR-T cell biology, and allow for optimization of next generation CAR-T cells.
Open Access
The role of vitamin A and mycobacterial lipids in the immunopathogenesis of Mycobacterium tuberculosis infections
(Colorado State University. Libraries, 2023) Harris, Macallister C., author; Podell, Brendan, advisor; Zabel, Mark, committee member; Henao Tamayo, Marcela, committee member; Ryan, Elizabeth, committee member; Basaraba, Randall, committee member
Until recently Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), was the deadliest and most ubiquitous infectious disease in the world, infecting an estimated 1.7 billion people as of 2018 and killing 1.6 million people in 2021. However, recently COVID-19 has displaced this disease as the most fatal disease, and in the process of doing so has disrupted integral structural apparatuses meant to monitor and treat TB in endemic countries. This degradation has led to an unprecedented rise in disease transmission with a 4.3% rise in infection, with 10.6 million new infections in 2021. Further compounding the problem of this wave of Mtb infections is the rise of multidrug resistant infections, accounting for 3.9% of all new infections and responsible for 14% of Mtb fatalities in 2017. With an upsurge in case incidence, faltering antibiotic regimens and a variably effective 100-year-old vaccine, there is a new push in understanding disease comorbidities and alternative treatments for TB. Although over 1 billion people are presumed to be infected with Mtb, though not all of these patients have clinical TB. In fact, the WHO estimates that 1/3 of infections results in latent disease, a non-clinical phase of the infection which can last in definitively or eventually progress into active, clinical apparent TB. Patient comorbidities have become an intense area of study for further defining the delineating factors that direct infected individuals into either active infection, latent infection, or overcoming the infection. Two defining comorbidities, HIV status and diabetes have been correlated to an increased risk for having progressive TB. HIV infections in itself has been demonstrated to cause a 20 to 30 fold increase risk in clinical manifestations of a Mtb infection, leading to 187,000 deaths in 2021. An equally prolific area of Mtb research lies in developing new, efficacious prophylactic and active infection treatment regimens. As previously mentioned, current first line drugs regimens, such as rifampicin and isoniazid, are faltering against multidrug resistant Mtb. Additionally, the only widely available vaccine, the Bacillus Calmette-Guerin vaccine, has high efficacious against several forms of TB, like adolescent TB meningitis, but has variable to low efficacy in control of classic pulmonary TB. These factors are driving research of novel treatments and vaccines that utilize niche characteristics of Mtb, host immunity and overall TB disease pathogenesis to prevent clinical disease manifestation. This thesis centers at the intersection of these two areas of this immunologic research, Mtb comorbidities and investigating a novel branch of immunology, by investigating the immunopathogenesis of TB in relation to a newly recognized comorbidity, vitamin A deficiency, and examining the utility of harnessing a niche lipid-based branch of immunology, CD1 lipid restricted immunology, to combat TB disease progression. In chapter one, this thesis, stemming from the foundational epidemiological work of Dr. Megan Murray, endeavored to investigate the pathophysiology of vitamin A deficiency and TB in a highly translatable guinea pig model. This novel vitamin A animal model demonstrated that vitamin A deficiency leads to a dysregulated immune system with atypically granulomatous lesions, skewed inflammatory population and contradicting inflammatory gene profiles. These changes lead to progressive clinical disease with increased pulmonary bacterial burden and splenic lesion burdens. Additionally, this chapter was able to demonstrate the ability to partially alter the detrimental effects of vitamin A deficiency during the course of an active Mtb infection via reintroduction of a vitamin A sufficient diet. In chapter two, this thesis aimed to establish the kinetic expression of CD1 glycoproteins, the lipid antigen presenting molecule homologous to MHC I and the linchpin to lipid-restricted immunology, within tissues of infected guinea pigs. This work demonstrated CD1b upregulation during the early adaptive immunology phase of infection by profiling of CD1 glycoproteins via flow cytometric analysis, immunohistochemical tissue evaluation, and CD1 specific qPCR profiling. This study went further by not only establishing kinetic expression of this molecule but correlating that expression back to a functional cytolytic response driven by CD1-restricted T cells. Lastly, chapter three of this thesis aimed to build on the CD1 work in chapter two by assessing the role CD1 lipid restricted immunity plays in the disease pathogenesis of Mtb infections. To perform this work CD1 glycoprotein invivo modulation techniques were perfected via the use of CD1 ortholog specific synthetic antisense RNA, morpholinos, to down regulate CD1 protein translation. To stimulate CD1 expression upregulation, a pleotropic growth factor, FLT3-L, previously shown to upregulate CD1, was utilized. This chapter demonstrated that these compounds were able to alter CD1 expression in vivo when evaluated via flow cytometric analysis, immunohistochemically, and via altered qPCR profiles. Additionally, this chapter showed the ability to hamper the functional cytolytic activity of CD1-restricted T cells via CD1 down regulation. Although CD1 glycoprotein expression was altered, disease progression of Mtb in the guinea pig, as measured by lesion and bacterial burden, remained unaltered. Collective these aims serve as the beginning investigative studies into two unique and potential promising arenas of translational Mtb research. The discovery that vitamin A resupplementation mitigates vitamin A deficiency within chapter 1 has launched a host of different investigations examining the ability of this essential micronutrient to not only prevent clinical TB but to also serve as an immunologic boost to enhance the efficacy of a variety of Mtb treatment regimens. Chapters 2 and 3 reexamined CD1 immunity and the utility of the guinea pig for lipid immunologic work. Though much work has yet to be done in this niche branch of immunology, these chapters lay the ground work and provide the new tools for future studies into potential efficacious and translatable lipid-based vaccination schemes. Tuberculosis, caused by Mycobacterium tuberculosis, is a disease with global ramifications that requires innovative research to meet the new challenges on the horizon. This dissertation looked to meet those challenges by contributing new knowledge to two developing fields of immunologic research while also laying out the tools for future groundbreaking work.