Browsing by Subject "Immunology"

Now showing 1 - 16 of 16
  • Thumbnail Image
    Item
    Analysis of the pre-immune T cell repertoire.
    (2009-12) Chu, Hon Man Hamlet
    Cell-mediated immune responses are initiated when a population of pre-immune (or naïve) T cells recognize their cognate ligands in the form of a specific peptide bound to a self major histocompatibility complex molecule (pMHC). This recognition is made possible by highly specific T-cell receptors (TCR) on individual T cell clones specific for a given pMHC complex. The pre-immune T cell repertoire is comprised of populations specific for at least 100,000 different pMHC, each containing multiple clones. It is important to understand the composition of this repertoire because it is the repository of all the host's potential for future cell-mediated immune responses to microbes and tumors, and in some cases its own tissues. . However, the study of pre-immune tumor antigen-specific, or any other pathogen-specific T cell populations within such a diverse T cell repertoire have been extremely difficult due to their low frequency in the body. A novel soluble pMHC magnetic enrichment technique was therefore developed to analyze naive T cell populations in mice and humans,. Using this procedure, different pMHCII-specific naive CD4+ T cell populations could be identified and enumerated. The size of these populations was found to vary depending on pMHC specificity. Additionally, these differences were directly proportional to the magnitude and TCR gene usage diversity of the primary CD4+ T cell response after immunization with relevant peptide. Thus, variation in naive T cell frequencies can explain why some peptides give rise to greater T cell responses than others. We explored this issue by enumerating pMHCII-specific CD4+ T cell populations that normally number 20 or 200 cells per mouse. Thymic positive or negative selection was required for optimizing the absolute size pf each population but did not alter the 10-fold ratio between the two populations. Large naïve population size was related to the presence of certain amino acids at T cell receptor contact sites within the peptide. These results suggest that certain MHCII-bound peptides are immunodominant because they contain amino acids with chemical properties that foster binding to many TCRs.
  • Thumbnail Image
    Item
    Cd4 T Cell Activation And Ox40 Agonist Immunotherapy In Tuberculosis
    (2023-09) Gress, Abigail
    After Mycobacterium tuberculosis (Mtb) infection millions of effector T cells traffic to the lungs, but relatively few find antigen and become activated. We used an antigen receptor reporter mouse (Nur77-GFP) infected with Mtb to distinguish recently activated CD4 T cells from others in the lungs. Recently activated Nur77-GFPHI cells more often expressed protective markers and were enriched for expanded TCR clonotypes. Nur77-GFPHI cells differentially expressed co-stimulatory genes including Tnfrsf4/OX40 and were functionally more protective than Nur77-GFPLO. Nur77-GFPLO cells more often expressed markers of terminal exhaustion, cytotoxicity, and the trafficking receptor S1pr5, associated with vascular localization. A short course of immunotherapy with an agonist monoclonal antibody targeting OX40+ cells during early infection transiently expanded CD4 T cell numbers and shifted their phenotype towards parenchymal protective cells, which decreased the lung bacterial burden and extended host survival, offering an additive benefit to antibiotics. We have newly identified OX40 as a conserved marker of recently activated CD4 T cells at the infection site and a target for immunotherapy in tuberculosis.
  • Thumbnail Image
    Item
    Celebrating a Century of Microbiology and Immunology
    (Department of Microbiology and Immunology, 2019) University of Minnesota. Department of Microbiology and Immunology; Hasse, Ashley T.
  • Thumbnail Image
    Item
    Determining the Effect of Type I and Type III Interferons on Thymic Selection
    (2023) Heiland, H. W.; Ashby, K. M.; Hogquist, K. A.
  • Thumbnail Image
    Item
    Development Of Regulatory T Cells Capable Of Maintaining Immune Homeostasis
    (2020-09) Owen, David
    The adaptive immune response, comprised of both T cells and B cells, is essential to control infections and eliminate transformed cancer cells. The success of the adaptive immune system relies on the ability to discriminate self from non-self-antigens. The thymus is the site of selection for T cells, where self-reactive T cells are eliminated, generating a non-self focused T cell compartment. However, this selection process is leaky and potentially pathogenic cells do escape thymic, or central, tolerance. Thus, a population of suppressor cells termed regulatory T cells (Treg cells) co-evolved in order to keep these self-reactive escapees in check. Treg cells that develop in the thymus as part of central tolerance induction are a critical population of T cells that are required to maintain immune homeostasis and prevent autoimmunity. Without intervention, mice or humans that lack the ability to generate Treg cells die shortly after birth from widespread autoimmune-mediated tissue destruction. Further, neonatal thymectomy in mice causes the development of an autoimmune wasting phenotype. These observations highlight the importance of thymic Treg cell selection in immune homeostasis. Thymic Treg cell development occurs via a two-step process. Step one involves developing CD4+ thymocytes receiving strong T cell receptor (TCR) stimulation via engagement of thymic self-antigens, leading to upregulation of CD25, the high affinity subunit of the IL-2 receptor, or FOXP3, the lineage defining transcription factor of Treg cells, generating either CD25+ or FOXP3lo Treg cell progenitors (TregP). Step two is driven by encounters between TregP cell and intrathymic STAT5 activating cytokines, predominantly IL-2, leading to co-expression of CD25 and FOXP3. These CD25+FOXP3+ cells represent fully mature Treg cells that disseminate from the thymus to mediate immune tolerance. While the framework of this two-step development process is understood, many details of each step remain incompletely understood. This thesis addresses several aspects of thymic Treg cell development. First, we identify that T cells are the critical source of IL-2 required to drive Treg differentiation. Second, we provide evidence that CD25+ and FOXP3lo TregP arise via distinct selection programs and contribute functionally distinct TCRs to the mature Treg compartment. Third, using single-cell RNA-sequencing analysis of conventional and Treg lineage thymocytes we provide a more detailed analysis of transcriptional signatures and intermediates of thymic Treg development. Finally, we gathered preliminary data to better understand the heterogeneity and function of recirculating or resident thymic Treg cells. Developing a holistic understanding of Treg development is essential to discern the etiology of immune disorders and properly modulate Treg cells to treat autoimmune disease, infections and cancer.
  • Thumbnail Image
    Item
    The Divergent Role of Co-receptors and Cytokines in the Immune Response to Cancer and Self
    (2020-08) Tucker, Christopher
    The mammalian immune system has evolved over millennia to respond to countless biologic factors and environmental cues. This exquisite system has been shaped over time, to destroy hostile invaders all while sparing healthy surrounding tissue. The immune system is vital to survival, a fact that is readily apparent when immune components are mutated in human disease or mouse models. When immune components are lost, this balance can be distorted in both directions. Alterations to pro-inflammatory molecules, such as Tumor Necrosis Factor alpha (TNFα) can increase infection and cancer risk. Conversely, mutations in immunoregulatory pathways such as programmed death-1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) have been associated with increased autoimmune disease incidence. This balance is uniquely apparent in the PD-1 pathway and CTLA4 pathways. Blockade of the PD-1 pathway can elicit potent anti-tumor responses, as evidenced by monoclonal antibody therapy Nivolumab and Pembrolizumab response rates, but also can unleash off-tissue pathology such as type one diabetes (T1D). T1D in these cancer patients is rapid and severe. This is perhaps not surprising, as Non Obese Diabetic (NOD) mice treated with anti-PD-1 or anti-PD-L1 therapy rapidly succumb to fulminant T1D. In common, CTLA4 blockade has been shown to halt tumor growth and even cure patients of metastatic cancer, however it has also unleashed potent autoimmune responses in the gut, brain, heart and eyes. Being able to better dissect these pathways, determine who will respond poorly to therapy and how these therapies can be maximized for tumor destruction, are key questions to be answered by the field of immunology. Complementary to the idea of blocking an inhibitory signal, positive signals are also used to invigorate the immune system. OX40, a TNF receptor family member, has been shown to be integral in provide co-stimulation to effector and naïve T-cells, and agonism through this receptor can cause potent anti-tumor effects. Interconnected and also important, the cytokine IL-12 has been shown to induce profound changes in T-cells, driving cytolytic T-cell programming, higher cytokine production, better tumor trafficking, proliferation and survival in the tumor environment while preventing tumor induced T-cell exhaustion. Like PD-1 and CTLA4, both OX40 and IL-12 are associated with their own unique set of autoimmune conditions. The focus of this chapter and this thesis as whole will be on these two immunoregulatory pathways, PD-1 and CTLA-4 as well as two immunostimulatory pathways OX40 and IL-12 and their relative importance in autoimmunity and cancer immunity.
  • Thumbnail Image
    Item
    The functional role of the activating receptors Tim-3 and CD16 in human natural killer (NK) cell biology
    (2012-09) Gleason, Michelle Kathleen
    Human natural killer (NK) cells are lymphocytes that develop in the bone marrow from hematopoietic progenitor cells (HPCs) and are also found in the lymph nodes, spleen and peripheral blood (PB), where they comprise 10-15% of the mononuclear cell fraction. PB NK cells are phenotypically defined as expressing the surface receptor CD56 (NCAM, neural cell adhesion molecule) and lacking expression of CD3. They mediate their function through the exocytosis of granules that contain lytic enzymes such as perforin and granzymes, the expression of death receptor ligands, the expression of FcRgammaIIIA (CD16, a mediator of antibody-dependent cell-mediated cytotoxicity or ADCC), and the secretion of cytokines and chemokines. As a result, NK cells take part in both the innate and adaptive immune responses and have critical roles in the control of early viral infection, hematopoietic cell transplantation (HCT) and tumor immunosurveillance. The ability of NK cells to differentiate normal healthy cells (self) from infected or transformed (non-self) cells is regulated by a sophisticated repertoire of cell surface receptors that control their activation, proliferation and effector functions. The net balance of inhibitory and activating signals transmitted by these receptors determines whether an NK cell will eliminate its target. The work presented in this manuscript focuses on the modulation of NK cell effector function by two receptors found in their activating repertoire, namely Tim-3 and CD16, and their potential for enhancing the therapeutic effects of NK cells for the treatment of human hematopoietic malignancies.
  • Thumbnail Image
    Item
    Injury-induced neuroinflammation alters addiction behavior
    (2022-12) Emmitt, Nicole
    Traumatic brain injury (TBI) affects 64-74 million people every year worldwide. A history of mild brain injury increases the risk of substance use disorder (SUD) by 2-6 times that of the general population. With the rise of the opiate epidemic, it is imperative to understand the link between TBI and opiate use disorder. Therefore, a model of mild TBI was developed in mice. Mice that received mild TBI had a transient motor deficit at the acute stage of injury and a long-term spatial learning deficit at the chronic stage of injury. The innate immune response in the brain was active at the acute and chronic stage, with macrophage and neutrophil infiltrate peaking at 3 days post injury (DPI), and an increase in the activated macrophage phenotype at both 15 and 30-DPI. Next, to understand how drug seeking behavior is modified, mice with mild TBI or sham injury were subjected to intravenous (IV) self-administration and conditioned place preference (CPP) behavioral assays to evaluate drug seeking behavior. Injured mice had increased consumption of and preference to morphine. The innate immune response was also altered after IV self-administration, with increased macrophage and neutrophil infiltrate on the side ipsilateral to injury. Furthermore, both IV self-administration and CPP with morphine resulted in increased lymphocyte infiltrate on the side ipsilateral to injury. Considering the increased inflammation observed due to morphine, the neuroinflammatory response to a consistent clinical dose morphine was examined, since the majority of people are first exposed to opiates through prescription for pain relief. After TBI or sham injury, mice received morphine twice daily (5mg/kg) for up to 15 days. A biphasic immune response was observed to this dose of morphine with increased macrophage infiltrate and a lymphocyte infiltrate at 15-DPI. This was accompanied by a dynamic, temporally distinct cytokine response, a decrease in the phagocytic activity of macrophages and microglia at 15-DPI and increased leakage of the blood brain barrier in the mid brain. Results from these studies demonstrate a novel increase in the proinflammatory response at the chronic stage of traumatic brain injury when exposed to morphine. The models developed here will provide new insights to help identify new targets to reduce the risk of SUD following TBI.
  • Thumbnail Image
    Item
    Investigation of pancreatic tumor cell intrinsic differences in capacity to activate tumor-specific T cells
    (2018-12-11) Olson, Margaret A; Stromnes, Ingunn M
    Pancreatic ductal adenocarcinoma (PDA), is the most common form of pancreatic cancer which can be modeled in mice. There are two district morphological types of PDA cells: cuboidal and mesenchymal. In this line of experimentation, it has been found that both cell types do not activate T cells from endogenous antigen sources, however, when a peptide is presented to these cells they can activate T cells. Based on this information future experiments need to be conducted, including determining if peptide processing machinery is present in PDA cells. With this information novel immunotherapy techniques can be developed to destroy PDA cells and eliminate the cancer.
  • Thumbnail Image
    Item
    The major histocompatibility complex of the turkey.
    (2010-05) Chaves, Lee D.
    The ability to identify between self and foreign pathogens is a key function of the vertebrate immune system. To achieve this, vertebrates have developed complex genetic and epigenetic mechanisms to provide sufficient variability to respond to continuously evolving pathogens or transformed cells they face. One of these systems is the large and highly polymorphic major histocompatibility complex (MHC), responsible for the presentation of endogenous and exogenous peptide antigens to T cells. The degree of polymorphism, number of gene copies, and the co-dominant expression of genes of the mammalian MHC allows for a great number possible antigens to be presented. Compared to mammals, the chicken MHC (MHC-B) is greatly condensed, containing the major histocompatibility antigens in a region of just 50 kb with a reduction in gene copy numbers and a lack of co-dominant expression. In addition to the MHC-B, the chicken has a second MHC-like region (MHC-Y) located on the same microchromosome as B yet it is genetically unlinked. The work presented in this dissertation physically and genetically maps the homologous MHC regions in the turkey and identifies genes within these regions. Further work surveyed the polymorphism content within commercial and wild turkeys. A final study assayed the genome-wide diversity of the individual from which the MHC was sequenced to determine the appropriateness of this source DNA for whole genome sequencing. These data provide helpful background information to advance turkey whole genome sequencing and develops genomic resources for the study of the effect of MHC alleles on the outcome of pathogenic infections in the turkey
  • Thumbnail Image
    Item
    Mechanisms of early hemato-endothelial development from human pluripotent stem cells
    (2016-09) Angelos, Mathew George
    Hemogenic endothelium is a highly specialized population of vascular endothelial cells that produces hematopoietic stem cells (HSCs) during embryonic development. This process, referred to as the endothelial-to-hematopoietic transition (EHT), is critical to establish a functional hematopoietic system that persists throughout adulthood. The underlying genetic and cell signaling mechanisms that regulate human EHT remain poorly defined. Human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) provide a well-defined cellular platform that can be used to study these mechanisms. In this work, functional human hemogenic endothelium was identified and isolated from the earliest hemato-endothelial cells differentiated from hESCs. Analysis of hemogenic endothelial cells at a single-cell resolution found hESC-derived hemogenic endothelium was transcriptionally distinct from vascular endothelial cells lacking hematopoietic potential. Novel genetic markers distinguishing human hemogenic endothelium are also presented. Contributions from the aryl hydrocarbon receptor (AHR), an important cell signaling molecule in HSC biology, were also assessed at the level of human EHT. Small molecule inhibition and gene deletion of AHR significantly improved functional hematopoietic stem and progenitor cell development from hESCs. Importantly, a novel role for AHR in the development of hESC-derived innate lymphoid cells is also presented. Collectively, this dissertation identifies and describes key transcriptional and signaling mechanisms that support human EHT. This information will be useful to optimize the development of HSCs and other hematopoietic lineages that are suitable for future clinical application.
  • Thumbnail Image
    Item
    Microbiome and Immune Response to Salmonella enterica serovar Typhimurium and Lawsonia intracellularis Infection in Swine
    (2018-05) Leite, Fernando Lopes Leivas
    Salmonella enterica is a leading cause of foodborne illness world-wide. In the US alone Salmonella is responsible for over 1 million cases of disease a year in humans and causes an estimated loss of more than 3.5 billion dollars annually. Pork is frequently associated with food borne illnesses caused by S. enterica in humans, many of which are attributed to Salmonella enterica serovar Typhimurium. Efforts to reduce the incidence of salmonellosis due to meat consumption have mainly remained ineffective. This study extends previous findings that pigs are more susceptible to colonization by Salmonella enterica when co-infected with the pathogen Lawsonia intracellularis. We determined the composition of the porcine gut microbiome in response to co-infection to determine how potential disturbances caused by L. intracellularis could favor S. Typhimurium. This analysis revealed that L. intracellularis led to a decreased abundance of Clostridium species and Clostridium butyricum in addition to other changes that may favor S. Typhimurium. We also investigated if vaccination against L. intracellularis could have an effect on the shedding S. Typhimurium and found that vaccination significantly reduced S. Typhimurium shedding in animals co-infected with L. intracellularis. To better understand the host response to L. intracellularis, we performed transcriptome analysis of infected mucosal tissue and found that infection induced a signature of genes associated with inflammation and proliferation in the gut. We then tested zinc supplementation, which is known to impact immune function, and found that zinc amino acid supplementation led to a significant reduction of lesions caused by L. intracellularis. Finally, we investigated whether co-infection of enterocytes in vitro caused increases in certain inflammatory cytokines. We found that L. intracellularis up regulated expression of IL-8 and TNFα, two pro-inflammatory cytokines crucial to the pathogenesis of S. Typhimurium infection. This research suggests that increased inflammation mediated by L. intracellularis along with changes in microbiome composition are likely responsible for enhancement of S. Typhimurium infection in swine. We have also identified that L. intracellularis vaccination and zinc amino acid complex supplementation are two promising alternatives to the use of antimicrobials in swine.
  • Thumbnail Image
    Item
    Regulation of Anti-Tumor Immunity and Immunotherapy Response In Colorectal Cancer
    (2020-06) Zhao, Xianda
    Immune checkpoint blockade therapy (ICBT) has revolutionized the treatment and management of numerous cancers, yet a substantial proportion of colorectal cancer (CRC) patients are resistant. Most importantly, the mechanisms that cause ICBT resistance in CRC patients are mostly unclear. Both clinical and laboratory studies implied that both tumor cell-intrinsic factors and traditional cancer therapies (e.g., chemotherapy and oncogenic pathway-targeted therapy) have regulatory effects on anti-tumor immunity and ICBT efficacy. In the present thesis, we first characterized the pathological and immunological features of different pre-clinical CRC models. We reported the feasibility of using small animal endoscopy to establish mouse orthotopic colon tumors. We found that tumors grown orthotopically in the colon microenvironment develop better immune infiltration than tumors with the same genetic background growing in a subcutaneous microenvironment. These data indicated that the tissue microenvironment impacts anti-tumor immunity. Meanwhile, we observed that the endoscopy-guided cancer cell line-originated orthotopic CRC model is much more sensitive to ICBT over the subcutaneous model, making it not suitable for experiments that require ICBT-resistant tumors. This observation made us decide to use the subcutaneous tumor models, which are ICBT-resistant, for understanding cancer immunotherapy resistance. In the second section, we evaluated the impact of tumor-draining lymph nodes (TdLNs) and chemotherapy on ICBT efficacy. Specifically, we demonstrated TdLNs are critical for tumor antigen-specific T-cell response in early-stage tumors. However, TdLNs shift from an immunoreactive to an immunotolerant environment during tumor development. In mice with advanced primary tumors, TdLNs are not the major reservoir of tumor antigen-specific T cells. To evaluate the impacts of those immunotolerant TdLNs on ICBT response, we established a surgical model to mimic tumor recurrence in situ. We surgically removed the established primary tumors with or without concurrent TdLNs resection. Then, we inoculated secondary tumors, which are in the same lymphatic drainage area as the primary tumors, to mimic tumor recurrency. Notably, removing those immunotolerant TdLNs concurrently with established primary tumors did not affect the ICBT response on localized secondary tumors. In another set of experiments, we evaluated the impacts of chemotherapy (5-FU) on ICBT efficacy. We revealed that using 5-FU as induction treatment for ICBT increased tumor visibility to immune cells, decreased immunosuppressive cells in the tumor microenvironment, and limited chemotherapy-induced T-cell depletion. However, sustained chemotherapy impaired the efficacy of ICBT by suppressing the host immune system and depleting tumor-infiltrating T cells. Therefore, the sequential combination of chemotherapy with ICBT may result in a better response than the sustained chemotherapy and ICBT combination. Finally, we investigated how tumor cells regulate T-cell activation via intercellular communication based on extracellular vesicles (EVs). Specifically, we revealed that tumor cells-secreted EVs (TEVs) containing microRNA miR-424 suppressed the CD28-CD80/86 costimulatory pathway in tumor-infiltrating T cells and dendritic cells. Modified TEVs with miR-424 knocked down enhanced T-cell mediated antitumor immune response in CRC tumor models and increased the response to ICBT. Intravenous injection of modified TEVs induced tumor antigen-specific immune responses. Moreover, injections of modified TEVs boosted the ICBT efficacy in CRC models that mimic treatment-refractory late-stage disease. Collectively, the present study improves our understanding of CRC anti-tumor immune regulation and proposed novel treatment for ICBT resistant human CRC.
  • Thumbnail Image
    Item
    Revolutionizing T cell Therapy for Pancreatic Cancer: Harnessing the Power of T cell Receptor Exchange Mice
    (2023-03) Rollins, Meagan
    Pancreatic ductal adenocarcinoma (PDA) is a lethal cancer characterized by a suppressive tumor microenvironment (TME) including elevated levels of TGF. The adoptive transfer of T cell receptor (TCR) engineered T cells specific to mesothelin (Msln) can effectively target PDA, but efficacy is limited by the suppressive TME that promotes engineered T cell dysfunction. T cell receptor (TCR) transgenic mice represent an invaluable tool to study antigen-specific immune responses. In the pre-existing models, a monoclonal TCR is driven by a non-physiologic promoter and randomly integrated into the genome. Here, we create a highly efficient methodology to develop T cell receptor exchange (TRex) mice, in which TCRs, specific to the self/tumor antigen mesothelin (Msln), are integrated into the Trac locus, with concomitant Msln disruption to circumvent T cell tolerance. We show that high-affinity TRex thymocytes undergo all sequential stages of maturation, express the exogenous TCR at DN4, require MHC class I for positive selection and undergo negative selection only when both Msln alleles are present. By comparison of TCRs with the same specificity but varying affinity, we show that Trac targeting improves functional sensitivity of a lower affinity TCR and confers resistance to T cell functional loss. By generating P14 TRex mice with the same specificity as the widely used LCMV-P14 TCR transgenic mouse, we demonstrate increased avidity of Trac-targeted TCRs over transgenic TCRs, while preserving physiologic T cell development. To test the hypothesis that TGFβ is a major driver of engineered T cell dysfunction in PDA, we knocked out Tgfbr2 using CRISPR/Cas9 in in vitro activated Msln-specific TRex cells. The loss of Tgfbr2 signaling in high affinity (1045) Msln-specific TRex T cells drive increases in markers of effector T cells such as Klrg1, Cxcr3, and CD44. When transferred into orthotopic PDA tumor-bearing mice, both Tgbr2-WT and Tgbr2-/- engineered T cells traffic to tumors driven by increased frequency and number of cDC1 and cDC2 dendritic cells. With vaccination, the engineered T cells cause a 10-fold reduction in tumor weight at day 13 post tumor and are highly proliferative. Tumor-infiltrating Tgbr2-/- cells upregulated IFNg, TNFa, and Granzyme b and decreased markers of terminal exhaustion PD-1 and Lag3. Our studies suggest, interfering with TGFβ signaling can alter T cell fate prior to transfer and maintain effector differentiation within the TME promoting cytotoxic Klrg1+ T cells at the expense of PD-1+ exhausted T cells and leading to tumor control.
  • Thumbnail Image
    Item
    Toward the Construction of a Vascularized, Hydrogel-Based Lymph Node Model for In Vitro and In Vivo Therapeutic Applications
    (2021-04) Harff, Caleb
    Clinical trials for drugs and vaccines often suffer from the use of culture or animal models that do not accurately recreate the microenvironment of human tissues, including the lymph nodes. Furthermore, insufficient immune function resulting from genetic deficits, cancer or auto-immune disease, and the loss of lymph nodes due to surgical resection or radiation therapy may benefit from the implantation of therapeutic immune cells. These needs could be served through the development of a biocompatible, vascularized, 3D hydrogel scaffold that supports leukocytes and recreates lymph node function by providing a biomimetic microenvironment. While other lymph node models exist, their complexity and function are limited in that they incorporate few of the bioactive molecules from the lymph node microenvironment and do not contain vasculature. Preliminary viability studies were performed to determine the optimal choice of hydrogel type and density for the 3D culture of peripheral blood mononuclear cells (PBMCs). Fibrin hydrogels were found to better maintain PBMC viability over three days compared to gelatin methacrylate hydrogels. The inclusion of ECM from decellularized porcine lymph nodes into fibrin hydrogels was met with technical challenges with regard to solubilization and peripheral blood cell (PBC) viability assessment, indicating that additional steps or different approaches were required. However, successful decellularization was demonstrated by the sufficient removal of DNA content as determined by DNA quantification and histological staining. Cytokine and growth factor analysis showed significant depletion of many of the analytes but retention of IFNα2, IL-3, IL-9, IL-13, IL-17A, and VEGF. To construct a perfusable model, pin molds were used to create channels that would undergo eventual endothelialization and angiogenesis. Few of these channels maintained structural integrity. While continued experimentation is required to implement all of the features desired for the construction of a biomimetic and functional human lymph node model, these results indicate that it is feasible to improve upon previous designs by incorporating decellularized ECM and introducing vascularization in fibrin hydrogels.
  • Thumbnail Image
    Item
    Untitled
    (2022-05) Sunil Arvindam, Upasana
    Natural Killer (NK) cells are cytotoxic innate immune cells that are important components of anti-tumor immunity. They make up 5-15% of total lymphocytes and are derived from the common lymphoid progenitor. Their phenotype is determined by the marker CD56 that divides them into two groups, CD56bright and CD56dim cells. CD56bright cells have a lower cytotoxic capacity but show high proliferation and cytokine secretion upon stimulation. CD56dim cells display high cytotoxic capacity. NK cells can recognize and killer tumor cells through a wide array of germline encoded receptors. They can facilitate broader immune system activation through the secretion of pro-inflammatory cytokines. NK cell-based treatments can be divided into two main areas; biologics that enhance endogenous NK cell function and adoptive transfer of NK cells. Both these treatments are being developed to increase targeting of therapeutic NK cells towards tumor cells. We have established a series of NK cell biologics called trispecific killer engagers (TriKETM). To target acute myeloid leukemia (AML) cells, we developed a TriKE containing an anti-CD16 portion that activates NK cells, an IL-15 molecule that drives NK cell priming, expansion and survival, and a portion against the AML antigen, CLEC12A (CLEC12A TriKE). The CLEC12A TriKE induced robust NK cell proliferation, enhanced killing of both AML cell lines and primary patient derived AML blasts and reduced tumor burden in pre-clinical mouse models. For TriKE based treatments to be successful in solid tumors, we need to address the additional mechanisms of immune suppression that exist in the solid tumor microenvironment (TME). We focused on the role of low oxygen or hypoxia (1% O2) on NK cell function and found that hypoxia strongly inhibits NK cell proliferation and cytotoxicity. There is a decrease in the expression of activating receptors, cytotoxicity markers and key transcription factors (Tbet and Eomes). There is change in NK cell metabolism with a switch to glycolysis. Hypoxia results in a remodeling of the transcriptome and epigenome. We investigated the impact of exogenous therapeutics on NK cells under hypoxia with the addition of recombinant IL-15 or TriKE and the use of NK cells that have been expanded on K562 feeder cells containing 4-1BBL and IL-21. These therapeutics improved NK cell killing under hypoxia. Future directions include combination therapies with biologics and adoptive transfer of genetically modified NK cells that function better in suppressive TMEs.