Browsing by Subject "T cell"
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Item Open Access Abnormal Adaptive Immunity in Bacterial Bladder Infections(2020) Wu, JianxuanBacterial bladder infection, also known as cystitis, is one type of urinary tract infections (UTIs). These infections typically initiate when uropathogens, especially uropathogenic E. coli (UPEC), invade into the bladder through the urethra. Those bacteria could attach to the uroepithelium of the bladder and invade into bladder epithelial cells (BECs). The bladder is critical for the control of these bacteria by activating BECs and recruiting immune cells. If bacteria are not promptly cleared in the bladder, they will further invade ureters and kidneys causing pyelonephritis, which is another common form of UTIs. Nowadays, bacterial bladder infection is one of the most common bacterial infections and a big clinical burden. Its annual incidence rate among human population was estimated to be around 3%-12.6% in female and 0.5%-3% in male. Besides the high incidence rate, the recurrence rate is also very high, up to 44%. As a comparison, the recurrence rate of bacterial infections in the respiratory tract is only around 10% and that rate in the gastrointestinal tract is about 1.5% to 12%. The high incidence rate and high recurrence rate pinpoint the inefficiency of immunity in bladder. However, it is unclear which component of the bladder immunity is inefficient in clearing bacteria and preventing recurrence. By using cutting edge models and techniques, such as genetic knock-out mice, newly developed cytokine reporter mice, optimized flow cytometry and microscopy, I identified that the bladder immunity, especially CD4 T cell mediated adaptive immunity, is focusing on repairing damaged uroepithelium rather than clearing UPEC. This response is modulated by antigen presenting cells (APCs) in bladder. This abnormal bladder immunity also leads to bladder dysfunction featured by urinary frequency. By applying a vaccination strategy, I successfully improved the anti-bacteria ability of CD4 mediated bladder adaptive immunity in mouse model. In summary, this study identified that an abnormal adaptive immunity induced by the uroepithelium-APC-T cell signaling axis is responsible for the suboptimal clearance of bacteria and infection recurrence in bladder. With proper vaccination, the adaptive immunity in bladder can be tuned to be protective against UPEC infections.
Item Open Access Antibody-Redirected T-Cell Immunotherapy for Brain Tumors(2014) Choi, Bryan DaehahnThe most common primary malignant brain tumor, glioblastoma, is uniformly fatal. Current therapy provides only incremental benefits in survival and is often incapacitating owing to limits defined by nonspecific toxicity. By contrast, immunotherapy offers a particularly promising approach, and has the theoretical potential to target and eliminate malignant cells with unprecedented specificity. The goal of this dissertation is to apply recombinant technologies to develop a new immune-based therapy for patients with malignant glioma. This work will span the design, production, and preclinical testing of a novel bispecific antibody designed to redirect T cells against a tumor-specific mutant of the epidermal growth factor receptor, EGFRvIII.
Chapters 1 and 2 will provide an overview of broad topics in antitumor immunotherapy and immune biology, with special focus on concepts as they relate to tumors of the central nervous system. In addition, the history and current state of bispecific antibodies, particularly those of the bispecific T-cell engager (BiTE) subclass, as well as their potential role in the treatment of malignant disease, will be considered in detail. Data presented in Chapter 3 will describe our approach to generating novel bispecific tandem single-chain antibody reagents, while experiments in Chapter 4 will demonstrate the capacity of one of these molecules, an EGFRvIII-specific BiTE, to achieve antitumor efficacy both in vitro and in vivo using murine models of glioma. Addressing a major barrier to the translation of immune therapies for cancer, chapter 5 will establish a potential role for BiTEs in overcoming cell-mediated immune suppression associated with malignant disease. Lastly, Chapter 6 and 7 will report on emerging areas of study, including the use of syngeneic, transgenic murine systems, and strategies by which BiTEs may be propelled rapidly into early phase clinical trials.
In summary, separating BiTEs from other available immunotherapeutic approaches, our work in this field suggests that BiTEs are (1) highly-specific molecules that greatly reduce the risk of toxicity, (2) have the ability to penetrate the blood-brain barrier and accumulate in intracerebral tumors, and (3) may potentially overcome multiple mechanisms of immunosuppression present in patients with glioblastoma. Together, these studies have the potential to improve the clinical management of patients with glioblastoma through the generation of a novel therapeutic.
Item Open Access CAR T-cell Immunotherapy for Brain Tumors(2017) Suryadevara, CarterGlioblastoma (GBM) is the most common and deadly primary malignant brain tumor. Despite an aggressive multimodal standard of care, prognoses and patient quality of life remain exceptionally poor, due in part to the non-specific and toxic nature of conventional treatment options. By contrast, adoptive cell transfer of T cells genetically modified to express tumor-specific chimeric antigen receptors (CARs) has emerged as a promising approach to targeting brain tumors, given that T cells have migratory capacity within the brain parenchyma, a mechanism to discriminate between normal and neoplastic tissue, and can develop immunological memory. This work spans the development of an effective CAR T-cell immunotherapy strategy targeting the tumor-specific driver mutation, EGFRvIII, which is expressed exclusively by GBM and other cancers but not normal tissue.
Chapters 1 and 2 provide an overview of GBM and the current clinical standard of care, the role of the immune system as it relates to the development and eradication of cancer, and an introduction to various immunotherapy platforms under active preclinical and clinical investigation. Chapter 3 details the historical context of adoptive T-cell immunotherapy and its evolution to present day, detailing our early proof-of-principle studies that led to the inception of the original research described herein. Data presented in Chapter 4 summarizes our translational objectives in implementing CAR T-cell immunotherapy clinically for patients with newly-diagnosed GBM. Chapter 5 addresses a perennial limitation to the immunotherapy of solid tumors by demonstrating an ability of modified CARs to circumvent intratumoral immunosuppression mediated by regulatory T cells. In Chapter 6, we present data that demonstrate, for the first time, a novel role for host lymphodepletion in cellular immunotherapy delivered directly into the brain. Lastly, Chapter 7 contains concluding remarks on the current state of CAR technology and important future directions.
In summary, our work here demonstrates that CAR T cell immunotherapy 1) has curative potential against highly established, orthotopic and syngeneic murine GBM, 2) can be strategically implemented within the current clinical treatment paradigm for GBM, and 3) can overcome a major mechanism of immunosuppression, demonstrating the versatility of gene-modified T cells for the treatment of malignant brain tumors. Together, these studies have paved way for the rationale design of two phase I clinical trials in patients with newly-diagnosed and recurrent EGFRvIII-positive GBM at Duke University.
Item Open Access Dendritic cells in the intestine: sensing of microbiota and inducing of inflammatory bowel disease(2017) Liang, JieDendritic cells (DCs) are potent antigen presenting cells (APC) that sense microbes and induce T cell activation and functional differentiation. The APC function of DCs is upregulated by the signaling pathway downstream of the microbial sensing receptor, a process well studied during pathogen infection and immunization. Multiple lines of evidence suggested that DCs in the intestine lamina propria (LP-DCs) frequently interact with the innocuous microbiota, and through these interactions LP-DCs support intestinal immune homeostasis. However, DC responses to microbiota, if not regulated, can give rise to inflammatory T cells and trigger inflammatory bowel disease (IBD). The DC subsets, DC functions and signaling pathways that induce inflammatory T cells remain incompletely characterized. Here, we demonstrated that mice lacking signaling attenuator A20 (A20cko mice) in DCs develop spontaneous small intestine inflammation that is dependent of microbiota, DCs and T cells. LP-DCs induce inflammatory T cells and that the signals perceived and APC functions are unique for three distinct LP-DC subsets. Thus, while CD103+CD11b- DCs exclusively upregulate their ability to instruct IFNγ+ T cells, CD103+CD11b+ DCs exclusively upregulate their ability to instruct IL-17+ T cells. Of note, APC functions of both DC subsets are upregulated in a MyD88-independent fashion. In contrast, CD103-CD11b+ DCs instruct both IFNγ+ and IL-17+ T cells, and only the IL-17-inducing APC functions require MyD88. In disease pathogenesis, both CD103-CD11b+ and CD103+CD11b+ DCs expand pathologic Th17 cells.
Although MyD88 pathways are potent inducer of intestinal inflammation in the colitis of IL-10 knockout mice and upon transferring of naïve T cells into Rag-deficient hosts, MyD88 pathways are not required for the inflammation of small intestine in A20cko mice. Among the MyD88-independent signaling pathways that could mediate host interaction with microbiota, Dectin-1 pathway is of particular interest because both the receptor Dectin-1 and the downstream signaling molecule CARD9 are IBD-associated genes. Additionally, the defect in either molecule influences the severity of the intestinal inflammation in mouse. We established that the production of inflammatory cytokines downstream of the Dectin-1 pathway is restricted by A20. Mechanistically, A20 inhibits TRAF6 ubiquitination downstream of the Dectin-1 pathway, thereby controlling NFκB and Jnk activation. Although we showed that CD103-CD11b+ and CD103+CD11b+ DCs express Dectin-1 and CARD9, the Dectin-1 pathway is not required for the upregulation of DC function and expansion of inflammatory T cells in the intestine of A20cko mice. Thus, our studies have unveiled a critical role of MyD88-independent pathways in mediating the interaction of the microbiota and LP-DCs. MyD88-independent pathway is capable of driving functional maturation of LP-DCs, pathological expansion of CD4 T cells, and the inflammatory disease in the small intestine.
Item Embargo Developing Strategies to Evaluate Autochthonous Tumor-Specific Immune Responses(2024) Himes, JonathonThe adaptive immune system plays a crucial role in combating tumors through immunosurveillance and responding to immunotherapies. However, many studies investigating the anti-tumor immune response focus primarily on tumor-infiltrating lymphocytes (TILs), which often lack specificity for the antigenic epitopes presented on tumor cells, making them less relevant for effective anti-tumor immunity. To gain insights into novel immunotherapeutic targets and biomarkers of response, it is essential to characterize the phenotypic features and dysfunctional mechanisms of tumor-specific T cell populations. This requires the use of tumor models that express known neoantigens in order to study tumor-specific T cell responses in vivo. While transplant models with known neoantigen expression are widely used, there is a limitation in the availability of autochthonous tumor models where the tumor coevolves with the immune system. In this dissertation, various approaches to studying the tumor-specific immune response in the autochthonous setting are presented and discussed. One such approach that has been developed involves combining CRISPR/Cas9 and sleeping beauty transposase technology to create an autochthonous orthotopic murine sarcoma model. This model incorporates key genetic elements such as oncogenic KrasG12D, functionally impaired p53, and the expression of known MHCI and MHCII sarcoma neoantigens. By utilizing MHC tetramer flow cytometry, a tumor-specific immune response in the peripheral blood was identified as early as 10 days after tumor induction, leading to effective tumor clearance. Interestingly, when CD8 and CD4 T cells were co-depleted, tumors developed at a high penetrance. However, depleting either CD8 or CD4 T cells alone was insufficient to permit tumor growth. These findings indicate that both CD8 and CD4 T cells can independently contribute to immunosurveillance and participate in the clearance of sarcomas expressing MHCI and MHCII neoantigens. Understanding the tumor-specific immune response in autochthonous models is crucial for uncovering new targets for immunotherapy and identifying biomarkers of response. The development of the autochthonous orthotopic murine sarcoma model described in this dissertation provides a valuable tool for investigating the mechanisms and characteristics of tumor-specific T cell responses in an in vivo setting.
Item Open Access Effects of Dysregulated Diacylglycerol-Mediated Signaling on T Cell Function(2013) Krishna, SrutiDiacylglycerol (DAG), a lipid messenger generated upon T cell receptor (TCR) engagement, mediates signaling through the IKK/NF-κB and Ras/ERK pathways. Further downstream of the Ras/ERK pathway are mammalian target of rapamycin (mTOR) and MAP kinase signal integrating kinases Mnk1 and Mnk2. While mTOR acts as a critical regulator of T cell metabolism, homeostasis and function, Mnk1 and Mnk2 phosphorylate the initiation factor eIF4E that plays an important role in cap-dependent mRNA translation. Diacylglycerol kinases (DGKs) terminate DAG-mediated signals by phosphorylating DAG into phosphatidic acid. T cells that lack both α and ζ isoforms of DGK accumulate excess DAG upon activation, resulting in hyper-activation of the IKK/NF-κB, Ras/ERK and mTOR pathways, hypersensitivity to TCR stimulation, and loss of self-tolerance. Here, we have examined the mechanisms by which dysregulated DAG-mediated signaling affects T cell function. To this end, we studied the effects of hyper-activating individual DAG-mediated pathways (IKK/NF-κB and TSC/mTOR) on T cell function. We also examined the role of ERK-activated kinases Mnk1 and Mnk2 in T cell function.
Using mice with T cell-specific expression of a constitutively active form of IKKβ (`IKK' mice), we found that uncontrolled IKKβ/NF-κB signaling promotes T cell apoptosis and attenuates responsiveness to TCR stimulation. Defective IL-2 production and increased FasL expression contributed to enhanced IKK T cell apoptosis. Impaired IKK T cell activation and proliferation were associated with defects in TCR signaling, and upregulation of the cell surface inhibitory receptor PD1. In vivo, IKK T cells mounted a compromised antigen-specific CD8 T cell response with curtailed expansion and exaggerated contraction phases. Notably, expression of transcriptional repressor Blimp1 (a regulator of T cell exhaustion) was increased in IKK T cells, and conditionally deleting Blimp1 was able to largely restore responsiveness to TCR stimulation.
Investigating Mnk1/2 double knockout (DKO) mice, we found that Mnk1 and Mnk2 are dispensable for T cell development and function, but important for the pathogenesis of experimental autoimmune encephalomyelitis (EAE). TCR engagement activated Mnk1/2 in a Ras/ERK-dependent manner in primary T cells, and was inhibited by DGK α and ζ. Mnk1/2 deficiency did not affect the development of conventional αβ T cells, regulatory T cells, or invariant NKT cells. Mature T cells from DKO mice showed normal activation and CD4 TH differentiation ex vivo, but DKO mice developed lower clinical scores than WT counterparts in an EAE model, correlating with a smaller pool of MOG-reactive IL-17-producing and IFNγ-producing CD4 cells. These results suggest that Mnk1/2 may play a minimal role in T cell development and function but may control non-T cell lineages to regulate TH1 and TH17 differentiation in vivo.
To determine the effect of constitutive mTOR complex 1 activity on anti-bacterial CD8 responses, we investigated mice with T cell-specific deletion of TSC1, a suppressor of mTOR complex 1 activity. Using an established model system of transgenic (OT1) CD8 cell adoptive transfer and challenge with Listeria monocytogenes expressing a cognate antigen, we found that TSC1 deficiency impairs antigen-specific CD8 responses. Fewer TSC1-deficient OT1 cells were present in the peripheral blood and spleen at the peak of the response and fewer memory cells were found at later time points, in individual and competitive adoptive transfer experiments with WT counterparts. Weak expansion of TSC1-deficient cells was correlated with defects in survival and proliferation in vivo, while exaggerated contraction was associated with an increased ratio of SLECs to MPECs in the effector cell population. This perturbation in effector-memory differentiation was concomitant with enhanced T-bet expression and decreased Eomes expression among activated TSC1 KO cells. Upon competitive adoptive transfer with WT counterparts and antigen re-challenge, TSC1-deficient memory cells showed moderate defects in expansion but not cytokine production. Taken together, these findings provide direct evidence of a CD8 cell-intrinsic role for TSC1 in regulating antigen-specific primary and memory responses.
In sum, findings from these studies provide deeper insight into the regulation of T cell function by DAG-mediated pathways, and may have implications for the design of immune-modulation strategies during vaccination, autoimmunity and cancer immunotherapy.
Item Open Access Glutaminase Modulates T Cell Metabolism and Function in Inflammation and Cancer(2018) Johnson, Marc ODuring the immune response, helper T cells must proliferate and upregulate key metabolic programs including glucose and glutamine uptake. Metabolic reprogramming is imperative for appropriate T cell responses, as inhibition of glucose or glutamine uptake hinders T cell effector responses. Glutamine and glutaminolysis use in cancer cells has partially been explored. However, the role of glutamine and its downstream metabolites is incomplete and unclear in T cells. The first step of glutamine metabolism is conversion to glutamate via the hydrolase enzyme glutaminase (GLS). To target glutaminolysis, two different methods were employed: 1) genetic knockout of GLS using a CRE-recombinase system specific for CD4/CD8 T cells, and 2) pharmacological inhibition of GLS via the potent and specific small molecular CB839. These two models of glutaminase insufficiency were used as a tool to target glutamine metabolism during T cell activation and differentiation both in vitro and in vivo.
GLS-deficient T cells had decreased activation at early time points compared to control. Over several days, these GLS-deficient T cells differentiated preferentially to Th1-like effector cells. This was reliant on increased glucose carbons incorporating into Tri-Carboxylic Acid (TCA) metabolites. This increased effector response in vitro occurred in both CD4+ T helper cells and CD8+ cells (Cytotoxic lymphocytes, or CTLs). Differentiation of CD4+ T cells to Th1 or Th17 subsets showed decreased Th17 differentiation and cytokine production, while Th1 effector responses were increased. This increased Th1 function was dependent on IL-2 signaling and mTORC1, as reducing IL-2 or inhibiting mTORC1 with rapamycin prevented GLS inhibition-induced Th1 effector function. Th17 cells, meanwhile, were inhibited by changes in reactive oxygen species, and recovery of Th17 function was achieved with n-acetylcysteine treatment.
T cells lacking GLS were unable to induce inflammation in a mouse model of Graft vs Host disease, an inflammatory bowel disease model, or in an airway inflammatory model. Importantly, Chimeric Antigen Receptor (CAR) T cells made from GLS knockout cells were unable to maintain B cell aplasia in recipient mice. Contrary to this, temporary inhibition of GLS via small-molecule inhibition increased B cell killing in vitro and enhanced T cell persistence in both the B cell aplasia and in a vaccinia virus recall response. These results indicate a balance, where permanent deficiency of GLS is detrimental to T cell responses, but acute inhibition can actually promote T effector responses and survival. Overall, this work aims to understand how perturbations in glutamine metabolism in T cells affects differentiation and function and the role of glutaminolysis and improve therapies for inflammatory disease and cancer.
Item Open Access IL-12 CAR T cell Immunotherapy for Heterogeneous Brain Tumors(2023) Shen, Steven HaochengGlioblastoma (GBM) is the most common and deadly primary malignant brain tumor with a median survival of <20 months. Despite aggressive standard of care therapies, GBM remains lethal. Alternatively, immunotherapy in the form of adoptively transferred T cells expressing chimeric antigen receptors (CARs) has emerged as a promising approach to targeting brain tumors. Preclinically, CARs for GBM-specific epidermal growth factor receptor variant III (EGFRvIII; CARvIII) have been successful combined with total body irradiation (TBI) in treating tumors exclusively expressing EGFRvIII. While effective at the bench, this model does not translate clinically; therefore, next generation immunotherapy aims to enhance CARs to secrete immunomodulatory factors to better treat GBM. This work spans the development and success of this new CAR “armored” to secrete IL-12, a stimulatory cytokine that enhances T cell persistence and function, to treat orthotopic heterogeneous GBM.Chapters 1 and 2 provide an overview of GBM. Detailed in these chapters are the current clinical standard of care, immune privilege of the brain, and various immunotherapies under active preclinical and clinical investigation. Chapter 3 details the history of adoptive T cell therapy in the context of brain tumors. Specifically, it focuses on existing CAR T cell therapies for GBM. Chapter 4 summarizes the next generation of “armored” CARs currently being developed. In Chapter 5 we present the development of a new CAR T therapy and demonstrate, for the first time, its efficacy in treating an in vivo, heterogeneous brain tumor. Chapter 6 summarizes the data gathered from our single-cell sequencing of immune cells collected from CAR-treated, tumor-bearing brains and flow cytometry. Additionally, we briefly evaluated the toxicity of our CAR treatment. In Chapter 7, we evaluate numerous in vivo, immunological depletion models to better understand the mechanism of action of our CAR therapy. To conclude, Chapter 8 contains closing remarks on the current state of CAR T cell therapy and future directions. To summarize, we have engineered a fourth-generation CAR T cell that can cure homogeneous and kill heterogeneous brain tumors in immunocompetent mice without host lymphodepletive preconditioning through reprogramming endogenous immune cells in the tumor microenvironment.
Item Open Access Mechanisms of immune-related adverse events during the treatment of cancer with immune checkpoint inhibitors.(Rheumatology (Oxford, England), 2019-12) Weinmann, Sophia C; Pisetsky, David SImmune checkpoint inhibitors are novel biologic agents to treat cancer by inhibiting the regulatory interactions that limit T cell cytotoxicity to tumours. Current agents target either CTLA-4 or the PD-1/PD-L1 axis. Because checkpoints may also regulate autoreactivity, immune checkpoint inhibitor therapy is complicated by side effects known as immune-related adverse events (irAEs). The aim of this article is to review the mechanisms of these events. irAEs can involve different tissues and include arthritis and other rheumatic manifestations. The frequency of irAEs is related to the checkpoint inhibited, with the combination of agents more toxic. Because of their severity, irAEs can limit therapy and require immunosuppressive treatment. The mechanisms leading to irAEs are likely similar to those promoting anti-tumour responses and involve expansion of the T cell repertoire; furthermore, immune checkpoint inhibitors can affect B cell responses and induce autoantibody production. Better understanding of the mechanisms of irAEs will be important to improve patient outcome as well as quality of life during treatment.Item Open Access MicroRNA and Epigenetic Controls of CD4+ T cells' Activation, Differentiation and Maintenance(2014) Li, ChaoranAs a major component of the adaptive immune system, CD4+ T cells play a vital role in host defense and immune tolerance. The potency and accuracy of CD4+ T cell-mediated protection lie in their ability to differentiate into distinct subsets that could carry out unique duties. In this dissertation, we dissected the roles and interplays between two emerging mechanisms, miRNAs and epigenetic processes, in regulating CD4+ T cell-mediated responses. Using both gain- and loss-of-function genetic tools, we demonstrated that a miRNA cluster, miR-17-92, is critical to promote Th1 responses and suppress inducible Treg differentiation. Mechanistically, we found that through targeting Pten, miR-17-92 promotes PI3K activation. Strong TCR-PI3K activation leads to the accumulation of DNMT1, elevated CpG methylation in the foxp3 promoter, and suppression of foxp3 transcription. Furthermore, we demonstrated that an epigenetic regulator, methyl CpG binding protein 2 (MeCP2), is critical to sustain Foxp3 expression in Tregs, and to support Th1 and Th17 differentiation in conventional CD4+ T cells (Tcons). In Tregs, MeCP2 directly binds to the CNS2 region of foxp3 locus to promote its local histone H3 acetylation; while in Tcons, MeCP2 enhances the locus accessibility and transcription of miR-124, which negatively controls SOCS5 translation to support STAT1, STAT3 activation and Th1, Th17 differentiation. Overall, miRNAs and epigenetic processes may crosstalk to control CD4+ T cell differentiation and function.
Item Open Access microRNA Regulation of Cellular Immunity(2016) Lykken, Erik AllenImmunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.
T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.
In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.
In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.
The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.
Item Embargo Orthogonal screens to decode human T cell state and function(2024) McCutcheon, Sean RIn the last decade, the paradigm for cancer therapy has incrementally transitioned away from non-specific cytotoxic therapies (radiation, chemotherapy) and targeted therapies (small molecules, biologics) and towards immune cell-based therapies. Immune cell-based therapies such as adoptive T cell therapy (ACT) harness the intrinsic ‘sense and respond’ functions of immune cells to selectively target and eliminate cancer cells. Nevertheless, more than half of cancer patients either do not respond or relapse to existing ACTs. Several studies have defined specific transcriptional and epigenetic signatures of the infused T cell product associated with clinical response, indicating that T cell state and fitness is linked to ACT efficacy. Thus, epigenetically reprogramming T cells with enhanced potency and durability has the potential to improve ACT. However, this potential has yet to be fully realized due to technical challenges of adapting CRISPR-based epigenome editing technologies for applications in primary human T cells. To overcome these challenges, we developed and rigorously characterized compact and robust CRISPR repressors and activators for endogenous gene regulation. Next, we leveraged these technologies to systematically interrogate the effects of >100 transcriptional and epigenetic regulators on human CD8+ T cell state and function through complementary CRISPR interference (CRISPRi) and activation (CRISPRa) screens. These CRISPRi/a screens converged on basic leucine zipper ATF-like transcription factor (BATF3). Subsequent assays revealed that BATF3 overexpression promotes specific features of memory T cells (such as increased expression of IL7R and glycolysis), counters T cell exhaustion, and enhances CAR T cell potency in both in vitro and in vivo tumor models. In addition, BATF3 programs a transcriptional profile strongly associated with positive clinical response to CD19 CAR T cell therapy. Given that BATF3 is a compact transcription factor (TF) without any transactivation or epigenetic domains, we speculated that BATF3 achieves its widespread effects by interacting with other TFs. To identify these factors, we conducted parallel CRISPR knockout screens targeting all TFs with or without BATF3 overexpression. Using IL7R expression as a proxy for BATF3 activity, we identified both BATF3-independent and dependent transcriptional regulators of IL7R expression. For example, JUNB and IRF4 were uniquely enriched in the low IL7R population in the screen with BATF3 overexpression, suggesting BATF3 heterodimerizes with JUNB and interacts with IRF4 to regulate gene expression. Finally, these CRISPR knockout screens illuminated other candidate therapeutic targets for future exploration and characterization. Overall, we have developed a widely applicable synthetic biology toolkit of orthogonal epigenome editors, which we used to systematically identify regulators of human CD8+ T cell state and function. This catalogue of regulators could serve as the basis for engineering next generation T cell therapies for cancer.
Item Open Access Roles of CTCF and YY1 in T Cell Receptor Gene Rearrangement And T Cell Development(2016) Chen, LiangDiversity of T cell receptors (TCR) and immunoglobulins (Ig) is generated by V(D)J recombination of antigen receptor (AgR) loci. The Tcra-Tcrd locus is of particular interest because it displays a nested organization of Tcrd and Tcra gene segments and V(D)J recombination follows an intricate developmental program to assemble both TCRδ and TCRα repertoires. However, the mechanisms that dictate the developmental regulation of V(D)J recombination of the Tcra-Tcrd locus remain unclear.
We have previously shown that CCCTC-binding factor (CTCF) regulates Tcra gene transcription and rearrangement through organizing chromatin looping between CTCF- binding elements (CBEs). This study is one of many showing that CTCF functions as a chromatin organizer and transcriptional regulator genome-wide. However, detailed understanding of the impact of specific CBEs is needed to fully comprehend the biological function of CTCF and how CTCF influences the generation of the TCR repertoire during thymocyte development. Thus, we generated several mouse models with genetically modified CBEs to gain insight into the CTCF-dependent regulation of the Tcra-Tcrd locus. We revealed a CTCF-dependent chromatin interaction network at the Tcra-Tcrd locus in double-negative thymocytes. Disruption of a discrete chromatin loop encompassing Dδ, Jδ and Cδ gene segments allowed a single Vδ segment to frequently contact and rearrange to diversity and joining gene segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrowed the TCRα repertoire, which, we believe, followed as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. In addition, we showed that insertion of an ectopic CBE can modify chromatin interactions and disrupt the rearrangement of particular Vδ gene segments. Finally, we investigated the role of YY1 in early T cell development by conditionally deleting YY1 in developing thymocytes. We found that early ablation of YY1 caused severe developmental defects in the DN compartment due to a dramatic increase in DN thymocyte apoptosis. Furthermore, late ablation of YY1 resulted in increased apoptosis of DP thymocytes and a restricted TCRα repertoire. Mechanistically, we showed that p53 was upregulated in both DN and DP YY1-deficient thymocytes. Eliminating p53 in YY1-deficient thymocytes rescued the survival and developmental defects, indicating that these YY1-dependent defects were p53-mediated. We conclude that YY1 is required to maintain cell viability during thymocyte development by thwarting the accumulation of p53.
Overall, this thesis work has shown that CTCF-dependent looping provides a central framework for lineage- and developmental stage-specific regulation of Tcra-Tcrd gene expression and rearrangements. In addition, we identified YY1 as a novel regulator of thymocyte viability.
Item Open Access The Elucidation of the Mechanisms of CD8+ T Cell-Mediated Suppression of Human Immunodeficiency Virus Type 1 Replication(2010) Saunders, Kevin O'NeilHerein we detail the progress made at understanding the overall process of CD8+ T-lymphocyte noncytolytic antiviral response (CNAR). This response is comprised of 3 key components, the virus, the effector cell and the target cell, each of which contribute to noncytolytic suppression. During the course of CNAR, the effector cells express antiviral factors that induce intracellular events in the target cell resulting in host-pathogen interactions that inhibit HIV-1 gene expression. The goal of this work was to clarify each step of the process of noncytolytic suppression.
The effector cell was examined to understand the regulation of antiviral factors and to construct a profile of the factors expressed during CNAR. CD8+ T-lymphocytes from HIV-1 infected individuals express unidentified factors that suppress viral replication by inhibiting HIV-1 gene expression. Understanding the regulation of these antiviral CD8+ T cell-derived factors can provide important insights into how to elicit these factors with therapeutic regimens. For a small subset of human genes, histone deacetylases (HDACs) are epigenetic regulators that condense chromatin to repress transcription. We examined the role of epigenetics in modulating the HIV-1 suppressive factors expressed by primary CD8+ T cells from subjects naturally controlling virus replication. HIV-1 suppression by CD8+ T-lymphocytes from virus controllers was reversed up to 40% by the addition of an HDAC inhibitor. Therefore, histone deacetylation within CD8+ T-lymphocytes was necessary for potent suppression of HIV-1 infection.
Blocking HDACs impairs the ability of CD8+ T-lymphocytes to repress HIV-1 transcription, demonstrating the expression of the suppressive factors is regulated by epigenetics. We used this tool to identify the potential antiviral factors that result in decreased noncytolytic suppression. Through real-time PCR analysis of 164 genes we identified 4 genes in primary CD8+ T-lymphocytes from a virus controller, and 12 genes in a CD8+ T-cell line that were greatly downregulated in response to a HDAC inhibitor. Additionally, we analyzed the chemokine and cytokine profile of these two cell types to characterize what molecules these cells secrete during CNAR. MIP-1 Beta, MIP-1 Alpha, IP-10, and MIG correlated most strongly with the magnitude of CNAR (p < 0.0001).
The response of the target cell to the antiviral factors was analyzed to better understand how CD8+ T cell antiviral factors exert suppressive activity on the HIV-1 genome in an infected cell. Noncytolytic suppression was not dependent on epigenetic changes within the target cells, as HDAC1 within the target cell was dispensable, and histone acetylation at the HIV-1 LTR remained unchanged in the presence of CD8+ T-lymphocytes.
The genetic elements within HIV-1 and the viral protein Tat were investigated to provide insight into resistance to CNAR. Two virus isolates from the same individual with contrasting sensitivities to CNAR were investigated to identify the genetic elements that confer these phenotypes. Sequence analysis of the two isolates identified mutations in the exon splicing silencers (ESS) 2 and 3 in these viruses. ESS2 and 3 are thought to control splicing of HIV-1 Tat, however levels of spliced Tat RNA levels did not differ between the two isolates. The introduction of the ESS2 mutation into a heterologous HIV-1 isolate moderately boosted resistance to CNAR, suggesting a function for the mutation apart from spliced Tat RNA levels.
In total, a comprehensive analysis of each component of CNAR is discussed here to enhance the overall understanding of the mechanisms of CNAR.
Item Open Access The Role of Glucose Metabolism in T Cell Stimulation and Homeostasis(2009) Jacobs, Sarah RuthThe role of two cell extrinsic signals, T cell receptor (TCR) ligation and interleukin-7, in promoting glucose uptake and survival of T lymphocytes is examined in this work. Both of these signals are capable of regulating the uptake and fate of glucose, but the requirement of this regulation for T cell homeostasis and functionality remains unclear. To examine the role of TCR mediated increases of glucose metabolism and the signals involved, primary murine T cells were activated in vitro and the role and regulation of glucose uptake was examined. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required for maximal glucose uptake following TCR stimulation by upregulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active Akt (mAkt) increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent upregulation of Glut1 protein and Akt-dependent Glut1 cell surface trafficking. In support of this model, co-expression of Glut1 and mAkt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism necessary for T cell activation and proliferation.
Glucose uptake is dramatically increased in response to TCR and costimulation signaling, however, glucose uptake must be maintained at a low level in naive T cells to promote survival and homeostasis. Interleukin-7 (IL-7) plays a central role in maintaining naive T cell homeostasis, and mediates this effect in vivo at least in part through control of homeostatic proliferation and inhibition of apoptosis. IL-7 can promote glucose uptake and glycolysis in vitro and may also promote glucose metabolism in vivo to maintain T cell survival. To determine if IL-7 regulates T cell metabolism in vivo, we generated a transgenic model for conditional IL-7 receptor (IL 7R) expression on IL-7R-/- T cells. T cells in this model developed normally and, consistent with previous work, deletion of the IL-7R transgene in vivo led to cell death even in an otherwise normal lymphoid compartment. Importantly, in vivo deletion of IL 7R also led to decreased cell size and glycolytic flux. However, glucose uptake was not altered following deletion of the IL-7R indicating that while not essential for glucose uptake, IL-7 is required for maintenance of glycolysis. These data are the first to identify a signal required in vivo to regulate lymphocyte metabolism and demonstrate that in addition to its well-defined roles in homeostatic proliferation and cell survival, IL-7 plays a key and non-redundant role to maintain T cell glycolysis. Together, these data concerning the role of TCR, costimulation, and IL-7 in the regulation of glucose uptake and metabolism exemplify the importance of cell extrinsic signals and the regulation of glucose utilization.
Item Open Access The Role of Tcrb Subnuclear Positioning in V(D)J Recombination(2014) Chan, Elizabeth Ann WilcoxT cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.
The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.
Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, Tcrb alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However Tcrb alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of Tcrb alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.
In search of the mechanism by which recombination is suppressed on peripheral Tcrb alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral Tcrb alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.