Identifying the immunotherapy driven epigenetic landscape that underlies transcriptional changes in tumor infiltrating CD8 T cells”
University of Pittsburgh Melanoma SPORE Grant
Checkpoint blockade immunotherapy for melanoma has been revolutionary for patients. However, not all patients respond, prompting investigations into the mechanism of immune checkpoint blockade and pro-tumor resistance. Believed to be a primary target of checkpoint blockade, CD8 T cells exhibit profound dysfunction (termed exhaustion) in the tumor, limiting their cytotoxic potential for tumor clearance. Exhaustion is a heterogenous state of differentiation, which we now understand to be progressive in response to both persistent antigenic stimulation and tumor microenvironmental signals. Initially thought to reverse exhaustion, the checkpoint blockade therapy anti-PD-1 is now understood to have several cellular targets in both the tumor and draining lymph node. Early studies investigating anti-PD-1 immunotherapy in settings of exhaustion mediated by chronic viral infection determined that exhausted T cells retained epigenetic features after anti-PD-1 despite increased effector function and transcriptional changes. However, a comprehensive study of the epigenome in a melanoma that assess changes across exhaustion subsets has not been reported. In this application, we propose to revisit this question in the context of melanoma. First, using the anti- PD-1 sensitive murine melanoma cell line YUMMER1.7, we will dive deep into the epigenome using CUT&Tag to profile 5 histone modifications and DNA binding proteins. Prior studies from our group using B16 melanoma revealed dysfunctional chromatin landscapes in exhaustion using this system. Second, we will interrogate changes in the epigenome after anti-PD-1 therapy exploring both CD8 T cells from draining lymph node as well as precursor and terminal exhausted populations from the TIL. Finally, we will use a new adaptation of the CUT&Tag technology termed Multi-Cut&Tag to simultaneously profile epigenetic modifications of CD8 TIL isolated from melanoma at the single cell level. This study will carefully assess epigenetic changes in response to immunotherapy and utilize an innovative tool to understand the single- cell epigenetic landscape that underlies the progression to exhaustion. These fundamental questions will provide deep knowledge of melanoma driven exhaustion and how anti-PD-1 therapy impacts the chromatin landscape. Finally, the outcome of this study will have important implications on the combination therapies that may better prevent exhaustion or increase the proliferative burst of anti-tumor CD8 T cells.
National Cancer Institute R01 Multi-PI with Dr. Delgoffe and Dr. Najjar
Immunotherapeutic treatments for cancer, especially the use of monoclonal antibody mediated blockade of `checkpoint' molecules like PD-1, has changed the treatment paradigm for patients with solid tumors like melanoma. However, only a subset of patients benefit from these therapies, due to several resistance mechanisms concentrated within the tumor microenvironment (TME), the site of action of cytotoxic T cells. T cells must contend with physical barriers to infiltration, immunosuppressive cell types, the expression of co- inhibitory ligands on target cells, and a harsh metabolic environment produced by cancer cells. In addition to T cell-extrinsic immunosuppression, T cells within tumors have a distinct differentiation trajectory, resulting in acquisition of an alternative, dysfunctional fate termed exhaustion. Exhausted T cells are terminally differentiated, hypofunctional upon stimulation, and possess poor capacity to proliferate, a crucial component of immune memory. We and others have shown that exhausted T cells have severe metabolic deficiencies, and that metabolic stress within the TME, most notably hypoxia exposure, potentiates differentiation towards exhaustion. In line with this, we and others have shown that melanoma patients with more oxidative, hypoxic tumors are more likely to progress on anti-PD1. Thus, the hypoxic TME and the intrinsic functional deficiency of exhausted T cells are linked. However, how hypoxia and resultant oxidative stress alter T cell differentiation remain unclear. Our hypothesis is that hypoxia exposure promotes T cell exhaustion, by driving aberrant chromatin bivalency and loss of transcription, such that hypoxia mitigation treatments will alter T cell differentiation and support increased T cell function. AIM 1: How does hypoxia drive epigenetic changes that bias T cell differentiation and function? Hypoxia drives several cellular adaptations, including transcriptional reprogramming via HIF-1α, induction of reactive oxygen species (ROS), and metabolic shifts. We will A) use in vitro systems to identify mechanisms of hypoxia contributing to altered histone methylation and bivalency in murine T cells; and B) determine contributions of hypoxia to the T cell epigenome and explore potential mitigation strategies in murine tumor models. AIM 2: How do hypoxia reducing regimens alter intratumoral T cell differentiation in melanoma patients? We and other have shown that targeting tumor cell metabolism or angiogenesis can increase the oxygen tension within tumors in both mouse models and melanoma patients. In this Aim, we will take advantage of two investigator-initiated clinical trials in melanoma utilizing metformin or axitinib in combination with anti-PD-1, and deeply explore transcriptional, epigenetic, metabolic, and functional outcomes associated with reduction of hypoxia coincident with anti-PD-1. We expect these studies to address knowledge gaps in the fields of epigenetics, immunology, and cancer immunotherapy, uncovering how tumor hypoxia can bias T cell differentiation and response to anti-PD-1, with the goal of identifying novel targets that mitigate hypoxia driven T cell exhaustion and overcome barriers to immunotherapy for cancer.
Lung-specific expression and function of Blimp-1 in T cells impacting allergic asthma
National Institute of Allergy and Infectious Diseases R01
Allergic asthma is a chronic inflammatory disease of the lung that drives a type 2 cytokine response (such as IL- 4, IL-5, and IL-13) predominately produced by activated CD4 T cells of a Th2 phenotype in response to exposure with environmental allergens. The master transcription factor GATA3 is known to drive Th2 development and promote type 2 cytokine production, however, the molecular pathways that drive Th2 cells to allergens at both the initiation and recall stages are not well understood. We have reported that Blimp-1 is an unexpected driver of allergic asthma that is critical to promote Th2 cell development in the lung and subsequent airway inflammation. Blimp-1 is a transcriptional repressor that is pleiotropically expressed by effector T cells and can regulate effector cell responses to constrain T cell-mediated autoimmunity. The purpose of this proposal is to understand the tissue-specific environmental niche of the lung that promotes Blimp-1 expression to drive Th2 development in the lung and subsequent inflammation in response to allergens. We have shown that the IL-10- STAT3-Blimp-1 axis is critical for Blimp-1 to promote Th2 cells in response to inhaled allergens. We now hypothesize that inhalation of allergens creates a lung-specific inflammatory environment to promote Blimp-1 and drive differentiation Th2 cells in both primary and memory responses to allergen leading to cycle of chronic lung inflammation. We will explore this hypothesis in three aims: (1) Demonstrate that expression of Blimp-1 requires IL-10 from lung-derived migratory cDC2s produced in response to inhaled allergens. (2) Determine if the kinetics of Blimp-1 expression in T cells underlie its context-dependent function to drive Th2 differentiation or constrain effector T cells. (3) Demonstrate that allergen-specific memory T cells require Blimp-1 for Th2 driven recall responses in the lung. Overall, we will determine the role of Blimp-1 in de novo generation of Th2 cells to drive a type 2 niche and promote persistent inflammation. This study is significant because of its potential to identify pathways regulating Th2 cells in response to chronic allergens, as well as to elucidate the context- dependent function of Blimp-1, an important regulator of effector T cell differentiation and function. This work therefore will demonstrate how tissue-specific and context-dependent immunity must be considered in therapeutic approaches for diseases associated with tissues such as allergic asthma.
Tissue-specific mediators of allergen-driven type 2 inflammation
National Institute of Allergy and Infectious Diseases R21
Allergens are a class of innocuous environmental antigens that drive an inappropriate inflammatory immune response in susceptible individuals that is often characterized by type 2 cytokine production from helper T cells (Th2). Inhalation of allergens such as house dust mite leads to allergic inflammation of the lung, yet precisely how allergens drive a type 2 immune response in the lung tissue is not clear. We have previously demonstrated that Blimp-1 plays a central role in Th2 cell differentiation in the lung in response to inhaled allergens. However, administration of the same allergens subcutaneously does not require Blimp-1 for the formation of Th2 cells, suggesting the route of entry and the resident tissue specific immune cells determine the necessity for Blimp-1 in Th2 cell differentiation. We have found in response to inhaled antigens that IL-10 is a key cytokine that promotes Blimp-1 in Th2 cells. Blimp-1 acts by repressing Bcl6, itself a potent suppressor of the canonical transcription factor associated with Th2 cells, GATA3. Based on these data, we propose that unique signals driven by lung cells draining to the lymph node from the local tissue environment during T cell priming impact lung specific responses to allergens by promoting Blimp-1 and thus type 2 immunity. In this proposal we will 1) Determine how allergens support Blimp-1 and Th2 cells via IL-10 using an innovative technology that combines unbiased gene expression analysis with spatial location in the tissue to identify IL-10 producing cells in the lung and lymph nodes and their relationship to T cells expressing Blimp-1. In addition, we will 2) perform scRNAseq and scATACseq to identify how lung-specific environments established prior to allergic sensitization impact the immune response upon allergen challenge. We expect these studies to identify fundamental early steps in T cell priming in the lung draining lymph node in response to lung-derived allergens, demonstrating that tissue-specific environmental signals at homeostasis can shape subsequent immune responses to antigen challenge driving unique T cell differentiation pathways. In addition, our unbiased approaches have the potential to elucidate both the spatial location of early T cell priming pathways but also identify novel mediators that promote type 2 immunity to allergens. These studies therefore will have a fundamental impact on future studies that could identify potential therapeutic targets for the treatment of allergic asthma.
Identifying the expression and function of Blimp-1 in allergen-driven ILC2 activation
UPMC Mellon Scholars Pilot Award
The major goals of this award are to determine the extent of Blimp-1 expression in ILC2s in allergic disease and identify the function of Blimp-1 in ILC2s using a mouse model to delete Blimp-1 from ILCs.
Metabolic regulation of epigenetic dysfunction in CD4 T cells in lupus
Pittsburgh Autoimmunity Center of Excellence in Rheumatology (PACER) Catalytic Award
The goals of this award are to interrogate the role that metabolic dysfunction has in driving epigenetic abnormalities observed in lupus T cells that underlie hyperactivation, effector function and subsequent disease.
Learning Protein’s Role in Allergic Asthma Could Lead to New Treatments
American Lung Association Innovation Award
Allergic asthma is a chronic lung disease with no cure, with cases rising in adults and children. Inhalation of allergens such as house dust mite leads to allergic inflammation of the lung, yet precisely how allergens drive an immune response in the lung tissue is not clear. We are studying the role of a protein called Blimp-1 in promoting development of an inflammatory cell called Th2, which is inappropriately activated in asthma. This process may be critical to promoting allergic asthma. We will identify the lung-specific environmental factors that promote Blimp-1 dependent Th2 cells in allergic asthma. We expect these studies to identify novel drug targets for therapies for allergic asthma.