Influenza virus is a constant threat to international public health. The current, seasonal vaccine generates a response to epitopes of influenza virus that have a high rate of mutation leading to antigenic drift and antigenic shift leaving current memory responses useless and potentially resulting in a pandemic. The need for a universal influenza vaccine is evident. High affinity, broadly neutralizing antibodies (bnAbs) against a conserved epitope of the influenza virion, such as the stem of hemagglutinin, show potential to protect against a wide breadth, if not all influenza viruses. Identifying germline precursors of these bnAbs and generating immunogens that drive the maturation of a heterosubtypic response could provide a universal answer to influenza. In this thesis, I show the development and characterization of a mouse model consisting of B cells containing the germline heavy chain of the influenza bnAb CR9114. Furthermore, I outline the responses of two germline targeting immunogens to influenza: an anti-idiotypic antibody known as K1-18 and an engineered variant of an H1-hemagglutinin protein known as 587H1-X. Both K1-18 and 587H1-X are shown via calcium flux and activation markers to simulate B cells of our 9114 gH mouse model. Immunization studies with both immunogens show their ability to generate an IgG response that cross binds to wild type hemagglutinin. 587H1-X in particular shows mutation capabilities that drive these gl9114 antibodies towards their mature stage. This study sheds light on a germline targeting approach to inducing a universal response to influenza.

Chapter I introduces the innate and adaptive immune systems, which serve as the context for my dissertation, as well as the protein of interest in both systems.

Chapter II is a reprint of a study on which I was the second author. In this study, we investigated the role of caspase-8 in T cell survival. Caspase-8 deficient T cells were found to accumulate defectively in response to antigenic stimulation in vitro, and mice with caspase-8 deficient T cells (tCasp8-/- mice) were unable to mount a response to an acute viral infection. Inhibition of Ripk1 kinase was shown to rescue this accumulation defect in vitro, and this form of death was termed necroptosis due to its dependence upon Ripk1 and lack of shared morphological characteristics with apoptosis. However, it was unclear whether other proteins were also required for necroptosis. Both programmed necrosis (as defined by a requirement for cyclophilin D) and autophagy (as defined by a requirement for Atg7) had been proposed to play a role in T cell necroptosis. Ripk3 had also been shown to interact with Ripk1 and trigger programmed necrosis. We used genetic studies to show that while neither cyclophilin D nor Atg7 could rescue the accumulation defect of caspase-8 deficient T cells, Ripk3 did. Only mice with caspase-8 deficient T cells and an additional loss of Ripk3 (DKO mice) were able to mount a response to an acute viral infection. We also found that DKO mice developed a lymphoproliferative disease with similarities to lpr mice, which have a lupus-like autoimmune disease.

Chapter III examines the role of caspase-8 in another type of immune cell, dendritic cells (DCs). Caspase-8 was shown to negatively regulate the Rig-I innate immune signaling pathway, which produces type I interferons in response to RNA viruses. I found that mice with caspase-8 deficient DCs (dcCasp8-/- mice) develop an age-dependent autoimmunity characterized by hyperactivated DCs and T cells, organ immunopathology, and helper T cells that skew towards a Th1 phenotype. Since DC activation is considered required for a robust T cell response to viral infection, we next infected dcCasp8-/- mice with a chronic virus and found that they had an enhanced virus-specific T cell response characterized by less exhausted T cells and lower viral loads. Caspase-8 deficient DCs appeared to hyperactivate in response to Rig-I stimulation, which was likely dependent on endogenous activation of both IRF3 and NF-kB in the absence of caspase-8.

Upon infection, naive T cells proliferate and differentiate into highly specialized effector cells to combat the invading pathogen. Naive CD4+ T cells have the potential to differentiate into multiple functionally distinct T helper (TH) subsets based on the type of infection. Differing pathogens elicit distinct infection milieux that help direct the differentiation of naive CD4+ T cells to ensure that each class of pathogen is countered with the appropriate immune response. The majority of effector T cells will die as the infection wanes, while a small proportion of cells will survive to established a long-lived memory population. This memory population is essential for improved antibody responses and also enables a rapid and robust secondary response against recurring pathogens, thus conferring lasting cellular immunity. However due to the functional breadth of CD4+ T cell lineages, the identification of a conserved memory CD4+ T cell precursor and memory population across TH lineages has proved challenging. Lack of such knowledge impedes the ability to investigate conserved mechanisms of memory CD4+ T cell formation and regulation. To better understand the biology of CD4+ memory T cells, we sought to identify a conserved marker of memory CD4+ T cells across different TH subsets. Utilizing fluorescent reporter mice, we found that expression of Id3, an inhibitor of E protein transcription factors, identified a population of cells within both the CD4+ TFH and TH1 helper lineages that exhibited memory potential in response to secondary infection. Notably, a subset of TH1 memory cells expressing Id3 exhibited enhanced expansion upon response to pathogen, generating both TH1 and TFH secondary effector cell populations, and displayed enrichment of key molecules associated with memory potential when compared to Id3lo TH1 cells. Relative to Id3lo TH1 memory cells, Id3hi TH1 cells exhibited a transcriptomic profile more akin to that of memory T lymphocytes. Thus, we found that Id3 expression serves as an important marker of multipotent memory CD4+ T cells.To investigate novel regulators of CD4+ memory T cells, we took a computational ap- proach by using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and bulk RNA sequencing (RNA-seq) of effector and memory CD4+ T cell popula- tions. We leveraged the PageRank algorithm to first predict putative regulators based on changes in transcriptomic expression as well as chromatin accessibility between effector and memory CD4+ T cells. Validation of predicted targets utilized the electroporation of CRISPR/Cas9 RNP complex to achieve loss-of-function disruptions of target genes in CD4+ T cell prior to LCMV- Armstrong infection. Although initial testing of predicted targets Srebf2 and Rorb did not reveal significant effects in CD4+ memory T cell formation, the optimization of the CRISPR/Cas9 RNP system has provided an efficient and reliable method for gene-disruption in T cells that undoubtedly expands our ability to investigate T cell biology. Future experiments utilizing this workflow have the potential to identify conserved regulators of CD4+ precursor and memory T cell populations across TH lineages, shedding light on possible mechanism for CD4+ T cell memory formation.

The clearance of enteric bacteria by phagocytes plays an essential role in the host response of infection. Previously, we have reported that BAI1 (Brain Angiogenesis Inhibitor 1) binds bacterial Lipopolysaccharide (LPS) and facilitates engulfment of Gram-negative bacteria by activating ELMO1 (Engulfment and cell motility protein 1). Using Salmonella as a model organism, we hypothesized that ELMO1 regulates bacterial clearance, activates autophagy and modulates the endosomal lysosomal pathway. The data from Salmonella infection in J774 murine macrophages with depleted ELMO1 expression, by genetic modification, suggest that ELMO1 is important for LC3BII accumulation. There was a compounded decrease in LC3BII accumulation when ELMO1 and ATG-5 were simultaneously inhibited. These observations suggest that ELMO1 is upstream of ATG-5 and important for its function. The effect of ULK-1 depleted macrophages on LC3BII accumulation was insignificant when compared to control macrophages. Therefore, it appears that Salmonella is cleared mostly by LC3-associated phagocytosis in an ELMO1-dependent manner. ELMO1-depleted macrophages showed delayed bacterial clearance post infection. ELMO1 was found to interact with the late endosomal protein Rab9, which is involved in lysosome biogenesis and cellular trafficking. The accumulation of the lysosomal enzyme cathepsin B varried in a time-dependent manner and decreased in ELMO1-depleted macrophages compared to control macrophages 12 h post infection. The expression of genes associated with the Salmonella Pathogenicity Island-2 (SPI-2), important for bacterial survival inside macrophages, decreased in ELMO1-depleted macrophages compared to control macrophages at 6 hours post infection. The understanding of the host response to enteric infections is important because they persistently reoccur.

Pancreatic cancer is the third leading cause of cancer death in the United States. Pancreatic ductal adenocarcinoma is the most prevalent type of pancreatic cancer and is characterized by dense fibrosis (termed desmoplasia) in the tumor microenvironment. Cancer associated fibroblasts (CAFs), which are thought to derive from pancreatic stellate cells (PSCs) and fibroblasts (PFs), play a large part in this desmoplasia. Signaling from cancer cells activates CAFs, which in turn can signal to promote tumorigenesis. Previous work done in the Insel lab had identified a GPCR, Orphan 1, as one of the most highly up-regulated GPCRs in CAFs compared to normal PSCs and PFs. This thesis project identifies TGFβ and TNFα as factors that, increase, respectively, the expression in PSCs of fibrotic markers and Orphan 1. Other work in this project shows that Orphan 1 in CAFs regulates the expression and secretion of Interleukin-6 (IL-6). Orphan 1 signals through a Gs-coupled pathway (via cAMP and protein kinase A [PKA]) to increase IL-6 formation and secretion by CAFs; IL-6 can aid in the development of PDAC. Thus, inhibiting the secretion of IL-6 through Orphan 1 may be a novel strategy for the treatment of pancreatic cancer.

Asthma is a heterogeneous disease with distinct features of airway hyper-responsiveness (AHR) and mucus secretion. In general, long-acting b2 adrenergic agonists (LABAs) are commonly recommended for asthma patients to reduce these symptoms. When LABAs act on airway smooth muscle (ASM), they bind to ASM’s b2 adrenergic receptors (b2ARs) to increase cAMP, which induces bronchodilation effect to ameliorate the AHR. In our previous study, we observed that the G protein-coupled receptors (GPCRs) in dendritic cells (DCs) triggers the Th17 differentiation of naïve T lymphocytes via cAMP signaling. Such a pro-Th17 activity is a serious response because it induces neutrophilic asthma with more severe inflammation. Thus, LABAs, which directly target b2AR of both ASM and lung DCs, need re-evaluation for their risk of causing severe inflammation. Therefore, we examined whether LABAs trigger b2AR of lung DCs and activate Th17 differentiation in asthma. Our results demonstrated that LABA treatment induced Th17 response in both murine DCs and human DC-like cell lines in vitro. Also, we observed that LABA inhalation triggered neutrophilic asthma in vivo. After we had confirmed the side-effect of LABAs, we further examined the combination of LABAs with Inhaled corticosteroids (ICS). Our results from LABA-ICS combination administration showed that the LABA-ICS combination inhibited both Th2 and Th17 response in asthma, supporting the anti-inflammatory effect of ICS with LABAs. Conclusively, we showed that LABAs have a side-effect of inducing neutrophilic asthma with its Th17 response, which suggests that the continuing use of LABAs may worsen the asthmatic symptoms of LABAs users.