Methylation controlled J protein (MCJ) is a negative regulator of mitochondrial metabolism that has a substantial impact on overall cell metabolism and function. MCJ is highly expressed by naïve CD8+ T cells, however its role in their immune effector functions was unknown. In this dissertation, it will be demonstrated that MCJ restricts the mitochondrial metabolism of CD8+ T cells, in part by reducing respiratory supercomplex formation. MCJ deficiency enhances the immune effector functions and memory responses of CD8+ T cells in a mitochondrial ATP dependent manner. As a consequence, protection to influenza virus infection is substantially improved. Reduced expression of MCJ therefore promotes viral immunity, however the loss of MCJ is not always beneficial. In cancer, decreased MCJ expression is correlated with ATP binding cassette (ABC) transporter mediated chemotherapy resistance and poor patient responses. This dissertation will also address the role of MCJ in chemoresistance. Increased mitochondrial ATP production due to MCJ deficiency is sufficient to fuel ABC transporter activity, thereby directly promoting chemoresistance. This can be reversed by restoration of MCJ function in chemoresistant cells. Overall, the results presented in this dissertation identify MCJ as a potential therapeutic target, as modulating MCJ expression can significantly affect the severity of viral infections and the responses to chemotherapy.

Semi-invariant natural killer T (iNKT) cells are critical components of the host immune response in peripheral tissues such as the lung, liver, and gut, and they play important roles in cancer, bacterial infections, autoimmunity, wound repair, and atherosclerosis. Tissue-resident iNKT cells exert their effects early in the developing immune response by rapidly producing a wide variety of cytokines and chemokines, and it was recently discovered that different tissues possess iNKT cell subsets that preferentially produce IFN-γ (NKT1), IL-4 (NKT2), or IL-17 (NKT17). Despite their critical role in the immune response, the mechanisms that regulate iNKT cell function in the periphery remain unclear. Signaling lymphocyte activation marker (SLAM) proteins are cell surface-expressed molecular switches that are expressed on all hematopoietic cells. The nine SLAM family receptors serve a variety of functions including promotion of cell-cell adhesion, regulation of cytokine production, co-stimulation, and inhibition. Importantly, SLAM family receptors are critical for the development of iNKT cells. Yet, numerous efforts to ascribe discrete roles of SLAM family receptors in iNKT cell function has proven difficult. We conducted a comprehensive analysis of SLAM family receptor co-expression on iNKT cell subsets in the lung, spleen, liver, and thymus and identified co-expression profiles that varied in a tissue and strain-dependent manner. Interestingly, we found that SLAM family receptor expression profiles varied among different iNKT cell subsets. In particular, we noted a close association of SLAMf6 expression with the NKT2 and NKT17 subsets in both the periphery and in the thymus. Further investigation using SLAMf6-deficient mice revealed a critical role for SLAMf6 in NKT2 and NKT17 subset development, and in iNKT IL-4 and IL-17 cytokine production in the periphery. This investigation also revealed that the SLAMf6high NKT2 and NKT17 subsets exhibited significantly higher proliferative capacity than the NKT1 subset and the NKT2 and NKT17 proliferation was dependent, in part, on SLAMf6 expression. Since Slam family genes are highly polymorphic, we next investigated whether these polymorphisms regulated iNKT function. We employed a B6.129 congenic mouse exhibiting impaired NKT cell function, in which a 6.6 Mbp 129/SvJ locus encompassing Slam genes was introgressed onto the C57BL/6 background. To test the hypothesis that Slam gene polymorphisms regulate iNKT cell function, we refined this genetic interval by generating B6.129 subcongenic lines and assessing iNKT cell function. Unexpectedly, we found that while Slam gene polymorphisms in this model do regulate iNKT cell function, the dominant regulator was in a 0.14 Mbp interval centromeric to the Slam genes. Further experimentation revealed that impaired iNKT cell development and function was associated with changes in the expression of Fcgr3 (Fc gamma receptor III) on iNKT cells, suggesting it as a novel candidate gene regulating iNKT cell function. Taken together, these data reveal for the first time a specific role for SLAMf6 on NKT2 and NKT17 subset development and function. In addition, these data identify Fcgr3 as a novel candidate gene that regulates iNKT cell subset development and cytokine production. Cumulatively, these data reveal the presence of discrete regulatory mechanisms at work in different iNKT subsets, a finding that has broad implications for our understanding of iNKT-cell mediated immunity.

CD4 and CD8 T cells play a vital role in mediating the clearance of viral pathogens following infection. Mice deficient- or depleted of their CD4 and/or CD8 T cells exhibit a diminished ability to control viral replication following infection and in some cases develop a persistent viral infection. CD8 T cells upregulate cytotoxic effector molecules such as granzyme B, Fas and TNF-related apoptosis-inducing ligand (TRAIL) that them to directly kill virus-infected cells. Following a systemic virus infection the CD8 T cell response is primed within secondary lymphoid organs, such as the spleen and lymph nodes (LNs). Although, it has been shown that the LNs are important for the generation of optimal CD8 T cell responses following systemic viral infections, the relative role of the spleen versus the LN in priming the CD8 T cell response is unknown. Studies in this thesis demonstrate that LNs, but not the spleen, are critical for the optimal generation of a CD8 T cell response following a systemic intraperitoneal (i.p.) lymphocytic choriomeningitis virus (LCMV) infection. Using adoptively transferred naïve LCMV-specific CD8 T cells, we demonstrate that the mediastinal LN (MedLN) serves as the initial draining LN and is responsible for priming the majority of the virus-specific CD8 T cell response following an i.p. LCMV infection. Moreover, the draining MedLN exhibits an increased frequency of CD62L- effector memory (TEM) CD8 T cells for up to 8 weeks following viral clearance. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells is not due to residual viral antigen. Furthermore, a similar increase in CD62L- TEM CD8 T cells is found in the ipsilateral popliteal LN following a footpad LCMV infection. I demonstrate that the increased frequency of CD62L- TEM CD8 T cells in the draining LN is due to increased recruitment. CD4 T cells promote the generation of both effector and memory CD8 T cells either indirectly through their CD40-CD40L-dependent maturation of dendritic cells or through the production of cytokines such as IL-2 and IFN-γ that directly interact with CD8 T cells. CD4 T cells are also critical for the generation of germinal center B cells and promote the differentiation of activated B cells into memory B cells and plasma B cells. However, CD4 T cells often recognize epitopes derived from a broad array of pathogen-encoded proteins, making it difficult to accurately quantify the magnitude of virus-specific CD4 T cell responses. Therefore, I evaluated a large panel of activation and/or memory markers to determine a combination that could be used to reliably identify antigen-specific CD4 T cells following viral infection. I show that the integrins CD11a and CD49d are upregulated in an antigen-dependent manner on virus-specific CD4 T cells following LCMV infection. Furthermore, memory LCMV-specific CD4 T cells retain their CD11ahiCD49d+ expression pattern. Using CD11a and CD49d as surrogate makers for antigen-specific CD4 T cells, I show that approximately 50% of the CD4 T cells following LCMV infection are virus-specific, indicating that the virus-specific CD4 T cell response is substantially larger than previously recognized. Furthermore, I demonstrate that CD11a and CD49d can be used to accurately track newly-activated CD4 T cells following a heterologous virus challenge. In addition to LCMV, respiratory syncytial virus (RSV)-specific CD4 T cells are CD11ahiCD49d+. The two previously identified RSV CD4 T cell epitopes only account for ~3% of the CD11ahiCD49d+ CD4 T cell population during the peak of RSV infection, indicating that additional RSV-derived epitopes remain to be identified. Therefore, I used an overlapping peptide library spanning each of the RSV-derived proteins to identify novel RSV-specific CD4 and CD8 T cell epitopes. Using this approach, I identified 5 novel RSV-derived CD4 T cell epitopes and 4 novel CD8 T cell epitopes. Furthermore, I demonstrate that stimulation of CD4 T cells with 17-mer peptides results in over a 2-fold increase in the frequency of responding CD4 T cells as compared to stimulation with the commonly used 15-mer peptides. Collectively, the data shown here provides new insight into where and how the CD8 T cell response is initiated following a systemic virus infection, as well as provide a novel approach to track the endogenous CD4 T cell response following viral infections.

Demyelination in the CNS is known to involve several immune effector mechanisms, including complement proteins. For this dissertation project the central hypothesis that C3 and downstream effector complement proteins exacerbate demyelination through activation of glial cells was tested. To investigate the role of C3 and downstream complement proteins in demyelination and remyelination pathology in vivo we utilized the cuprizone model. We used C3 knockout mice (C3-/-), which are lacking the central C3 protein and subsequently all downstream complement effector proteins, and transgenic mice expressing C3a or C5a under the control of the glial GFAP promoter. Interestingly, we found no changes in demyelination or remyelination pathology between C3-/- and control mice. However, C3a and C5a transgenic mice had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to WT mice. Transgenic mice had increased cellularity in the corpus callosum due to increased activation and/or migration of microglia. There was also evidence of T cells in the corpus callosum during demyelination in C5a transgenic mice, suggesting C5a may modulate BBB permeability. During early remyelination oligodendrocytes migrated to the corpus callosum in higher numbers in C3a and C5a transgenic mice, thus enabling these mice to remyelinate as effectively as WT mice by the end of the ten week study. To determine the effects of anaphylatoxins on individual glial subsets, we created murine recombinant C3a and C5a proteins. We found that the MAPK pathway proteins JNK1 and ERK1/2 were activated in glia upon stimulation with recombinant anaphylatoxin proteins. When microglia and mixed glial cultures were stimulated with C3a and/or C5a, we observed an increase in the production of proinflammatory cytokines and chemokines. In contrast, anaphylatoxin-treated primary astrocytes had suppressed cytokine and chemokine production compared to untreated astrocytes. In vitro, primary microglia and astrocytes did not significantly migrate in response to stimulation with C3a or C5a proteins, suggesting migration may not be a primary anaphylatoxin-mediated function in the CNS. Overall, our findings show that anaphylatoxin production in the brain plays a negative proinflammatory role during demyelination and that anaphylatoxin proteins can activate individual subsets of glia, initiating the production of inflammatory mediators.

The availability of attenuated, bitypic, genetic recombinant strains of herpes simplex virus (HSV) made possible the following investigations. The recombinant virus, D5E1, exhibited limited, short-lived replication in the central nervous system of mice and guinea pigs. Nevertheless, this virus was sufficient to stimulate substantial levels of neutralizing antibody and localized cellular immunity in genital tissue. Immunization with D5El protected mice and/or guinea pigs against (HSV) type l and 2 infections when the challenge virus was given by a variety of pathways. In particular, it reduced vaginal virus shedding, inflammation, and acute and latent infection of the regional ganglia. The degree of protection in mice was influenced by the strain of rodent, the route of challenge and type of wild virus. Vaccination of newborn mice with live, attenuated virus also resulted in protection against HSV-2 subsequently inoculated into the footpad. The ability of recombinant HSV strains to establish latent ganglionic infection was related to their virulence, the route of inoculation and the genetic strain of the mouse. The D5E1 vaccine failed to establish latent disease by itself, but nevertheless conferred good protection against HSV type 1 or 2 challenge of immunized mice or guinea pigs.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality in the U.S. and worldwide. Atherosclerosis, the buildup of plaque in the arteries, is a common cause of CVD. For many years, research in atherosclerosis was focused on lipid metabolism and the accumulation of low-density lipoprotein in the arteries. While this research set public health guidelines for lipid management, lipid concentration was not the only factor influencing atherosclerosis and CVD events. Many scientists, as far back as the 1850’s recognized the role of inflammation in the progression of atherosclerotic disease. The continuous low levels of immune activation in the body contribute to atherosclerosis. Research in animal models and epidemiologic studies have shown the involvement of both the innate and the adaptive immune systems in plaque development and to elucidate the roles of monocytes and T cells. In addition to animal studies and epidemiologic research, CVD and atherosclerotic research has extended to genetic analysis in the search for associations with risk factors and outcomes. The first chapter is a review of the literature studying the immune system’s involvement in atherosclerosis. Beginning with an examination of the impact of CVD and atherosclerosis, the basic pathophysiology, and the involvement of the innate and adaptive immune systems through animal models and epidemiology. Some of the significant cohort studies in CVD and genome wide association studies are also discussed. Chapter 2 examines the associations of soluble interleukin 2 receptor alpha (sIL-2Rα) with clinical events in the Cardiovascular Health Study and genetic variants. Interleukin 2 (IL-2) and its receptor regulate both tolerance and immunity, IL-2 induces the proliferation and differentiation of T cells, part of the adaptive immune system. The results showed an association between sIL-2Rα and CVD events. The genome-wide association study found 52 variants to be significantly associated with sIL-2Rα in European Americans. Chapter 3 assesses the involvement of the innate immune system in atherosclerosis through the associations of soluble CD163 (sCD163). CD163 is a marker of macrophage activation, specifically associated with M2 macrophages. In CHS, sCD163 levels were analyzed for associations with cardiovascular events and genetic variants. sCD163 was found to be associated with CVD risk factors and with cardiovascular events. In a genome-wide association study six variants in European Americans and three variants in African Americans were found to be significant. Chapter 4 summarizes the results and discusses some bench to bedside translational science already seen in atherosclerosis treatment and prevention. Continued investigation of markers of T-cell and monocyte differentiation in animal models and cohort studies may lead to opportunities for the prevention of atherosclerosis and/or treatment through an increased understanding of the biology and genetics of the innate and adaptive immune.

The onset of immune response against microbial stimuli activates induction of many anti- inflammatory genes and ISGs for effective clearance of the pathogen. This response includes transcriptional activation of several non-coding transcripts such as miRNAs and long non-coding RNAs (lncRNAs). LncRNAs constitutes the largest class of non-coding genome and are arbitrarily described as transcripts greater than 200 base pairs. Similar to protein coding mRNAs, lncRNAs are RNA polymerase II transcripts and undergo mRNA processing such as capping, splicing and polyadenylation. In recent years, high throughput sequencing has enabled an in-depth exploration of the human genome and subsequent discovery of lncRNAs. Several studies have highlighted the crucial role of lncRNAs in many biological processes including as regulators of gene expression as well as molecular effectors of host-pathogen driven immune responses. To date, majority of lncRNAs have been studied in murine models with limited understanding in human cells. In order to elucidate the role of lncRNAs in human immune cell regulation, the goal of this thesis is to identify and characterize novel lncRNAs critical to host-pathogen innate immune responses. RNA sequencing in LPS, IAV and HSV stimulated cells revealed lncRNA LUCAT1 as most differentially regulated lncRNA. CRISPR-cas9 and shRNA mediated depletion of LUCAT1 showed enhanced IFN-I genes signature, which was suppressed upon overexpression of LUCAT1. Additionally, LPS stimulated hDCs showed enrichment of LUCAT1 in the nucleus and its association with the chromatin markers. Further, LUCAT1 depletion contributed to enhanced occupancy of transcriptional coactivators at the promoters of IFN-I genes. Global identification of RNA associated proteins revealed LUCAT1 association with STAT1 in the nucleus thus emphasizing its role in transcriptional regulation of Type I IFN genes in inflammatory responses. This thesis furthers the understanding about the molecular factors affecting immune regulation and describes the novel role of LUCAT1 as an attenuator of immune cell response to pathogens.

Numerous factors that affect herpes simplex virus 1 (HSV1)-mediated pathogenesis have been identified. Such factors directly impact the replication of HSV1 as well as modulate host immune responses following HSV1 infection. In this work, I characterize how HSV1 replication is impacted by expression of the protein encoded by C21orf91, or “Cold Sore Susceptibility Gene” (CSSG1), that has been linked to HSV1 reactivation in humans. I investigated expression of CSSG1 mRNA expression in various tissues and found that CSSG1 mRNA was present in several tissues of importance in HSV1 disease, including brain, trigeminal ganglia (TG), cornea and spleen. Western blot analysis demonstrated that CSSG1 protein is expressed in human cells. Subcellular fractionation analysis reveals that CSSG1 is predominantly found in the cell nucleus, where it colocalizes with chromatin and with Tip60, a chromatin-binding histone modifying protein that has been shown to be essential for the replication of herpesviruses. I also discovered that CSSG1 is present in the cytosol of cells where it forms large cytosolic aggregates in presence of TRAF6, a downstream adapter that plays an important role in innate immune receptor signaling. To determine if CSSG1 directly impacts viral replication, I generated CSSG1 knockdown human cell lines. I found that HSV1 replication was reduced in CSSG1 knockdown cells compared to control cells, whereas replication of the unrelated virus, vesicular stomatitis virus (VSV), was not affected by knockdown of CSSG1. I demonstrate that CSSG1 was necessary for efficient expression of HSV1 viral proteins during infection. Western blot analysis and measurement of expression of HSV1 proteins expressed at various stages of viral replication illustrates that CSSG1 was required for HSV1 replication at very early stage of infection. I also noted that CSSG1 expression impacted the DNA damage response in HSV1 infected cells. Levels of H2AX phosphorylation, a marker of the DNA damage response, were increased in HSV1-infected CSSG1 knockdown cells compared to control cells. DNA damage responses are thought to promote HSV1 reactivation from latency and HSV1 gene expression, indicating a potential mechanism for role of CSSG1 in HSV1 replication through modulating the DNA damage response. Overall, my work demonstrates that CSSG1 affects HSV1 replication and provides insight on how CSSG1 polymorphisms in humans could affect HSV1 reactivation and replication to promote cold sores. These discoveries may also lead to a better understanding of pathogenesis of other herpesviruses in humans.

Influenza is a seasonal acute respiratory infection, causing millions of illnesses worldwide on a yearly basis. A common subtype, the influenza A virus (IAV), is a single stranded RNA virus, that similarly to other subtypes, targets epithelial cells. The best way to protect against the virus is through vaccination. Vaccine induced protection is mediated through the generation of adaptive CD4 and CD8 T cells, as well as antibody producing B Cells. Although generally thought of as helper cells, previous research has highlighted additional roles of memory CD4 T cells in mediating protection against IAV beyond their helper function. More specifically they have been shown to enhance innate inflammatory responses and facilitate the recruitment of innate cell populations; including a recently discovered population of Innate Lymphoid Cells (ILC). Previous research has shown ILCs to have a key role in tissue repair and limiting tissue damage following infection. Whether memory cell response, during protective recall, modulates ILC repair function is currently not well understood. To better understand the possible regulation of ILCs by memory cells, we utilized a molecular technique called reverse transcription polymerase chain reaction (RT-qPCR), to assess select innate lymphoid cell associated protein expression following IAV challenge and rechallenge. We hypothesize, that memory cells drive differential expression of ILC associated repair proteins to assist in a faster and more efficient mobilization of repair processes following pathogenic invasion. Our goal is to highlight and better understand the regulatory and inflammatory responses memory cells provide following viral infection, as these may lead to key steps in the development of long lasting and efficacious vaccines.

Cell-mediated lysis of cultured tumor target cells by nonspecific cytotoxic cells (NCC) was examined in the oyster toadfish (Opsanus tau), an estuarine teleost. NCC activity was evaluated in cells taken from the head kidney, peripheral blood, spleen and peritoneal cavity. NCC activity was a property of plastic nonadherent cells which lacked phagocytic activity, indicating that in terms of their functional capacity, the cells which mediate nonspecific cytoxic immune responses in oyster toadfish do not appear to be monocytes or macrophages. However, light and electron microscopic examination of these cells revealed that morphologically, they resemble monocytes and macrophages. A new technique to assess in vitro phagocytic function of fish macrophages is described. This assay involves the spectrophotometric measurement of congo red-stained yeast cells that have been phagocytized by macrophages. The assay is simple, rapid and reproducible. Furthermore, it is less subjective than previously described methods that employ microscopic examination of cells. Using this technique with oyster toadfish, phagocytosis of yeast cells was found to increase with time, reaching a maximum between 60 and 90 minutes (as determined by absorbance at 510 nm). Sampling sites selected for this field investigation had a sediment-bound polycyclic aromatic hydrocarbon (PAH) concentration gradient of 55 ppb to 96,000 ppb total PAH. Results of this study suggest that varying levels of PAH contamination did not lead to significant between-site differences in terms of simple ability of oyster toadfish macrophages to phagocytize foreign particles. This technique has been used in studies designed to characterize the functioning of oyster toadfish NCC and in toxicological investigations comparing the effects of PAH and NCC and macrophage function in oyster toadfish. Studies were undertaken to determine the effect of the chemical carcinogen 7,12-dimethylbenz (a) anthracene (DMBA) on the function of oyster toadfish peritoneal nonspecific cytotoxic cells (NCC) and macrophages. The functioning of these two cell populations was assessed in terms of their ability to lyse cultured tumor target cells and to phagocytize yeast cells, respectively. In a time-course study, the effects of DMBA on NCC activity were found to be highly persistent. Following a single intraperitoneal injection of 10 mg/kg, NCC activity was again virtually eliminated, and did not recover throughout the 28 days of the exposure study. (Abstract shortened by UMI.).