The correct and timely delivery of immune cells is critical for protection against foreign antigen. In order for cells to access most organs, there are requirements that must be met to facilitate exit from the blood into extravasculature. The initial requirement is selectin-selectin ligand interactions that mediate tethering and rolling to allow shear resistance. For proper selectin-selectin ligand interaction, glycoproteins must be modified by fucosyltransferases –IV and –VII, which adds fucose to an acceptor substrate to form the sialyl-LewisX moiety. Using fucosyltransferase –IV and –VII double knockout (FtDKO) mice, we made several novel observations. Our first observation showed increased numbers of naïve T cells in non-lymphoid organs. To support this observation, we blocked chemokine-mediated entry into lymph nodes (LNs) with pertussis toxin and L-selectin mediated entry with anti-CD62L antibody in WT mice. We also treated WT mice with the S1P1 agonist, FTY720, to retain lymphocytes in LNs. Our results suggested that when access to LN is perturbed, lymphocytes accumulate in non-lymphoid organs. Our second observation showed an enrichment of effector/memory T cells in FtDKO LNs. To determine if effector/memory CD8 T cells were retained in LNs, we transferred naïve and memory CD8 T cells into WT mice then treated the recipient mice with anti-CD62L. We found that LN exit rates of naïve and memory CD8 T cells were similar, but slowed as T cell density decreased. To understand if memory CD8 T cells were using selectin ligand independent mechanisms, we transferred naïve and memory CD8 T cells into WT or FtDKO mice. We found reduced numbers of memory CD8 T cells in LNs, however, their frequency was increased. We explored this result by transferring CFSE labeled memory CD8 T cells. We found that memory CD8 T cells divide more in FtDKO mice compared to WT. These experiments suggested that selectin ligand deficiencies cause increased frequency of effector/memory T cells in LNs due to low density and increased emptiness induced proliferation. Taken together, these findings reveal how selectin ligand deficiencies contribute to T cell accumulation in non-lymphoid organs and elucidate mechanisms of retention in LNs.

Prevalence of HIV-associated cognitive impairment is rising, the worst form of which is HIV-associated dementia (HAD). The disease is fuiled by a chronic innate type pro-inflammatory response in the brain which is highly dependent upon the activation of microglia. We first created an in vitro model of HAD composed of cultured microglial cells synergistically activated by the addition of IFN-gamma and the HIV-1 coat glycoprotein, gp120. This activation, as measured by TNF-alpha and NO release, is synergistically attenuated through the alpha7nAChR and p44/42 MAPK system by pretreatment with nicotine, and the cholinesterase inhibitor, galantamine. As these medications have been FDA approved, and over time, have shown only minor improvement in neurodegenerative disease for a limited period, we next sought to explore natural compounds that may attenuate HAD mediated inflammation and related pathology. This inflammation is a key moderator of A-#914; plaque deposition in the brain. Indeed it is likely a contributing factor as epidemiological data suggests significant numbers of HIV survivors are at elevated risk of developing Alzheimer's disease (AD). HIV-1 Tat-induced A-beta deposition, tau phosphorylation, and subsequent neuronal death could be risk factors for subsequent AD and/or HAD. Recent reports suggest green tea-derived (-)-epigallocatechin-3-gallate (EGCG) can attenuate neuronal damage mediated by conditions such as brain ischemia. In order to investigate the therapeutic potential of EGCG to mitigate the neuronal damage characteristic of HAD, IFN- gamma was evaluated for its ability to enhance well-known neurotoxic properties of HIV-1 proteins gp120 and Tat in primary neurons and mice. Indeed, IFN-gamma enhanced the neurotoxicity of gp120 and Tat via increased JAK/STAT signaling. Additionally, primary neurons pretreated with a JAK1 inhibitor, or those from STAT1-deficient mice, were largely resistant to the IFN- gamma-enhanced neurotoxicity of gp120 and Tat. Moreover, EGCG treatment of primary neurons from normal mice reduced IFN-gamma-enhanced neurotoxicity of gp120 and Tat by inhibiting JAK/STAT1 pathway activation. EGCG was also found to mitigate the neurotoxic properties of HIV-1 proteins in the presence of IFN-#947; in vivo. To explore the mechanism by which HIV may augment AD-like pathology, we found HIV-1 Tat protein inhibits microglial uptake of A-beta-1-42 peptide, a process enhanced by IFN-#947; and rescued by EGCG. To mimic the HAD clinical condition, we generated mice with HIV-1 Tat-induced AD-like pathology by cross-breeding HIV-1 Tat expressing mice (expressed under control of GFAP, Doxycline inducible promoter) with the PSAPP mouse model of AD. To simulate chronic Tat secretion over we used an optimized dose of 54 mg/kg/day on a biweekly basis over three months Tat significantly induced neuron degeneration and tau phosphorylation in Tat transgenic mice, dox dependently (P<0.001). Similar effects at the chronic 54 mg/kg/day dose were observed in PSAPP/Tat mice induced with dox. These mice also showed significantly more A-beta deposition (P < 0.05), neurodegeneration, neuronal apoptotic signaling, and phospho-tau than PSAPP mice (P < 0.05). In conclusion, HIV-1 Tat significantly promotes AD-like pathology in PSAPP/Tat mice. This model may provide a framework in which to identify new mechanisms involved in cognitive impairment in the HIV infected population, and possible treatments. Additional works will be needed to fully characterize the mechanism(s) of HIV- induced amyloid deposition, and to uncover viral mechanisms promoting AD-like pathology in general.

The studies presented within this dissertation provide the first description of PRDM1 (also known as Blimp-1 or PRDI-BF1) function in natural killer cells. NK cells are major effectors of the innate immune response via antigen-independent cytotoxicity and link to the adaptive immune response through cytokine release. Molecular mechanisms mediating NK activation are relatively well-studied; however, much less is known about the mechanisms that restrain activation. In the first study, the transcriptional repressor PRDM1 is shown to be a critical negative regulator of NK function. Microarray analysis was used to characterize transcriptional changes associated with cytokine-mediated activation. PRDM1 is expressed at low levels in resting NK cells and three distinct PRDM1 isoforms are selectively induced in the CD56dim NK population in response to activation. PRDM1 coordinately suppresses the production of IFNγ, TNFα and TNFβ through direct binding to multiple conserved regulatory regions. Ablation of PRDM1 expression leads to enhanced production of IFNγ and TNFα but does not alter cytotoxicity, whereas over-expression blocks cytokine production. PRDM1 response elements are defined at the IFNG and TNF loci. To further delineate the targets of PRDM1-mediated regulation in NK cells, global approaches were utilized. Experiments utilizing chromatin immunoprecipitation coupled to promoter tiling arrays identified 292 novel direct targets of PRDM1 binding. These studies revealed widespread binding of PRDM1 to the genome, which was not limited to proximal promoter regions. Furthermore, microarray analysis of stimulated NK cells combined with PRDM1 knockdown has enabled the identification of genes responsive to PRDM1 knockdown using primary cells. Collectively, these experiments identify both direct and indirect targets of PRDM1 regulation and help define a PRDM1-centered gene regulatory network in NK cells. Data presented in the final chapter pertains to an independent project aimed at identifying small molecule inhibitors of the methyltransferase G9a, which is recruited by PRDM1 and is required for silencing of target genes. A mass spectrometry-based assay was developed and used to screen a small molecule library. Several hits were identified and combinatorial chemistry yielded several compounds with < 20µM IC50 values. In cell-based assays, however, treatment with the small molecules had limited efficacy, indicating additional chemical modifications are necessary to yield bioactive compounds. The data presented here demonstrate a key role for PRDM1 in the negative regulation of NK activation and position PRDM1 as a common regulator of the adaptive and innate immune response.

Nuclear factor-κB (NF-κB) is one of the main regulators of inflammatory and immune responses. It is a family of transcription factors composed of five members: RelA, RelB, cRel, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Homo- and hetero-dimers of family members are inhibited by inhibitor of &klappaB (IκB) family members and activated by IκB kinase (IKK) family members. The IKK family is comprised of IKKα, IKKΒ, and IKKγ. The focus of my dissertation delves into the role of NF-κB activation by IKKΒ in both an immunotherapy setting and its role in T cell mediated anti-tumor immune responses. A central focus of immunotherapy is to develop vaccine adjuvants that are capable of enhancing a protective adaptive immune response. Microbial adjuvants in vaccines activate key transcription factors, including NF-κB and interferon response factors (IRF). The individual role of these transcription factors in successful vaccines is not clear. We used constitutively active IKKΒ (CA-IKKΒ) expressed in an adenoviral vector (AdIKK) to determine the role of classical NF-κB activation in a vaccine-induced immune response. In an in vivo model, AdIKK induced rapid and sustained NF-κB-driven inflammation in the lungs compared to the control virus. AdIKK infection had no impact on the magnitude of T cell activation as measured by IFN-γ production; however pulmonary inflammation resulted in increased cellularity of draining lymph nodes (LN) at early timepoints resulting in increased early antibody and T cell responses. Taken together, these findings show that IKKΒ-induced NF-κB activation of an inflammatory response affects the kinetics, but not the magnitude of the adaptive immune response. NF-κB is activated in many tumor types and contributes to the progression of cancer by suppressing apoptosis, and enhancing proliferation, angiogenesis and metastasis. NF-κB is also activated in other cells within the tumor microenvironment and promotes inflammation initiated by neutrophils and macrophages. In addition to inflammatory cells, T cells can be found within the tumor microenvironment and are associated with improved patient survival. Using CA-IKKΒ, we sought to determine if NF-κB activation in tumor cells could promote T cell mediated tumor immunity. In both primary tumors and a metastatic tumor model, we found that NF-κB expression in tumors rendered immunogenic through expression of Kb-OVA led to tumor rejection or growth suppression. Tumor regression was mediated by increased CD8 T cell recruitment by chemokines. Microarray results showed increases in T cell chemokines, including CCL2 and CCL5. Knock-down of CCL2 by Lentiviral shRNA in LLC-OVA-IKK cells resulted in abrogation of tumor regression. These results suggest that NF-κB is capable of promoting immune surveillance in tumors through increased recruitment of T cells. Overall, my dissertation highlights beneficial roles of IKKΒ-induced activation of NF-κB in two separate systems: vaccine induced immune responses and tumor immune surveillance.

Cancer cells employ a litany of immunosuppressive and immunevasive strategies to avoid detection and elimination by the various arms of the innate and adaptive immune system. Many hematologic and solid tumors progressively develop a specialized microenvironment which promotes tissue restructuring inflammation while masking the immune signature of the tumor cells themselves. Chronic lymphocytic leukemia, a malignancy of mature B lymphocytes must constantly balance on the precipice of immune recognition. A mature antigen presenting cell themselves, CLL clonal growth is dependent on the very interactions which, if effective, could potentially lead to their demise. To circumvent this, CLL clones set up unique signatures which promote immune recognition yet provide diversionary signals to the remaining immune armament resulting in profound immune dysfunction. While the aforementioned immune dysfunction is widespread, the B cell and T cell repertoire are severely impaired during leukemic progression. The lack of healthy B cells due to displacement by malignant B cells results in the obvious loss of an important antigen presenting cell as well as antibody-based immunity. Additionally, deficient interactions with T cells result in anergy and the preponderance of improperly polarized T lymphocytes which are impotent to eliminate both pathogens and leukemic cells. The result of such severe immune dysfunction is chronic infection and progressive disease which is the primary cause of death in CLL patients. Our research was focused on the premise that alleviating immune dysfunction and providing immunotherapeutic tools will significantly benefit CLL therapy. To this end we developed methods to improve the cellular interaction between CLL cells and T cells a critical step towards improving the antigen presentation capacity of the diseased B cell repertoire. We also identified a therapeutic strategy which can revert the anergic or improperly polarized state of T cells already in circulation allowing those cells to more effectively perform the effector functions necessary to fight pathogenic attack and malignant transformation. Finally, we identified a number of novel targets in CLL which could be used in a vaccinate-induce method to license the elimination of CLL cells by the patient's adaptive immune system. To achieve our goals we utilized a relatively new class of drugs called epigenetic modifiers which specifically alter the chromatin structure resulting in novel genetic signatures which are heritable over cellular generations. The unique properties of these drugs allow for the elicitation of suppressed genetic programs which, when properly controlled, have the potential to reassert healthy lymphocyte functions. Our studies provide a comprehensive therapeutic initiative which, by simultaneously alleviating the major causes of immune dysfunction in addition to facilitating the use of novel active immunotherapeutic strategies could potentially impact clinical therapy for CLL.

The peripheral immune system plays a role in delayed neural injury after stroke. This response originates from the spleen as splenectomy prior to middle cerebral artery occlusion (MCAO) in rats significantly reduces infarct volume in the brain. This research is based on the hypothesis that inhibiting the splenic response will reduce neurodegeneration after stroke. Studies in animals have implicated lymphocytes as the immune cell type that is detrimental following MCAO. Interferon gamma (IFNγ) has been identified as a pro-inflammatory cytokine that is also detrimental following stroke. IFNγ is important because it activates microglia and macrophages in a pro-inflammatory nature that increases neural injury following stroke. Therefore IFNγ was examined in the brain and the spleen following MCAO. IFNγ protein was elevated at 24 h in the spleen and at 72 h in the brain post MCAO. Microglia/macrophages become maximally activated at 72 h in the brain after MCAO. Splenectomy decreases the levels of IFNγ in the brain following MCAO. Systemic administration of IFNγ reversed the protective effects of splenectomy. The cellular response to MCAO was examined next because of the difference in time between the spike in IFNγ in the spleen and the delayed increase in the brain. The cellular response from the spleen was studied by labeling splenocytes five days prior to MCAO with a fluorescein dye. Tissues were examined 48 and 96 h post MCAO or sham MCAO for fluorescence. These cells were released from the spleen into circulation at 48 h post MCAO and migrated to the brain where the cells produced IFNγ at 96 h post MCAO. IFNγ appears to play a role in the splenic response to stroke. One protein that is up regulated by cells that have been activated by IFNγ, interferon-inducible protein 10 (IP-10) is part of the inflammatory cycle driven by IFNγ. IP-10 recruits more IFNγ producing T helper (Th) cells to the site of injury. IP-10 has the unique ability to attract Th1 cells, the pro-inflammatory Th cells, and inhibit Th2 cells, the anti-inflammatory Th cells. This leads to more IFNγ production as IFNγ is the signature cytokine of a Th1 response. IP-10 is significantly increased in the brain at 72 h post MCAO, similar to IFNγ expression. In the spleen IP-10 increased at 24 h and remained elevated out to 96 h following MCAO. IFNγ signaling was inhibited by utilizing an IFNγ neutralizing antibody administered beginning 24 h post MCAO. The IFNγ antibody treated group had decreased infarct volumes, IP-10 levels in the brain, and appeared to have decreased T cells in the ipsilateral hemisphere at 96 h post MCAO. Following ischemic stroke splenocytes are released into circulation and migrate to the brain. They release IFNγ to activate microglia/macrophages in a proinflammatory phenotype causing an increase in IP-10 levels. IP-10 then potentiates the Th1 driven inflammation which inhibits the Th2 response. The elevated levels of IFNγ increase neural injury following MCAO. Blocking IFNγ selectively blocks the inflammatory facet of the immune response to reduce stroke induced neurodegeneration. This leaves the other immune responses intact and able to contribute to tissue repair, regeneration, and able to respond to infections. Selectively inhibiting IFNγ signaling is a promising stroke therapeutic.

Recent studies suggest that some cases of prenatal depression may be associated with reactivation of latent infections of the herpesvirus family. The possible relationships among stress, prenatal depression, and herpes viral reactivation in pregnancy are understudied and the molecular pathways such as the neuroimmune biogenic amine pathway are unidentified. Chronic stress shifts the T helper-1 cell (Th1) cytokine profile to a Th2 profile, which favors virus induced pathogenesis and survival. Pregnancy is also associated with a similar Th2 dominance. In non-pregnant individuals, exposure to psychological or physical stress may be associated with latent herpes viral reactivation and could result in behavioral deficits and depression. Normally, type-1 cytokines such as Interferon-gamma (IFN -gamma) and inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) induce indoleamine-2, 3-dioxygenase (IDO) activation which inhibits herpes virus replication and reactivation, decreases tryptophan production, and alters phenylalanine /tyrosine metabolism. Thus it is possible that prenatal depression may occur from tryptophan stealing through the IDO pathway which results in decreased serotonin as well as increased risk for latent herpes viral reactivation. The purpose of this study is to analyze the relationships among stress, herpes viral titers, depression, and metabolites of IDO activation, which involves tryptophan and guanosine-triphosphate-cyclohydrolase-1(GTP-CH1) pathways. This study builds on Influence of Lactation on Postpartum Stress and Immunity (Grant number: R01-NR05000) which investigated perinatal immune, endocrine, and inflammatory changes in pregnancy and the postpartum. A secondary data analysis was conducted on baseline data from women collected at 16 to 25 gestational weeks. This data set included some herpes viral titers, and additional ones were measured in stored plasma samples. The aim of this study is to examine relationships among stress, herpes viral reactivation, depression, and the IDO activation pathway. The results of this study provide information about the possible role of further relationships of prenatal stress, latent herpes viral reactivation, and depression mechanisms. The results will be important in health promotion and disease prevention during pregnancy.

Respiratory syncytial virus (RSV) infection causes ~64 million cases of respiratory disease and 200,000 deaths annually worldwide, yet there is no broadly effective prophylactic or treatment regimen. RSV can produce acute respiratory illness in patients of all ages but strikes the age extremes, infants and the elderly, with highest frequency presumably due to innate immune deficiencies. A higher morbidity and mortality has been reported for the elderly above 65 years of age, which has been attributed to immune senescence. Efforts to generate an effective vaccine have thus far been unsuccessful. The innate immune system provides the first line of defense against viral pathogens with a repertoire of anatomical barriers, phagocytic immune cells, pattern recognition receptors (PRRs) and antiviral cytokines like interferons (IFNs). The precise mechanism of subversion of innate immunity in young and aged is poorly understood. A better understanding of innate immune pathways is expected to aid in the development of appropriate vaccines or prophylactics for these high-risk groups. Previously, the RSV nonstructural protein 1 (NS1) was shown to antagonize IFN responses by disrupting components of the innate immune system, although the mechanism is not well defined. We hypothesized that NS1 targets constituents of the PRR pathways to evade innate immunity and thus ensure viral survival. Using microscopy and co-immunoprecipitation assays, we found that NS1 localizes to the mitochondria and binds to the mitochondrially associated adaptor protein MAVS, thus preventing MAVS interaction with the RNA helicase, RIG-I. Expression of NS1 was also correlated with upstream IFN-response regulator, LGP2, and its expression was inducible in the absence of a viral infection. Tetracycline-inducible expression of recombinant NS1 in a cell model also promoted viral replication and emphasizes the key contribution of NS1 to RSV survival. Through this study, we demonstrated a mechanism for RSV NS1 in the disruption of early innate responses through mitochondrial localization and alteration of the RLH signaling. Whereas the above studies showed the importance RSV-induced innate immune pathways, whether the expression and signaling of innate immune pathways were adversely affected upon RSV infection in the high-risk groups remains unknown. Since elderly individuals are at an increased risk for severe bronchiolitis and RSV-induced pneumonia, often resulting in hospitalization and medical intervention and adaptive immune cell functionality and responsiveness reportedly decline with age, we hypothesized a similar age-related deterioration of the innate antiviral system. In this investigation, we used an aged mouse model to correlate age-associated changes in innate immune gene expression with RSV pathology. Of 84 antiviral genes examined, five genes including RIG-I, IFNAR1, TLR8, IL-1Β, and osteopontin (OPN) were associated with both age and infection. In response to RSV infection, aged mice had delayed induction of antiviral genes and diminished ability to secrete IL-6 in response to TLR7/8 agonist in primary alveolar macrophages. Lungs from aged, RSV-infected mice had increased cellular infiltration and prolonged infection as compared to young mice. In summary, age-related decline in expression and functionality of antiviral defenses were correlated with enhance RSV-induced lung disease in aged mice. In the absence of infection, aged mice chronically overproduced IL-1Β and OPN relative to young mice. Upon infection, aged mice had impaired ability to secrete higher levels of IL-1Β and mucus. In contrast, OPN secretion remained high and prolonged in aged mice throughout infection. The age-related decline in host antiviral gene induction and delayed cytokine production correlated with enhanced disease pathology. Using a transgenic strain of mice deficient in OPN (OPN-KO), we observed greater resistance to RSV and enhanced secretion of mucus, but unaltered cellular infiltration into the lungs. Therefore, OPN overproduction and defective mucus production likely contribute to pathology in aged mice. These findings demonstrate that RSV targets the innate virus recognition and antiviral cytokine activation pathways but also that the antiviral defense system is significantly affected by age. Consequently, efforts to generate vaccines or develop therapies that stimulate IFN induction may prove unsuccessful in the elderly given that RSV virulence factors and age weaken these responses. This study contributes to our understanding of how aging relates to the RSV subversion of the host antiviral response and should help with the development of better antiviral therapies suited to the growing elderly population.

Cancer represents the second leading cause of death in the United States. For many malignancies, currently available treatment options offer little long-lasting survival benefits to patients. However, recent studies have shown immunotherapeutic approaches to be an attractive strategy to cancer treatment. While many current immunotherapeutic strategies convey durable responses, such responses are only seen in a minority of patients. An increased understanding of the mechanisms governing tumor immunogenicity and the biology of immune responses is crucial to improving upon the efficacy of current and future cancer immunotherapies. Histone deacetylases (HDACs), enzymes classically associated with regulation of gene expression, have been therapeutic targets in various cancers for several years due to their involvement in cell growth. However, it has become increasingly clear that HDACs are intimately involved in regulating both the immunogenicity of tumor cells and immune response of leukocytes and lymphocytes. In order to expand upon this growing knowledge, the therapeutic efficacy of the pan-HDAC inhibitor LBH589 in the treatment of melanoma was studied. The results presented here demonstrate that LBH589 is a potent inhibitor of growth in a wide variety of melanomas through induction of cell cycle arrest and apoptosis. Additionally, LBH589 increases the immune visibility of melanoma cells by increasing expression of several immune associated cell surface markers (e.g. MHC I, MHC II, CD80, CD86) in addition to upregulating expression of melanoma differentiation antigens. Furthermore, LBH589 treatment of immune cells results in an enhanced pro-inflammatory phenotype of both APCs and T-cells. These combined effects result in better activation of T-cells and ultimately prolonged survival in LBH589 treated, melanoma-baring mice. To further the understanding of the role of individual HDACs in the T-cell response, the biology of the newest HDAC, HDAC11, was further assessed. To this end, it is shown that HDAC11 is differentially expressed in T-cell populations, and expression is rapidly decreased following activation. Utilizing an HDAC11 knockout (HDAC11KO) mouse strain, it is found that both CD4+ and CD8+ T-cells lacking HDAC11 have an enhanced type 1 effector function characterized by increased proliferation and secretion of IL-2, TNF and IFN-γ. Additionally, HDAC11KO CD8+ T-cells have increased expression of both granzyme B and perforin. HDAC11KO T-cells also demonstrate enhanced resistance to inhibition by Tregs and anergy formation. As a possible mechanism for the observed phenotype, it is also demonstrated that HDAC11KO T-cells produce elevated levels of the transcription factors Eomes and T-bet, both at the basal state and post-activation. In vivo, T-cells lacking HDAC11 have a more potent and robust ability to cause GvHD and mediate an enhanced anti-tumor response. Collectively, these results demonstrate that targeting of HDACs is a viable approach to cancer immunotherapy, and that targeting of specific HDACs may be an attractive strategy for optimizing immunotherapy efficacy while minimizing side effects.

Over the past 15 years Monteverde, Costa Rica has undergone rapid economic, social, political, and environmental change due to a flourishing ecotourism economy. While the effects of ecotourism development in Monteverde are many, two important consequences have been: 1) the immigration of Nicaraguan nationals to the area seeking low-skilled wage labor; and 2) compromised water resources management due to pollution and rapid population growth. The objective of this research is to investigate and identify the inter-relationships between ecotourism development in Monteverde and its affect on infectious diseases outcomes within the context of immigration and water resources management. Specifically, this dissertation uses both anthropological and public health methods within a political ecology of health framework to compare prevalence rates of intestinal parasites between Nicaraguan immigrants and Costa Rican residents living in Monteverde. Results indicate that Nicaraguan immigrants suffer disproportionately from infections with intestinal parasites compared to Costa Rican residents. The results further indicate that community based water resources are not a significant source of infection. Instead, the prevalence of intestinal parasites is most likely the result of fecal-oral transmission at the household level and is related to indicators such as access to health care, underemployment, home ownership, and household sanitation infrastructure.