Dendritic cells (DCs) represent a heterogeneous set of antigen presenting cells linking the innate and adaptive immune systems. DCs are referred to as professional antigen presenting cells (APCs) with regards to their ability to activate naïve T cells. In this study, the role that DCs play in the pathogenesis of Type 1 Diabetes (T1D) was initially examined. We utilized an experimental model that allowed for the generation of bone marrow derived-dendritic cells (BM-DC) subsets that exhibit either a DC type 1 (DC1) or DC2 phenotype. We define DC1 as cells capable of producing high levels of IL-12 and inducing naïve CD4+ T cell differentiation into T helper 1 (Th1) cells while DC2 are cells that produce low levels of IL-12 and induce Th2 cell differentiation. During the course of this study we also utilized several toll-like receptor (TLR) ligands to activate DC function. In addition, we tracked the migration of DCs in vivo utilizing a novel perfluoropolyether (PFPE) contrasting agent in conjunction with a modified magnetic resonance imaging (MRI) instrument. With this methodology we tested the hypothesis that NOD DCs were predisposed to adopt a DC1 phenotype that contributed to the pathogenesis of T1D. The results from our experiments show that NOD BM-DCs induced into a DC1 or DC2 functional phenotype exhibit no significant intrinsic differences in their production of IL-10 or IL-12, in comparison to wild type mice. Additionally, NOD BM-DCs expressed similar levels of CD80, CD86, CD40, and MHC class II in comparison to wild type BM-DC. Our analysis did show that the production by BM-DCs of IL-10, induced by CpG ODN, is negatively regulated by IFN-gamma. The suppressed production of IL-10 may have implications for the development of T helper cell type 1 (Th1) and Th2 cells. Altogether our study has shown that, 1) NOD BM-DCs are not intrinsically skewed towards a DC1 phenotype, 2) IFN-gamma negatively regulates the production of IL-10 in BM-DCs, and 3) NOD BM-DCs can be successfully imaged in vivo using a novel PFPE labeling/MRI detection technique.

In the first project we demonstrate that IFNgamma alone can block HSV-1 reactivation in some latently infected neurons, and we identify points of intervention in the life cycle of the reactivating virus. Cell suspensions of TGs that were latently infected with recombinant HSV-1 expressing EGFP from the promoter for ICP0 or gC were depleted of endogenous CD8+ or CD45+ cells and cultured with or without IFNgamma. IFNgamma treatment reduced: (i) HSV-1 reactivation; and (ii) the number of neurons that express the ICP0 or gC promoter in ex vivo TG cultures. Moreover, those neurons that expressed the ICP0 or gC promoters in the presence of IFNgamma showed a reduced reactivation. Interestingly, we detected transcripts for ICP0, ICP4, and gH in neurons that expressed the ICP0 promoter in the presence of IFNgamma, but were prevented from fully reactivating. Thus, the IFNgamma blockade of HSV-1 reactivation from latency occurs at prior to expression of the ICP0 gene (required for reactivation) as well as at a very late stage in reactivation following expression of at least some structural genes. A second project tested the hypothesis that IFNgamma regulates the observed expansion, contraction, and homeostasis/memory phases of the HSV-1-specific CD8+ T cell response within the latently infected TG. We addressed this hypothesis by observing the effect of in vivo IFNgamma neutralization during the three phases of the response. IFNgamma neutralization significantly reduced the expansion phase through a 50% reduction in proliferation of HSV-1-specific CD8eff cells. IFNgamma neutralization also significantly accelerated the contraction phase through an as yet undefined mechanism. In contrast, IFNgamma neutralization did not affect the homeostasis/memory phase. A novel finding of these studies was that an HSV-1-specific CD8+ memory precursor population (CD8mp, defined by expression of CD127) exhibited a delayed expansion and contraction relative to the overall CD8eff population. We are currently determining if the disruption of the expansion and contraction of CD8mp through IFNgamma neutralization will influence the functional avidity of the CD8mem population that ultimately develops. Since HSV-1-specific CD8mem appear to play an important role in regulating HSV-1 latency, optimizing this population could have important implications for controlling recurrent herpetic disease.

Studies demonstrating the ability of in vitro generated dendritic cells (DCs) to successfully mediate anti-tumor efficacy when used as therapeutic vaccines suggest that treatments capable of promoting in situ DC-mediated cross-priming events may exhibit at least a comparable degree of clinical effectiveness. As a result, I assessed whether optimizing the number of DCs within the tumor microenvironment would improve the cross-priming of tumor-reactive T cells, resulting in improved therapeutic benefit. I observed that the treatment of CMS4-bearing BALB/c mice with the combination of Flt3 ligand (FL) and GM-CSF for five sequential days is sufficient to optimize the number of tumor-infiltrating DCs and to result in the enhanced systemic priming of tumor-specific CD8+ T cells that are consequently recruited into the tumor microenvironment. Despite these preferred immunologic endpoints, combinational FL and GM-CSF treatment failed to impact the growth of established CMS4, RENCA or CT26 tumors. The observation that large numbers of CD4+ T cells also infiltrate tumors in mice treated with combinations of FL and GM-CSF prompted me to explore whether CD4+ regulatory T cells might play an active role in mediating the suppression of co-infiltrating tumor-specific CD8+ T cells. Indeed, I found that nearly half of the tumor-associated CD4+ T cells expressed the Foxp3 protein and significantly suppressed the proliferation of naïve allo-reactive CD4+ T cells and the IFN- production by tumor-specific CD8+ T cells in vitro. Moreover, I found that combinational FL and GM-CSF treatment induced significant expansion of Foxp3+CD4+ T cells in the spleens of treated animals, regardless of tumor-bearing status. Consistent with the suppressive effects of tumor-associated CD4+ T cells on combined FL and GM-CSF-based therapy, the in vivo depletion of CD4+ T cells resulted in a marked inhibition of tumor growth in treated mice that was dependent on the presence of CD8+ T cells. My findings have important implications in cancer therapy as they demonstrate that suppression mediated by regulatory T cells can present a major roadblock for the successful implementation of immunotherapeutic approaches to treat cancers.

The immunostimulatory outcome of the interactions of many pathogens with dendritic cells (DC) has been well characterized. There are many fewer examples of similar interactions between DC and self-molecules, especially the abnormal self-proteins such as many tumor antigens, and their effects on DC function and the immune response. We show that human epithelial cell antigen MUC1 mucin, is recognized in its aberrantly glycosylated form on tumor cells by immature human myeloid DC as both a chemoattractant (through its polypeptide core) and a maturation and activation signal (through its carbohydrate moieties). Interestingly, MUC1 is chemotactic for immature DC but not for other cells of the immune system. Not only is this the first example of a tumor antigen that has chemotactic abilities but there are also no known receptors that are expressed uniquely on immature DC. However, we have determined that the MUC1 chemotactic receptor is a member of the chemokine receptor family based on its sensitivity to pertussis toxin, the formation of pseudopods upon ligand binding, and the Ca2+ flux in response to MUC1. Upon encounter with MUC1, similar to the encounter with LPS, immature DC increase cell surface expression of CD80, CD86, CD40 and CD83 molecules and the production of IL-6 and TNF-Ñncytokines, but fail to make IL-12. When these DC are co-cultured with allogeneic CD4+ T cells, they induce production of IL-13 and IL-5 and lower levels of IL-2, thus failing to induce a type 1 response. Our data suggest that in cancer patients in vivo, MUC1 attracts immature DC to the tumor through chemotaxis and then subverts their function by negatively affecting their ability to stimulate type 1 helper T cell responses important for tumor rejection.

Eukaryotic pre-mRNA is processed within the 3-untranslated region (3-UTR) resulting in cleavage and polyadenylation. The efficiency of the cleavage reaction is dependent on the binding activity of the 64-kDa subunit of CstF, CstF-64, to the pre-mRNA and is increased with elevated levels of CstF-64. There is evidence that alternative polyadenylation occurs in the presence of increased CstF-64. Our results showed that CstF-64 levels increased with LPS stimulation of RAW 264.7 macrophages while the expression of other pre-mRNA processing factors remained unchanged. Because of the evidence that several macrophage genes exhibit alternative polyadenylation and post-transcriptional regulation under LPS stimulation, we used a reporter mini-gene to identify alternative polyadenylation in LPS-stimulated RAW macrophages. Upon LPS stimulation we measured a 2.5-fold increase in proximal poly(A) site selection that correlated with elevated levels of CstF-64. Forced expression of CstF-64 demonstrated similar alternative polyadenylation. Microarray analysis demonstrated 515 genes changed expression with LPS stimulation, 15 of which also changed with CstF-64 over-expression. A closer analysis of 5 of these 15 genes demonstrated alternative polyadenylation within their 3-UTR. Closer analysis of the 3-UTRs showed putative AU-rich regulatory elements. There is also evidence that pre-mRNA processing is coupled with transcription. Previous work has shown that the carboxy-terminal domain (CTD) of RNAP-II is necessary for 3-end processing, that CstF binds to RNAP-II CTD and that this binding is CTD phosphorylation dependent. Because our lab has also shown that increases in CstF-64 binding activity upon B-cell differentiation causes alternative polyadenylation on the Ig heavy chain gene and occurs in the absence of CstF-64 increases, we believe that the local concentration of CstF-64 to the nascent pre-mRNA is increased in plasma cells through the phosphorylation-dependent recruitment of CstF-64 to RNAP-II CTD. Using chromatin immunoprecipitation (ChIP), we measured an increase in Serine-2 and Serine-5 phosphorylation of the RNAP-II CTD at the promoter and variable regions of the Ig heavy chain gene in plasma cells compared to memory B cells. We believe that this increase in RNAP-II CTD phosphorylation plays a role in either increased transcription of the Ig heavy chain gene or recruitment of pre-mRNA processing factors to the transcriptional machinery.

Abstract Dendritic cells (DCs) are professional antigen-presenting cells that are integral to the induction of primary, antigen-specific T cell responses. In the cancer setting, DCs mediate cross-priming of tumor-reactive T cells by presenting tumor antigens acquired from viable or dead cancer cells. Due to their unique functional properties, DCs have been utilized as both vectors and targets for immunological intervention in numerous diseases and are optimal candidates for vaccination protocols in cancer. In addition to their antigen presentation function(s), recent evidence suggests that DCs may also perform an innate immune effector function, with human DCs reported to mediate direct tumoricidal activity in vitro. However, the mechanism(s) by which DCs directly kill tumor cells remain unclear. The goal of this study is to further characterize the mechanism(s) associated with murine DC tumoricidal function and to determine whether and how this function may be enhanced to promote anti-tumor immune responses that translate into therapeutic effectiveness. One way we sought to enhance this DC effector function was through the genetic engineering of DCs themselves. After transduction with mIL-12 and/or mIL-18 cDNA using recombinant adenoviral vectors, DCs exhibited significantly elevated tumor killing activity. This was mediated, at least in part, by TNF ligand-receptor complexes, as demonstrated by antibody blocking assays. When injected in situ, these engineered DCs exhibited prolonged survival, in association with enhanced levels of tumor apoptosis proximal to imaged DCs and our capacity to image DC that had engulfed tumor apoptotic bodies. We also observed notable therapeutic benefits upon intratumoral delivery of these DCs in concert with an expanded in vivo repertoire of anti-tumor CD8+ T cells. In addition to DC modification, we also evaluated treatments applied to tumor cells that resulted in enhanced sensitivity to (control) DC-mediated killing. Specifically, we found that pretreatment of A20 lymphoma cells with a nitric oxide (NO) donor compound, PAPA-NO, markedly increased the sensitivity of tumor cells to consequent apoptosis mediated by DCs. This appeared to provide DCs with a preferred source of tumor antigens, with which, they were capable of activating specific T cells via a cross-presentation pathway. We have also discovered that multiple TNF family ligands participated in DC-mediated tumoricidal function and that tumor cell-expressed survivin may represent a critical downstream factor regulating the apoptotic sensitivity of tumor cells to DC-mediated apoptosis. When taken together, these studies provide novel details regarding mechanisms involved in DC anti-tumor effector function, and suggest two DC-based, combinational cancer therapies that target the effective cross-priming of therapeutic T cells. The results presented in this dissertation support an efficient model in which DCs may not only serve as the gatherers and presenters of antigens, but also the hunters as well, with tumoricidal activity mediated via TNF family ligands.

MUC1 is a large glycoprotein that is expressed on ductal epithelial cells and also the majority of epithelial adenocarcinomas. Dysregulated expression of aberrantly glycoslyated MUC1 in carcinomas allows tumor-specific recognition of MUC1-derived epitopes by antibodies and T lymphocytes. However, despite the ability of CTL to specifically recognize and kill MUC1+ tumor cells, immune responses in cancer patients fail to prevent tumor progression. The failure of patients immune systems to eradicate MUC1+ tumors has been linked with their inability to mount MUC1-specific helper T cell responses. Here we show that CD4+ T cells play a central role in both the enhancement and suppression of MUC1-specific immune responses. Using MUC1-Tg mice as a model for tolerance to self-expressed MUC1, we show that MUC1-specific regulatory T cells (Tregs) respond to stimulation with MUC1 in the absence of a CD4+ T helper response. The Treg:Th imbalance in MUC1-Tg mice causes the suppression of MUC1-specific immunity. This suppression can be overcome by providing functional Th cells from WT mice. To focus our studies on MUC1-specific CD4+ T cells, we created a TCR-transgenic mouse whose CD4+ T cells are specific for an MHC Class II-restricted epitope derived from unglycosylated MUC1. Using these mice, we have confirmed that adoptive transfer of MUC1-specific CD4+ T replaces the MUC1-specific T cell help that is missing in MUC1-Tg mice and restores their ability to respond to MUC1 vaccines. This work shows that the generation of CD4 helper T cell responses is critical to establishing effective immunity to MUC1+ cancers.

The influence of ethanol (EtOH) on multiple dendritic cell (DC) subsets, either in steady state or following mobilization in vivo, has not been characterized. Herein, the generation of mouse bone marrow (BM)-derived DC in fms-like tyrosine kinase 3 ligand was inhibited by physiologically-relevant concentrations of EtOH, with selective suppression of plasmacytoid (p)DC. EtOH reduced surface expression of costimulatory (CD40, CD80, CD86) but not coinhibitory CD274 (B7-H1) molecules on resting or CpG-stimulated DC subsets. IL-12p70 production by activated DC was impaired. Consistent with these findings, EtOH-exposed (E)BMDC exhibited reduced capacity to induce naïve allogeneic T cell proliferation and impaired ability to prime T cells in vivo. Further, T cells from animals primed with EBMDC produced elevated levels of IL-10 following ex vivo challenge with donor alloantigen. DC subsets freshly-isolated from EtOH-fed mice were also examined. Liver DC, inherently immature and resistant to maturation, exhibited little change in low surface cosignaling molecule expression, whereas splenic DC showed reduced expression of cosignaling molecules in response to CpG stimulation. These splenic DC elicited reduced naïve allogeneic T cell proliferation in vitro, while the stimulatory capacity of resting but not CpG-activated liver DC was reduced by EtOH administration. In vivo, hepatic EDC elicited increased capacity to prime T cells compared to control hepatic DC. Conversely, splenic EDC exhibited impaired ability to prime T cells in vivo. This differential capacity of hepatic versus splenic EDC compared to control DC to prime T cells in vivo is likely due to several factors including differential phenotype and migratory capacity. In fact, liver EDC migrate in greater numbers to secondary lymphoid tissue compared to control liver DC. Thus, EtOH impairs cytokine-driven differentiation and function of mDC and pDC in vitro. Hepatic DC from chronic EtOH-fed mice are differentially affected compared to splenic DC. Splenic DC exhibit impaired functional maturation following CpG stimulation while hepatic DC exhibit altered migration to secondary lymphoid tissue. In addition to examining the effects of chronic EtOH exposure on DC, we have evaluated cell-mediated and humoral responses in EtOH-fed mice. In total, these results indicate potential mechanisms by which alcohol consumption is associated with immunosuppression.

The immediate challenge of transplant immunology is to induce donor-specific immune tolerance to improve graft outcome and to eliminate the need for dependency on immunosuppressive therapy. Cell-based therapy employing dendritic cells (DC) for induction of transplant tolerance has been researched intensely due to their critical roles in controlling and regulating immunity. As the most potent antigen (Ag)-presenting cells (APC), DC are well-equipped to capture, process and present Ags, and their unique migratory behavior in vivo is central to the direct and indirect allorecognition pathways of transplantation. This dissertation examined two approaches to exploiting the inherent plasticity of DC with respect to their migration and function to regulate immune responses and enhance allograft survival. First, FTY720 is a novel immunomodulator pro-drug known to cause blood lymphopenia. Its active metabolite, FTY720-phosphate (FTY720-P) is a receptor agonist of sphingosine 1-phosphate (S1P) that regulates cell growth, differentiation, and migration. Our hypothesis was that FTY720 would modulate DC trafficking and function, an effect that could contribute to FTY720-induced immunosuppression. Herein, we show that FTY720 retained DC in the circulation, and concomitantly reduced their number in lymph nodes and spleen, by down-regulating surface intercellular adhesion molecule and homing receptor expression on DC, likely via the S1P1 signaling pathway. Second, we investigated the immunoregulatory capacity of alternatively-activated (AA) DC and their therapeutic efficacy in a fully MHC-mismatched cardiac allograft model. We hypothesized that AADC, generated in an immunosuppressive milieu and then ¡¥activated¡¦ via Toll-like receptor (TLR) ligation, could exhibit tolerogenic properties and offer potential as therapeutic agents to promote long-term vascularized organ allograft survival. We demonstrated that the anti-inflammatory cytokines, IL-10 and TGF-Β, impacted significantly on DC maturation in response to the TLR4 ligand LPS and impaired their ability to induce T cell proliferation. AADC prolonged allograft survival by induction of a tolerogenic cytokine environment and expansion/proliferation of CD4+ regulatory T (Treg) cells, an effect that was markedly potentiated by blockade of the B7-CD28 costimulation pathway. These novel data provide new insights into manipulation of DC migration and function for regulation of alloimmune reactivity and promotion of transplant tolerance by control of S1PR, cytokine, and TLR signaling.

<p>Macrophages play vital roles in pathogen clearance, initiation of immune responses, and maintenance of immune homeostasis; however, current understanding of the contributions of macrophages to these processes <italic>in vivo</italic> has been limited by the reagents and animal models available. Of the many macrophage-deficient mouse models that have been reported, none have specific, long-term loss of distinct macrophage populations, and most develop infections that complicate the study of immune homeostasis. By conditionally deleting the anti-apoptotic protein cellular FLICE-like inhibitory protein (c-FLIP) in myeloid cells, we have generated a novel mouse model that has proven useful in studying both the <italic>in vivo</italic> functions of macrophages under steady-state conditions and the requirements for macrophage survival at steady-state and during inflammation.</p><p>c-FLIP<super>f/f</super> Lysm-Cre mice specifically lack bone marrow macrophages and splenic marginal zone macrophages and develop severe neutrophilia, splenomegaly, extramedullary hematopoiesis, decreased body weight, and increased production of G-CSF and IL-1&#946;, but not IL-17 secondary to the loss of these macrophage populations. c-FLIP<super>f/f</super> Lysm-Cre mice exhibit delayed clearance of circulating neutrophils, suggesting that failure of macrophages to efficiently clear apoptotic neutrophils causes production of cytokines that drive excess granulopoiesis. Further, blocking G-CSF, but not IL-1R signaling in vivo rescues this neutrophilia, suggesting that a G-CSF-dependent, IL-1&#946;-independent pathway plays a role in promoting neutrophil production in mice with defective clearance of apoptotic cells.</p><p>Furthermore, using mice expressing only one c-FLIP isoform in myeloid cells (c-FLIP_S or c-FLIP_L), we have shown that although either isoform is sufficient to promote survival of macrophages under steady-state conditions, both c-FLIP_S and c-FLIP_L required for macrophage survival during inflammation. In contrast, c-FLIP_L is sufficient to promote survival of eosinophils in inflammatory conditions in the absence of c-FLIP_S. These data demonstrate distinct requirements for myeloid cell survival in the presence and absence of inflammation and point to a mechanism by which pathogenic organisms may target macrophages to evade the immune response.</p><p>Together, these findings demonstrate a critical role for c-FLIP in promoting macrophage survival, which is in turn required for neutrophil homeostasis, and provide an <italic>in vivo</italic> model system for continuing studies of the non-redundant functions of c-FLIP_S and c-FLIP_L in myeloid cells during infection and inflammation.</p> / Dissertation