Dendritic cells (DCs) function mainly as antigen presenting cells (APCs) and as such they play a significant role in activating the adaptive immune system. Dendritic cells express toll-like receptors (TLR), and when these receptors are engaged by their cognate agonists, they promote DC maturation, which is critical in the induction of potent T helper (Th) cell -1 responses. Due to the multifunctional abilities of DCs, they have been explored as vaccine carriers, largely in cancer immunotherapy and some infectious diseases including hepatitis C. Previous studies showed that DCs loaded with mRNA of hepatitis C virus (HCV) antigen(s) induced strong immune responses but immune protection was not complete. Therefore, I expected that adoptive transfer of DCs transfected with HCV NS3/4A and/or NS5A mRNA and further treated with TLR agonist(s) ex vivo would induce HCV-specific immunity in vivo. Bone marrow-derived DCs generated with Flt3L (FL-DCs) or GM-CSF (GM-DCs), and loaded with HCV NS3/4A and/or NS5A mRNA showed maturation characteristics and produced substantial amounts of IL-12 after ex vivo activation with CpG ODN or CpG ODN plus Poly I:C, when compared to their untreated counterparts. Treatment with a combination of CpG ODN and Poly I:C synergized to augment IL-12 production in comparison with stimulation with CpG ODN alone. IL-12 secretion by DCs is pivotal in directing immune responses towards a Th1-bias response, which is needed to eliminate HCV. However, the ex vivo responses of stimulated DCs bearing HCV antigen(s) were not efficiently translated in mice to potentiate vigorous antigen-specific T cell responses. This resulted in a lack of protection after challenge with recombinant vaccinia virus expressing HCV NS3/NS4/NS5 in immunized mice. In contrast, both antigen-specific humoral and cell-mediated immune responses were induced in mice vaccinated with HCV recombinant NS3 or NS5A protein co-formulated with CpG ODN, host defense peptide and polyphosphazene. These responses, however, did not mediate viral clearance, as vaccinated mice remained unprotected from infection with recombinant vaccinia virus expressing HCV antigens. Taken together, these results suggest HCV recombinant protein co-formulated with triple adjuvant to be a better vaccine candidate than the DC-based vaccine.

Improvements in the use of traditional breast cancer therapies have improved the overall survival of women with early stage disease. Remarkable advances in research have created a unique opportunity for developing active vaccination strategies that engage the bodys own immune system in the fight against breast cancer. Human Epidermal Growth Factor Receptor 2 (HER-2/neu) is a breast tumor antigen (Ag) commonly overexpressed in 30% of breast cancer cases. HER-2/neu-targeted DNA-based and fiber-modified dendritic cell (DC)-based vaccines are both analyzed as potent elements in eliciting HER-2/neu specific antitumor immune responses. A HER-2/neu-expressing DNA plasmid (pcDNA/neu) coadministered with the appropriate adjuvant vector was the first study looking at improving vaccine efficacy and enhancing immune responses. Various protection and prevention studies, using FVB/N (wild-type) and FVB/neuN [transgenic (Tg)] mice and Tg1-1 tumor cells, derived from a spontaneous tumor from Tg mice, are used to help narrow down the large panel of adjuvant vectors. Results showed the adjuvant vector pcDNA/TNF-α, when coadministered with pcDNA/neu, induced more efficient protective tumor-specific immunity and significantly delayed breast cancer development in Tg mice.<p>Another study utilized an<i>in vivo</i> murine tumor model expressing the rat neu Ag to compare the immunization efficacy between DC transduced with replication-deficient fiber-modified adenovirus (AdV) containing neu (AdV(RGD)neu), to form DC(RGD)neu, and non-modified DCneu. DC(RGD)neu displayed an upregulation of immunologically important molecules and inflammatory cytokine expression through FACS Analysis, and more importantly increased expression of neu, when compared to DCneu. DC(RGD)neu stimulated a higher percentage of HER-2/neu-specific CD8+ T cells, a stronger neu-specific CTL response, and induced a much stronger Th1- and Th2-type immune response than DCneu. Furthermore, vaccination with DC(RGD)neu induced enhanced protective tumor-specific immunity compared to DCneu in wild-type and Tg mice.<p>Overall the construction of recombinant vectors containing two transgenes (HER-2/neu and TNF-α), can not overcome the induction of HER-2/neu-directed immune tolerance. The fiber-modified (RGD) DCneu vaccine induced enhanced anti-HER-2/neu immunity compared to non-modified DCneu in the prevention of breast cancers.

We report herein that IL-10-treated dendritic cells (DC) can be used effectively to reverse established severe asthma-like disease in a mouse model. Our lab had shown previously that allergen-presenting splenic CD8¦Á+ DCs could ¡Ö50% reduce airway hyper responsiveness (AHR), eosinophilia, and Th2 responses in asthma-phenotype mice, but only marginally reduce IgE/IgG1 levels. We now show that bone marrow-derived DCs that have been differentiated in the presence of IL-10 (DCIL-10) are effective in reversing the asthma phenotype. Co-culture of DCIL-10 with T memory (TM) cells from asthma-phenotype mice was associated with lack of Th2 responses, and this was partially reversed by IL-2. Immunostimulatory DC activated these Th2 cells. <i>In vivo</i>, delivery of allergen-pulsed DCIL-10, either into the airway or intraperitoneally abrogated AHR from weeks 3-10 post-treatment, and ameliorated lung eosinophilia and Th2 (IL-4, -5, -9, & -13, IgE) responses, as well as circulating allergen-specific IgE responses for at least 32 weeks following treatment. Repeated OVADCIL-10 treatments kept AHR normalized for 8 weeks as well as Th2 responses significantly low. In vivo, delivery of anti-IL-10R, but not anti-TGF-¦Â from day 12-21 after treatment had moderate effects on DCIL-10-driven tolerance, but 1-methyl tryptophan (inhibitor of indoleamine-2,3-dioxygenase) treatment had significant effects on Th2 responses. The mechanisms mediating tolerance in vivo are likely complex, but we speculate that infectious tolerance sustains this regulatory activity during the 32-week period in which we have observed tolerance to be in place.

<p><p>The receptor-recognized form of &alpha;₂-macroglobulin (&alpha;₂M*) targets antigens (Ag) to professional Ag-presenting cells (APCs) for rapid internalization, processing, and presentation. When employed as an Ag delivery vehicle, &alpha;₂M* amplifies major histocompatibility complex (MHC) class II presentation as demonstrated by increased antibody (Ab) titers. Recent evidence, however, suggests that &alpha;₂M*-encapsulation may also enhance Ag-specific cytotoxic T lymphocyte (CTL) immunity. In these studies, we demonstrate that &alpha;₂M*-delivered Ag (ovalbumin, OVA) enhances the production of specific <italic>in vitro</italic> and <italic>in vivo</italic> CTL responses. <br><p>Murine splenocytes expressing a transgenic T cell receptor (TCR) specific for CTL peptide OVA_257-264 (SIINFEKL) demonstrated up to 25-fold greater IFN-&gamma; and IL-2 secretion when treated <italic>in vitro</italic> with &alpha;₂M*-OVA compared to soluble OVA. The frequency of IFN-&gamma; -producing cells was increased ~15-fold as measured by ELISPOT. Expansion of the OVA-specific CD8<super>+</super> T cells, as assayed by tetramer binding and [<super>3</super>H]thymidine incorporation, and cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also significantly enhanced by &alpha;₂M*-OVA. Furthermore, CTL responses were observed at Ag doses tenfold lower than those required with OVA alone. <br><p>We also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with &alpha;₂M*-OVA. These &alpha;₂M*-OVA-immunized mice displayed increased protection against a subcutaneously implanted OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The anti-tumor response observed with &alpha;₂M*-mediated Ag delivery was comparable to that of an accepted vaccine adjuvant (CpG 1826) and appeared superior to a cell-based vaccine technique. <br><p>To further understand the mechanism underlying this enhanced CTL immunity, the subsets of professional APCs capable of cross-presenting &alpha;₂M*-encapsulated Ag were investigated. Although both dendritic cells (DCs) and macrophages appear to stimulate some degree of cross-priming in response to &alpha;₂M*-encapsulated Ag, CD8<super>+</super>CD4<super>-</super> and CD8<super>-</super>CD4<super>+</super> DCs appear to do so with the greatest efficiency. The implications of this finding to the ongoing debate regarding the relative contributions of APC subsets to Ag cross-presentation and the determinants of which cells cross-present with high efficiency are discussed. <br><p>These observations demonstrate that &alpha;₂M*-mediated Ag delivery promotes cross-presentation resulting in enhanced Ag-specific CTL immunity. Considered in the context of previous work, these results support &alpha;₂M* as an effective Ag delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.</p> / Dissertation

<p>Orthopoxviruses encode many genes that are not essential for viral replication, which often account for differences in pathogenesis among otherwise closely related orthopoxviruses. Although dendritic cells (DCs) are essential to the generation of an effective anti-viral immune response, the effects of different orthopoxviruses on DC function is poorly understood. The objective of these studies was to determine the effect of different orthopoxviruses on DCs. Cowpox virus (CPXV) is ideally suited to this purpose because it encodes the largest and most representative set of accessory genes among orthopoxviruses, it is endemic in mouse populations, and can infect humans. </p><p>We hypothesized that CPXV would have novel mechanisms of evading the immune response that other orthopoxviruses lack, which may exert maximal effect in the context of antigen presenting cells such as DCs, allowing for discovery of novel viral strategies of immune evasion. To test this, CPXV was used to infected mouse bone marrow-derived DCs (BMDCs), and the effect of the virus on DC survival, expression of T-cell costimulatory molecules and cytokine production was determined. The effects of vaccinia virus strain Western Reserve (VV), the prototype of the species, and modified vaccinia virus strain Ankara (MVA), a promising vaccine vector, on mouse BMDCs were also determined. Confirming the hypothesis that CPXV would have different effects on mouse BMDCs from other orthopoxviruses, BMDCs infected with CPXV survived longer in culture than those infected with MVA or VV. In addition, CPXV specifically downregulated MHC I, MHC II, CD40, and CD86, and induced production of significant levels of IL-6 and IL-10.</p><p>Because IL-10 has many suppressive effects on the immune system, inducing IL-10 may provide a selective advantage to CPXV in vivo. To examine the role of IL-10 in a CPXV infection, wild type and IL-10 deficient mice were infected intranasally with CPXV. The effect of CPXV infection on disease morbidity, viral loads, inflammation and the protective immune response was determined. As expected, IL-10 was important in controlling inflammation during CPXV infection, but there was no effect on viral replication or clearance. Surprisingly, IL-10 was important in generation of a protective memory response to CPXV, which may reflect a novel role for IL-10 in the immune response.</p> / Dissertation

<p>Monocytes and monocyte-derived cells are important in providing innate immunity against various pathogens. Monocytes become macrophages or dendritic cells after they enter tissues during inflammation. Macrophages phagocytose microbes and kill them intracellularly in lysosomes. After macrophages are activated, they secret a variety of cytokines as part of innate defense. However, such cytokines have been implicated in causing autoimmune diseases and influenza-induced pathology. For these reasons, we have investigated the role of monocytes and monocyte-derived cells in inducing immune pathology. Moreover, monocytes are also thought to affect adaptive immunity by shaping T cell responses. Yet the enterity of their contributions to adaptive immune response remains to be determined. </p><p>CCR2 is the chemokine receptor required for inflammatory monocytes to enter tissues, and its deficiency in mice has been shown to be protective for influenza-induced immune pathology. We hypothesized that cells that depend on CCR2 to migrate into inflammaed lungs are the cells that induce immune pathology during influenza infection. First, we identified cell types that are recruited to the lungs by CCL2. Similar myeloid cell types, monocytes, monocyte-derived DCs (moDCs), and exudated macrophages (exMAC), also accumulate in the lungs during influenza infection. We then show that these myeloid cells types are derived from monocytes, and that they produce high levels of TNF-&#945; and NOS2. Finally, we show a strong correlation between reduced accumulation of myeloid cells and decreased influenza-induced pathology and mortality in CCR2-deficient mice, suggesting that CCR2 inhibition may be a viable therapy for highly pathogenic influenza infection.</p><p>In the second part of this work, we focus on monocyte-derived dendritic cells in lymph nodes (LN). Inflammatory DCs in LN can arise from moDCs recruited via lymphatics (peripheral moDCs) and from inflammatory monocytes that enter LN directly from the blood (blood-derived moDCs). We examine the role of blood-derived moDCs in inducing LN T cell activation and polarization after immunogenic stimuli. We find that, following viral infection or immunization, inflammatory monocytes are recruited into LN directly from the blood to become CD11c<super>+<super>CD11b<super>hi<super>Gr-1<super>+<super> inflammatory DCs, which produce high levels of IL-12 (p70) and potently stimulate Th1 responses. This monocyte extravasation requires CCR2 but not CCL2 or CCR7. Thus, inflammatory DCs accumulation and Th1 responses are markedly reduced in CCR2<super>-/-<super> mice, preserved in CCL2<super>-/-<super> mice, and relatively increased in CCL19/21-Ser-deficient <italic>plt<italic> mice, in which all other LN DC types are reduced. </p><p>Our findings provide important insights into mutiple roles that monocytes play in both innate and adaptive immunity. Monocytes provide an early response against pathogens. As we now demonstrate, this response can be excessive, leading to a significant immune pathology during influenza infection that has been previously attributed to neutrophils. We also provide the first demonstration that monocytes play an important role in regulating adaptive immune responses. We find that monocyte-derived DCs are both sufficient and necessary for the development of Th1-polarized immune responses within LNs. Taken together, our results demonstrate that the roles played by monocytes in innate immunity adaptive immunity, and immune pathology are much greater than previously appreciated and that regulating monocyte function may be an effective means to regulate certain types of immune responses.</p> / Dissertation

<p>The immune system is constantly faced with the daunting task of protecting the host from a large number of ever-evolving pathogens. In vertebrates, the immune response results from the interplay of two cellular systems: the innate immunity and the adaptive immunity. In the past decades, dendritic cells have emerged as major players in the modulation of the immune response, being one of the primary links between these two branches of the immune system.</p><p>Dendritic cells are pathogen-sensing cells that alert the rest of the immune system of the presence of infection. The signals sent by dendritic cells result in the recruitment of the appropriate cell types and molecules required for effectively clearing the infection. A question of utmost importance in our understanding of the immune response and our ability to manipulate it in the development of vaccines and therapies is: "How do dendritic cells translate the various cues they perceive from the environment into different signals that specifically activate the appropriate parts of the immune system that result in an immune response streamlined to clear the given pathogen?"</p><p>Here we have developed computational and statistical methods aimed to address specific aspects of this question. In particular, understanding how dendritic cells ultimately modulate the immune response requires an understanding of the subtleties of their maturation process in response to different environmental signals. Hence, the first part of this dissertation focuses on elucidating the changes in the transcriptional</p><p>program of dendritic cells in response to the detection of two common pathogen- associated molecules, LPS and CpG. We have developed a method based on Langevin and Dirichlet processes to model and cluster gene expression temporal data, and have used it to identify, on a large scale, genes that present unique and common transcriptional behaviors in response to these two stimuli. Additionally, we have also investigated a different, but related, aspect of dendritic cell modulation of the adaptive immune response. In the second part of this dissertation, we present a method to predict peptides that will bind to MHC molecules, a requirement for the activation of pathogen-specific T cells. Together, these studies contribute to the elucidation of important aspects of dendritic cell biology.</p> / Dissertation

<p>Pin1 is a ubiquitously expressed phosphorylation-specific prolyl isomerase that regulates substrate function by catalyzing the cis-trans isomerization of prolyl bonds. Through this modulation, Pin1 has been shown to influence the stability, localization, and/or activity of a diverse set of protein substrates that participate in a variety of cellular responses, such as cell cycle progression, modulation of cell stress, and apoptosis. In addition to extensive studies in non-hematopoietic cells, Pin1 has also been shown to regulate immune cell function. Indeed, Pin1 participates in germinal center B cell development and eosinophil granulocyte survival. It also facilitates cytokine production in T cells, eosinophil granulocytes, and plasmacytoid dendritic cells. Through specific activities such as these, Pin1 has been demonstrated to modulate responses to viral challenge, respiratory allergens, and organ transplantation. </p><p>Due to previously described functions of Pin1 in regulating cells of both the innate and adaptive immune system, we predicted that Pin1 would participate in systemic inflammatory responses. Upon inducing systemic inflammation in mice, we observed a profound reduction in circulating cytokine concentrations in Pin1-null mice compared to WT mice. This result prompted further investigations, which are described in chapter 3 and chapter 4 of this dissertation. In chapter 3, we evaluate the potential contribution of macrophages to the defects we observe in LPS-challenged Pin1-null mice. Using primary macrophages, bone marrow-derived macrophages, and MEF, we ultimately exclude a role for Pin1 in modulating LPS-induced production of pro-inflammatory cytokines in these cells. In chapter 4, we uncover a defect in the accumulation of conventional dendritic cells (cDC) in LPS-challenged Pin1-null mice. Upon more careful examination of spleen cDC subsets in Pin1-null mice, we discovered a defect in the CD8+ subset. Experiments described in this chapter collectively indicate a role for Pin1 in preferentially modulating late stages of development of the CD8+ subset of cDC. Consistent with such a defect, the expansion of adoptively transferred WT CD8+ T cells was less robust in Pin1-null mice than WT mice upon infection with the bacterium Listeria monocytogenes . At the end of chapter 4, we provide evidence that Pin1 facilitates the degradation of the hematopoietic transcription factor PU.1, and propose that deregulation of PU.1 expression may be one mechanism by which Pin1 modulates CD8+ cDC development. The work described in this dissertation began by evaluating a potential role for Pin1 in modulating pro-inflammatory cytokine production in macrophages; ultimately, however, we uncovered a novel role for Pin1 in preferentially modulating the development of the CD8+ subset of cDC. The results presented herein expand the current understanding of DC development and further implicate Pin1 as an important modulator of both innate and adaptive immune responses.</p> / Dissertation

We apply a molecular dynamics (MD) simulation technique to complex molecules at interfaces. Partitioning of dendritic surfactants into clay gallery and Ab protein behavior near hydrated lipids are chosen for the purpose. Using a full atomistic model of dendritic surfactants, the confinement force profiles featuring oscillatory fashion at moderate layer separation of 10 to 25 Å were observed. Integration of the confinement forces led to free energy profiles, which, in turn, were used to determine the final morphology of the nanocomposite. From the free energy profiles, smaller and linear surfactants (G1 and G2L) are expected to intercalate into the clay comfortably, while larger surfactants (G2 and G3) are expected to form frustrated intercalated structures due to the location and depth of the free energy minima. This would agree with the previous observations. As primary steps to understand the Ab protein behavior under biological conditions, simulations of bulk water and hydrated lipids were performed and the results were compared with the literature. Hydrated lipids were simulated using a full atomistic model of lipids (dipalmitoylphosphatidylcholine) and water with a cvff force-field and it was found that structural properties such as the molecular head group area and membrane thickness were accurately produced with MD simulation. Systems of the protein Ab(1-42) in bulk water were simulated and some secondary structural change, with loss of part of the a-helical structure, occurred during the 1 ns of simulation time at 323K. The fragment Ab(31-42) with b-sheet conformation was also simulated in bulk water, and the extended b-sheet structure became a bent structure. Simulations of Ab(1- 42) or Ab(31-42) near lipid bilayers have been performed to investigate the structural property changes under biological conditions. The different nature of structural change was observed from the simulations of the protein or fragment in water and near lipid bilayers due to the different solvent environment. The protein has close contacts with the membrane surface. It was impossible to observe the conformational change to b-sheet and protein entrance into the lipid bilayer within 1 ns simulations.

NONE.