published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Dendritic cells (DC) are a heterogeneous population of hematopoietic cells that play a versatile role in orchestrating immune responses against an array of invading pathogens, including influenza virus. These cells reside in lymphoid organs as well as in non-lymphoid tissues such as mucosal surfaces of respiratory and gastro-intestinal system. Recent investigations have suggested that in the steady state, dendritic cells are derived mainly from bone marrow precursor cells without a monocytic intermediate whereas during inflammation or infection, monocytes readily differentiate to generate monocyte derived dendritic cells (MoDC). The ability of virus infected monocytes to differentiate into MoDC was investigated and the results demonstrated that in vitro infection of monocytes with influenza virus impaired their development into MoDC. It was also observed that influenza infection of monocytes, pre-treated with GM-CSF and IL-4 for DC differentiation, was minimally-productive and non-cytopathic. In spite of successful viral genome transcription, viral protein synthesis was restricted at an early stage. However, despite of the limited replication, influenza virus infected monocytes failed to develop the distinctive DC- like morphology when cultured with GM-CSF and IL- 4 as compared to their mock infected counterparts. Infected cells, after 4 days in culture, expressed reduced amounts of CD11c, CD172a (myeloid marker), CD1w2 (CD1b) and CCR5. Influenza virus infected monocytes also retained substantial non-specific esterase activity, a characteristic for monocytes and macrophages. Antigen presentation capability of infected cells was also affected as indicated by decreased endocytosis. Production of IL-12, a pro-inflammatory cytokine and IL-10, a reciprocal inhibitory cytokine, was coordinately modified in influenza virus infected monocytes in order to arrest their differentiation into DCs. At least limited viral replication was necessary to impede the differentiation process completely. However, viral NS1 protein activity, as evidenced with a mutant influenza virus, was not essential for this inhibition. This identified a new strategy by influenza virus to interfere with DC differentiation and evade a virus specific immune response.

Cancer is one of the most pervasive health problems in the world today. Despite major advances in its treatment in recent decades, conventional therapies have seen limited success. Aggressive, drug-resistant cancer cells can reemerge after treatment, resulting in relapse. Immunotherapy, a strategy that utilizes a patient's own immune system to specifically destroy cancer cells, is a potential solution to this problem. Immunotherapy, however, is limited by multiple mechanisms of cancer-induced immunosuppression. One of the most important of these mechanisms is the induction of Treg, which are capable of suppressing multiple arms of the anti-cancer immune response. In the current study, we evaluated strategies to hinder the deleterious function of Treg on cancer immunotherapy. First, we determined that imatinib mesylate could inhibit Treg function in vivo and in vitro and increase the efficacy of dendritic cell-based immunization against an imatinib-resistant lymphoma. Then, searching for further methods to inhibit Treg, we found that Th-1 cells were capable of inhibiting Treg function and synergizing with a tumor lysate vaccine to treat leukemia. This process was dependent on IFN-γ secretion by the Th-1 cells. While investigating the influence of Th-1 on Treg and the resulting immunomodulatory effects of these cells in vivo, we discovered that they were capable of promoting the non-conventional direct tumor killing function of DC. We determined that Th-1 induce the cytotoxic function of bone marrow-derived DC generated with GM-CSF and IL-4 by a mechanism dependent on IFN-γ. Finally, because our results indicate that the antigen presenting function of KDC may depend upon their cytotoxic ability, and since DC generated with IL-15 have been reported to be more efficient APC than those generated with IL-4, we evaluated their ability to also function as direct tumor cell killers. We found that while IL-15 DC can indeed kill tumor cells, only LPS and not IFN-γ was capable of inducing this capability. These findings contribute to both arms of anti-cancer immunity by impairing immunosuppression with imatinib and Th-1, and promoting anti-tumor immunity with KDC. This double-pronged approach may contribute to further strategic advances in the field of cancer immunotherapy.

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Early during an immune response, dendritic cells (DC) interact closely with CD4+ T cells, and cross-talk between these cells can come in the form of tumour necrosis factor (TNF) superfamily ligand-receptor interactions. These signals are critical for the maturation, function and survival of DC, and thereby dictate the capacity of DC to prime a robust T cell response. Among these cues, helper T cell-expressed CD40L interaction with DC-expressed CD40 is required to fully mature DC for cross-priming of help-dependent CD8+ T cell responses. The lymphotoxin-beta receptor (LTβR) is another TNF family receptor on DC, and it’s ligands LTα1β2 and LIGHT are expressed on activated T cells. Since abrogated LTβR signaling impairs T cell immunity, we have examined whether LTαβ represents another possible helper T cell-derived cue for full DC maturation. However the LT pathway controls lymphoid tissue organization and DC homeostasis, a second possible mechanism explaining the necessity of LTβR signaling for T cell immunity. Here we dissect the role of helper T cell-expressed LTβR ligands and DC-intrinsic LTβR signaling, independent of DC homeostasis or lymphoid organization, in DC function and T cell immunity. Absence of LTα1β2 and not LIGHT on helper T cells results in compromised T cell priming by DC ex vivo, and LTβ-/- CD4+ T cell responses are impaired in vivo. Ag-specific CD4+ T cell-expressed LTα1β2 and DC-intrinsic LTβR signaling are required for an optimal cytotoxic T lymphocyte (CTL) response in vivo. While CD40 induces IL-12 and is required for CTL function, DC-intrinsic LTβR signaling is necessary for CTL activation and expansion, early up-regulation of CD86 and IFNα/β production. Our results reveal non-redundant roles for distinct TNF family receptors in enabling DC to program different features in Ag-specific CD8+ T cells.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / In this study we examine the utility of using CD8+ T cell hybridomas to measure the ability of bone marrow dendritic cells (BMDCs) to internalize cancer proteins and display them to cytotoxic T cells, a process termed cross‐presentation. We test the ability of a newly generated T cell hybridoma called BUSA14 to detect cross‐presentation of the melanoma antigen gp100. BUSA14 produces a dose‐dependent response to human and mouse gp100 peptides. However, cross‐presentation of gp100 by BMDCs using SK‐MEL‐28 human melanoma cell lysates or direct MHC class I‐restricted presentation by B16 murine melanoma cells was not detected. Both SKMEL‐28 and B16 cells express gp100 protein by immunoblot, and gp100 as a membrane bound protein may be concentrated by cell fractionation techniques. We validated our crosspresentation assay with another T cell hybridoma B3Z to detect cross‐presentation of the model antigen ovalbumin. Lastly, we determined that although BUSA14 expresses the coreceptor CD8, BUSA14 lacks CD3 expression, which likely impairs the ability of this hybridoma to respond to engagement of the T cell receptor and contributes to the inability to detect presentation of native gp100 protein. To resolve these issues, we plan to use primary gp100‐specific T cells from pmel mice expressing the same T cell receptor as the BUSA14 hybridoma to detect presentation of gp100 protein. Ultimately, we plan to evaluate the requirements for cross‐presentation of gp100, including a role for gamma‐interferon‐inducible lysosomal thiol reductase (GILT), a disulfide bond reducing enzyme.

Diffusion controlled phase transformations are studied bymeans of the phase-field method. Morphological evolution ofdendrites, grains and Widmanst\"atten plates is modeled andsimulated. Growth of dendrites into highly supersaturated liquids ismodeled for binary alloy solidification. Phase-field equationsthat involve both temperature and solute redistribution areformulated. It is demonstrated that while at low undercoolingheat diffusion does not affect the growth of dendrites, i.e.solidification is nearly isothermal, at high cooling rates thesupersaturation is replaced by the thermal undercooling as thedriving force for growth. In experiments many crystals with different orientationsnucleate. The growth of randomly oriented dendrites, theirsubsequent impingement ant formation of grain boundaries arestudied in two dimensions using the FEM on adaptive grids. The structure of dendrites is determined by growthconditions and physical parameters of the solidifying material.Effects of the undercooling and anisotropic surface energy onthe crystal morphology are investigated. Transition betweenseaweeds, doublons and dendrites solidifying out of puresubstance is studied and compared to experimental data. Two-and three-dimensional simulations are performed in parallel onadaptive and uniform meshes. A phase-field method based on the Gibbs energy functional isformulated for ferrite to austenite phase transformation inFe-C. In combination with the solute drag model, transitionbetween diffusion controlled and massive transformations as afunction of C concentration and temperature is established byperforming a large number of one dimensional calculations withreal physical parameters. In two dimensions, growth ofWidmanstaetten plates is governed by the highly anisotropicsurface energy. It is found that the plate tip can beapproximated as sharp, in agreement with experiments. Keywords:heat and solute diffusion, solidification,solid-solid phase transformation, microstructure, crystalgrowth, dendrite, grain boundary, Widmanstaetten plate,phase-field, adaptive mesh generation, FEM. / <p>NR 20140805</p>

Dendritic cells are equipped with a range of different pattern-recognition receptors (PRR) aimed at recognizing foreign pathogens. Recent evidence has suggested that PRR signalling regulates the expression of microRNAs (miRNAs), important post-transcriptional regulators of gene expression, which have been shown to fine-tune innate immune responses. This thesis describes the discovery of miR-650, a novel PRR-responsive miRNA that is down regulated in monocyte-derived dendritic cells (DCs) on PRR stimulation. Chapter 4 describes the characterisation of miR-650 expression in DCs matured by exposure to a variety of different pathogen-derived ligands, or during Influenza A virus infection. When correlating the level of miR-650 to the induction of DC activation markers on the cell surface, an inverse correlation was observed, suggesting a relationship between miR-650 down regulation and the effective dose of the ligand. Work presented in this thesis further explores the potential function of miR-650 by using a multi-pronged approach encompassing computational biology, genome-wide expression profiling and individual reporter assays, to gain insight into the gene networks regulated by miR-650. While Chapter 5 focuses on the identification and confirmation of individual miR-650:target interactions, Chapter 6 investigates both direct as well as secondary effects exerted by miR-650 on a global level. The work in these two chapters identifies a number of novel miR-650 targets and suggests a dual role for miR-650 in the innate immune response. Firstly, it is shown that miR-650 directly regulates a group of interferon-stimulated genes with known antiviral activity. Supporting its role in antiviral host defence, miR-650 is also shown to directly target components of the autophagic machinery, and even more importantly, down regulation of miR-650 induces autophagosome formation. Secondly, identified targets also include negative regulators of innate signalling suggesting that, in addition to its antiviral function, PRR-mediated down regulation of miR-650 expression may also provide a negative feedback loop controlling inflammatory responses. Notably, miR-650 displays reciprocal target regulation with miR-155, a well-studied miRNA with established functions in the innate immune system, thus suggesting cooperativity between the two miRNAs. The original aim of this thesis was to examine the effect of HIV-1 infection on the global miRNAome of DCs using a genome-wide profiling method. However, as outlined in Chapter 3, the data generated suggest that HIV-1 infection has little or no impact on miRNA expression. Further work is needed to establish if this represents deliberate immune evasion by HIV-1, or just indicates the limits of the methodology employed.

In the brain, the dendritic spine is a point of information exchange that extends the neuronal surface on which synapses occur, as well as facilitates and stabilizes those contacts. Furthermore, dendritic spines dynamically change in shape and number in response to a variety of factors. Dendritic spine numbers are reduced in mental retardation, enhanced during development, sensory enrichment or physical exercise, or fluctuate during the reproductive cycle. Thus, for a given neuron type, it might be expected that dendritic spine number might achieve a dynamic optimum. Indeed, many studies of spine density of pyramidal neurons in sensory cortex indicate that an average of ~1.4 spines/micron occurs is present (Briner et al., 2010). Most such studies examined dendritic spines from primary sensory areas which are dominated by inputs from a single sensory modality. However, there are a large number of neural regions that receive inputs from more than one sensory modality and it is hypothesized that spine density should increase to accommodate these additional inputs. To test this hypothesis, the present experiments used Golgi-Cox stained layer 2-3 pyramidal neurons from ferret primary somatosensory (S1) and auditory (A1) cortical regions, as well as from the higher-level rostral posterior parietal (PPr) and lateral rostral suprasylvian (LRSS) multisensory areas. Spine densities in S1 (avg 1.309 ± 0.247 spines/micron) and A1 (avg 1.343 ± 0.273 spines/micron) were measured to be significantly greater (p<0.05, t-test) than those observed in multisensory regions PPr (avg 1.242 ± 0.205 spines/micron) or LRSS (avg 1.099 ± 0.217 spines/micron). These results also indicate that spine densities are greater in primary (S1, A1) than in higher-level (PPr, LRSS) sensory areas. The functional consequences of such unexpected findings are discussed in light of potential biophysical differences between unisensory and multisensory neurons.

The anterior cingulate cortex (ACC) can influence emotional and motivational states in primates by its dense connections with many neocortical and subcortical regions. Pyramidal neurons serve as the basic building blocks of these neocortical circuits, which have been extensively studied in other brain regions, but their morphological and electrophysiological properties in the primate ACC are not well understood. In this study, we used whole-cell patch clamp and high-resolution laser scanning confocal microscopy to reveal the general electrophysiological properties and detailed morphological features of layer 2 and 3 pyramidal neurons in ACC (area 24/32) of the rhesus monkey. Neurons from both layers had similar passive membrane properties and action potential properties. Morphologically, dendrites of layer 3 ACC neurons were more complex than those of layer 2 neurons, by having dendrites with longer total dendritic lengths, more branch points and dendritic segments, spanning larger convex hull volumes. This difference in total dendritic morphology was mainly due to the apical dendrites. In contrast, the basal dendrites displayed mostly similar features between the two groups of neurons. However, while apical dendrites extend to the same layer (layer 1), the basal dendrites of layer 3 extended into deeper layers than layer 2 because of the difference in soma-pia distance. Thus, basal dendrites of the two groups of neurons receive different laminar inputs. Analysis of spines showed that more spines were found in neurons of layer 3 apical dendritic arbors than layer 2 neurons. However, the apical spine densities were similar between neurons in the two layers. Thus, while higher spine number suggests that layer 3 neurons receive more excitatory input than layer 2 neurons, the similar spine density suggests similar spatial and temporal summation of these inputs. The combined effects of increased number of excitatory input and higher dendritic complexity in layer 3 than in layer 2 ACC neurons suggest the additional information received by layer 3 neurons, especially in the apical dendrites, might undergo more complex integration.