<i>Staphylococcus aureus</i> is a versatile opportunistic pathogen causing a wide spectrum of diseases in both humans and animals. My research focused on characterization of the immune responses of monocyte derived dendritic cells (DC) to <i>S. aureus</i>. We initially evaluated the potential of circulating monocytes to serve as precursors for DC during <i>S. aureus</i> infection. The CD14⁺ monocytes, when stimulated with irradiated (ISA) or live <i>S. aureus</i> (LSA), differentiated into CD11c^high CD11b^high DC (MonoDC) in an autocrine fashion. This was associated with the up- regulation of granulocyte-macrophage colony stimulating factor (GMCSF) and tumor necrosis factor-α (TNF-α) gene transcription. We continued our studies to identify the role of TNF-α in the LSA induced differentiation of monocyte to MonoDC. Blocking TNF-α reduced the expression of CD11c and increased the expression of CD14 on LSA stimulated monocyte derived MonoDC. Stimulated monocytes were able to secrete monocyte chemotactic protein-1 (MCP-1), a chemokine that recruits monocytes to the site of infection/injury and induces the expression of β₂ integrins on DC. Characterization of the response of DC derived from monocytes using GMCSF and IL-4 revealed that, intact <i>S. aureus</i> rather than its purified structural components were efficient in DC activation. In response to ISA or LSA stimulation, DC induced proliferation of T cells collected from the peripheral circulation of cows with a history of <i>S. aureus</i> mastitis. Subsequent characterization of the proliferating T cells identified the presence of memory T cells. Finally, we identified a unique population of DEC205⁺CD8^a+ in monocyte derived DC. We further elucidated the role of DC DEC205, a C-type lectin, in <i>S. aureus</i> uptake. Blocking of receptor mediated endocytosis resulted in reduced uptake of <i>S. aureus</i> by DC. Confocal microscopy confirmed a role for DEC205 in <i>S. aureus</i> internalization and delivery to endosomes. DEC205 DC upon stimulation with <i>S. aureus</i> displayed enhanced maturation and antigen presentation. In conclusion, monocyte derived DC can uptake <i>S. aureus</i> and elicit cell mediated immune responses. / Ph. D.

Sphingolipid metabolites play a role in the initiation and perpetuation of inflammatory responses. Since intestinal inflammation is a driving force in the development of colon cancer, in the present study, we investigated the suppression of dextran sodium sulfate (DSS)-induced colitis by dietary sphingomyelin in mice that lack functional peroxisome proliferator-activated receptor γ (PPAR-γ) in intestinal epithelial and immune cells. Dietary spingomyelin decreased colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ. Using a real-time polymerase chain reaction array, we detected an up-regulation in genes involved in Th1 (interferon γ) and Th17 (interleukin [IL]-17 and IL-23) responses despite the reduced inflammation. However, the genes involved in Th2 (IL-4, IL-13 and IL-13ra2) and Treg (IL-10rb) anti-inflammatory responses were up-regulated in a PPAR-γ-dependent manner. In order to direct mechanistic studies of how PPAR-γ expression is involved in SM-induced suppression of DSS colitis, we investigated the effect of dietary SM in DSS-treated mice that lack PPAR-γ in the CD4+ T-cells. While the pathogenesis of colitis was independent of PPAR-γ expression in CD4+ T-cells, dietary SM decreased disease activity and colonic inflammation in mice of both genotypes but more efficiently in mice expressing PPAR-γ, indicating both PPAR-γ dependent and independent signaling pathways. In conclusion, in contrast to endogenous sphingolipid metabolites, dietary SM modulated both pro- and anti-inflammatory responses at the early stages of the disease in a partially PPAR-γ dependent manner resulting in a suppression of inflammation that may be critical for the suppression of inflammation-driven colon cancer. / Master of Science

CD4+ T cells are a group of lymphocytes that play critical roles in the immune system. By releasing cytokines, CD4+ T cells regulate other immune cells for maximizing the efficiency of the system. Naive CD4+ T cells are activated and become mature upon engagement with antigens, and the mature CD4+ T cells have several subsets, which play diverse regulatory functions. For the past two decades, our understanding of CD4+ T cells has been advanced through the studies on the differentiation process and the lineage specification of various subsets of these cells. Although in most experimental studies of CD4+ T cells, researchers focused on how transcription factors and signaling molecules influence the differentiation of a particular subset of these cells, many evidence have shown that the differentiation of CD4+ T cells can be heterogeneous in terms of the phenotypes of the cells involved. This dissertation describes a framework that uses mathematical models of the dynamics of the signaling pathways to explain heterogeneous differentiation. We show that the mutual inhibitions among the master regulators govern the formation of multi-stability behavior, which in turn gives rise to heterogeneous differentiation. The framework can be applied to systems with two or more master regulators, and models based on the framework can make specific predictions about heterogeneous differentiations. In addition, this dissertation describes an experimental study on CD4+ T cell differentiation. Being part of the adaptive immune system, the differentiation of CD4+ T cells was previously known to be induced by the signals from the innate immune cells. However, the expression of Toll-like receptor in CD4+ T cells suggests that microbial products can also influence the differentiation directly. Using an in vitro cell differentiation approach, we show that the differentiation and proliferation of CD4+ T cells can be influenced by lipopolysaccharide under the condition that would favor the differentiation of induced regulatory T cells. These theoretical and experimental studies give novel insights on how CD4+ T cells differentiate in response to pathogenic challenges, and help to gain deeper understanding of regulatory mechanisms of the complex immune system. / Ph. D.

CD4+ T cells mediate and orchestrate a tremendous panoply of lymphoid cell subsets in the human immune system. CD4+ T cells are able to differentiate into either effector pro-inflammatory or regulatory anti-inflammatory subsets depending on the cytokine milieu in their environment. This complex process is mediated through a variety of cytokines and soluble factors. Yet, the mechanisms of action underlying the process of differentiation and plasticity of this interesting immune subset are incompletely understood. To gain a better understanding of the CD4+ T cell differentiation and function, here we present an array of different strategies to model and validate CD4+ T cell differentiation and heterogeneity. The approaches presented here vary from ordinary-differential equation-based to agent-based simulations, from data-driven to theory-based approaches, and from intracellular mathematical to tissue-level or cellular modeling. The knowledge generated throughout this dissertation exemplifies how a combination of computational modeling with experimental immunology can efficiently advance the scene on CD4+ T cell differentiation. In this thesis I present i) an overview on CD4+ T cell differentiation and an introduction to which computational strategies have been adopted in the field to tackle with this problem, ii) ODE-based modeling and predictions on Th17 plasticity modulated by PPARγ, iii) ODE- and ABM-based cellular level modeling of immune responses towards Helicobacter pylori and the role of CD4+ T cell subsets on it, iv) Intracellular strategies to validate a potential therapeutic target within a CD4+ T cell to treat H. pylori infection, and finally v) data-driven strategies to model Th17 differentiation based on sequencing or microarray data to generate novel predictions on specific components. I present both mathematical and computational work as well as experimental work, in vitro and in vivo with animal models, to demonstrate how computational immunology and immunoinformatics can help, not only in understanding this complex process, but also in the development of immune therapeutics for infectious, allergic and immune-mediated diseases. / Ph. D.

Long-term non-progressors (LTPNs) in HIV infection target the structural protein Gag more frequently than individuals who progress to disease. However, the targeting of Gag per se does not always distinguish these two groups. Various factors have been put forth as likely explanations for this discrepancy including differences in the breadth and magnitude of observed responses, the HLA type of the host, the nature of the individual epitopes targeted and the ability of the virus to mutate these antigenic regions. The purpose of this thesis was to examine, using PBMCs isolated from HIV-2 infected LTNPs and CTL clones established in vitro, the clonotypic architecture and quality of an immunodominant HIV-2 Gag-specific response directed towards the HLA-B*3501-restricted epitope NPVPVGNIY (NY9: Gag245-253). The data presented in this thesis show that in spite of the expression of multiple inhibitory receptors on the surface of NY9-specific CD8+ T-cells, the NY9-response, which is a clonotypically 'private' response, bears a signature characterised by an increased cytotoxic sensitivity and the production of an array of cytokines, most notably IFN-γ and MIP-1β. Moreover, the results of this thesis indicate that the NY9-specific CD8+ T-cells are able to cross-recognise and lyse target B-cells pulsed with the corresponding HIV epitope PY9 and its variants at functional avidities (EC50) that are close to those exhibited by PY9-specific T-cells. However, not all mobilised TCR clonotypes are equally sensitive or equally cross-reactive. When individual CTL clones were studied it emerged that dominant clonotypes within the NY9-specific CD8+ T-cell memory pool possessed a higher avidity for tetramer and sensitivity for antigen than subdominant ones and demonstrated a better cross-reactive potential towards variants of the HIV-2 epitope. Hence, future HIV vaccine strategies may benefit from the inclusion of epitopes like NY9, the presentation of which appears to mobilise CD8+ T-cells with superior functional profiles.

The germinal centre (GC) response is critical for generating highly effective humoral immune responses and immunological memory that forms the basis of successful immunisation. Control of the output of the GC response requires Follicular regulatory T (Tfr) cells, a subset of Foxp3+ Treg cells located within germinal centres. Tfr cells were first characterised in detail in 2011 and because of this relatively little is known about the exact role of Tfr cells within the GC, and the mechanism/s through which they exert their suppressive function. At the outset of this work, the major barrier to understanding Tfr cell biology was the lack of appropriate tools to study Tfr cells specifically, without affecting Tfh cells or other Treg cell subsets. This thesis set out to develop a strain of mice that specifically lacks Tfr cells. A unique feature of Tfr cells is their CXCR5-dependent localisation within the GC. Therefore, genetic strategies that exclude Treg cells from entering the GC are a rational approach to generating a mouse model that lacks Tfr cells. To this end, I generated a strain of mice that lacks CXCR5 on Foxp3+ Treg cells. These animals show a ~50% reduction in GC localised Tfr cells, and a GC response that is comparable to control animals. These data indicated that redundant mechanisms are involved in Treg cell homing to the GC. I identified CXCR4 as a chemokine receptor that is also highly expressed on Tfr cells, and hypothesised that it may also be involved in Tfr cell localisation to the GC. Surprisingly, simultaneous deletion of both CXCR4 and CXCR5 in Treg cells resulted in a less marked reduction in Tfr cells compared to deletion of CXCR5 alone, suggesting that CXCR4 might be involved in negative regulation of Treg homing to the GC. These data identify both CXCR4 and CXCR5 as key regulators of Tfr cell biology. Bcl6 drives Tfr cell differentiation, but how this transcriptional repressor facilitates commitment to the Tfr cell subset is unknown. I hypothesised that Bcl6 drives Tfr cell differentiation by repressing Tbx21, the transcriptional regulator involved in the differentiation of Th1-like Treg cells. I tested this hypothesis in Bcl6fl/fl CD4cre/+ animals and unexpectedly found that loss of Bcl6 regulates Treg cell differentiation in the absence of immunisation or infection. I have demonstrated that thymic loss of Bcl6 results in an increase in activated effector Treg cells, which occurs very early in life. These data point to a novel role for Bcl6 in preventing early thymic Treg activation, indicating that Bcl6 has a global role in Treg development and differentiation that is not simply limited to Tfr cells.

T-cells are central to our success as a species. They confer specific and long-term immunity in a process known as adaptive immunity. During adaptive immune response, pathogen ingested by peripheral sentinel cells are brought to the local lymph nodes and presented to T-cells. T-cell recognizes the antigen via its receptor complex (TCR-CD3). The high affinity binding primes the cell for activation. With a positive costimulationary signal from CD28, the T-cell is fully activated, resulting in IL-2 secretions and cellular proliferation. Clinicians are increasingly harnessing the adaptive immune system to combat diseases such as cancer. Specifically, T-cells are activated and expanded ex vivo for adoptive immunotherapies. The ability to modulate T-cell activation is crucial in engineering appropriate effector cell populations for therapeutics. The focus of this thesis is to address the functional impact of CD28 spatial organization on T-cell activation. It has been observed that the spatial segregation of CD3 and CD28 by a few microns has resulted in poor activation of human T-cells. Lck, a Src family kinase (SFK) emerges as the instigator of the phenomenon. The kinase is associated with both CD3 and CD28 signal cascades. We propose a reaction diffusion model to describe the delicate balance between protein mobility and Lck de-activation. The work in this dissertation describes two probes to investigate Lck kinase activity, which permit real-time imaging of both the initiation of pLck activity and its duration. A FRET reporter is constructed to study the spatial and temporal initiation of the kinase activity. Embedded with the Lck membrane domain and contained a substrate for pLck to phosphorylate, the FRET biosensor reports the Lck kinase activity in real-time. Using microprinting to control CD3 and CD28 spatial organizations, the FRET reporter reveals that while T-cells require CD28 for significant IL-2 secretion, CD3 engagement is essential to initiate cellular activation through a spike in pLck kinase activity. Spatially, the reporter shows heightened kinase activity concentrated at the center of the cells upon CD3 engagement. To study the duration of pLck activity, a recruitment reporter is made. CD3 is found ubiquitously throughout the cellular membrane. And its activation by pLck induces the recruitment of a pair of tandem SH2-domain. The recruitment probe (also containing a pair of tandem SH2-domain) revealed curtailed pLck kinase activity due to CD3-CD28 segregation. Ultimately, understanding CD28 modulation of T-cell activation is clinically relevant as it provides new opportunities and targets for the development of therapeutics.

Regulatory T cells (Tregs), expressing the transcription factor Foxp3, form a key component of peripheral immune tolerance, guarding against auto-aggressive immune responses. Multiple Sclerosis is an inflammatory and demyelinating disease of the central nervous system (CNS) which is largely believed to be mediated by immune components reacting to the self myelin antigens that insulate the nerve fibres. Recent investigations have reported that regulatory T cells are dysfunctional in MS patients; therefore enhancing the regulatory T cell responses in MS is an attractive therapeutic target. Using the mouse model of MS, experimental autoimmune encephalomyelitis (EAE) we have attempted to develop disease-relevant Treg-based therapies to prevent disease induction. This required an understanding of the antigenic-reactivity of Tregs during disease. Results described in this thesis show that a proportion of Tregs in the draining lymph nodes and CNS were reactive to the disease initiating antigen(s) and could suppress in vitro responses of naïve T cells bearing transgenic T cell receptors, recognising the same antigen. Adoptive transfer of antigen-reactive Tregs suppressed disease induced with the same antigen, but also reduced disease induced with a distinct myelin antigen. Peptide-based tolerance using a high affinity MHC binding peptide analogue expanded and maintained antigen-reactive T cells which were tolerant to antigenic restimulation, although these cells did not express Foxp3. Peptide-treated mice showed reduced incidence of disease relapses during EAE induced against a distinct myelin antigen. Thus, while EAE and MS will involve a polyclonal effector T cell response to many antigens, therapeutic targeting of Tregs reactive against one CNS component may be sufficient to reduce disease. Endogenous expression of myelin autoantigen did not grossly alter the response of antigenreactive Tregs in the periphery. However, expression of endogenously derived viral superantigen enhanced the proportion of superantigen-reactive Foxp3+ Tregs in the periphery. This observation was extended using exogenous superantigen, suggesting that prolonged exposure to low dose (super)antigen tips the balance of the immune system in favour of regulation. This has implications for the ability to successfully fight infection, as well as for the limitation of autoaggressive responses and may contribute to the understanding of the hygiene hypothesis.

Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Vγ2Vδ2 cells have been used for adoptive cancer immunotherapy with some partial and complete remissions. Most trials have used continuous zoledronate exposure to expand Vγ2Vδ2 cells. Zoledronate inhibits farnesyl pyrophosphate synthase causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells. Because zoledronate exposure is toxic, we hypothesized that a short period of exposure would reduce T cell toxicity but still be sufficient for monocytes uptake. Supporting this hypothesis, pulse zoledronate exposure with IL-2 resulted in more uniform expansion of Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous exposure. These Vγ2Vδ2 cells also had higher levels of CD107a and perforin and slightly increased tumor cytotoxicity. Importantly, adoptive immunotherapy with Vγ2Vδ2 cells derived by pulse stimulation controlled human PC-3 prostate cancer cells in immunodeficient NSG mice significantly better than those derived by continuous stimulation. Pulse zoledronate stimulation of Vγ2Vδ2 cells with IL-15 also resulted in higher purity and cell numbers. Like with CD8 αβ T cells, IL-15 preserved early memory Vγ2Vδ2 T cell subsets better than IL-2. However, despite this fact, adoptive immunotherapy with Vγ2Vδ2 cells derived with IL-15 showed similar inhibition of PC-3 tumor growth as those derived with IL-2. Thus, pulse zoledronate stimulation maximizes the purity, quantity, and quality of expanded Vγ2Vδ2 cells. This simple modification to existing protocols would likely enhance the effectiveness of adoptively transferred Vγ2Vδ2 T cells.

The extent to which the progeny of one primary memory CD8 T cell differs from the progeny of one naïve CD8 T cell of the same specificity remains an important question. In order to explore cell autonomous functional differences between naïve and memory CD8 T cells that are not influenced by differences in the priming environment, an experimental model has been developed in which physiological numbers of both populations of cells were co-transferred into naïve host before antigen-stimulation. Interestingly, naïve CD8 T cells expand in numbers more than primary memory CD8 T cells after various infections or immunizations. The intrinsic ability of one naïve CD8 T cell to give rise to more effector CD8 T cells than one memory CD8 T cell is independent of the number of primary memory CD8 T cells present in vivo. The sustained proliferation of primary, but not the increased death of secondary effectors was shown to contribute to the differences in the observed magnitudes of expansion. In addition, longitudinal analysis of primary and secondary CD8 T cell responses revealed that the ability of naïve CD8 T cells to generate long-lived progeny (`memory generation potential') is better than for primary memory CD8 T cells despite the differences in overall kinetics of both responses after infection. Taken together, the data presented here revealed previously unappreciated differences between naïve and memory CD8 T cells and will help further define the functional potential for both cell types. The goal of immunization is to generate memory CD8 T cells of sufficient quality and quantity, and it has been shown that the naïve to primary memory CD8 T cell differentiation in vivo is controlled, at least in part, by the amount and duration of inflammation present early after the initiation of the response. In experiments where naïve CD8 T cells were co-transferred with increasing numbers of primary memory CD8 T cells, we observed a negative correlation between the number of primary memory present and the magnitude of primary CD8 T cell responses. Interestingly, the conversion of newly recruited (either TCR-Tg or endogenous) primary CD8 T cells into CD8 T cells with the phenotype (CD62Lhi, CD27hi) and function (tissue distribution, Ag-driven proliferation, cytokine production) of long-term memory was facilitated when they were primed in the presence of memory CD8 T cells of the same or unrelated specificity. Therefore, these data suggest that the presence of anti-vectorial immunity will not necessarily decrease the efficacy of CD8 T cell vaccination since newly recruited CD8 T cells, despite their decreased magnitude of expansion, might differentiate into functional memory cells faster.