archives@tulane.edu / Effective adaptive immune responses depend on the presentation to CD4+ T cells antigen peptides bound to major histocompatibility complex class II proteins. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity and abundance of peptides that are presented to T cells. The dissertation presented here sought to expand our understanding of how antigen structure and stability influence adaptive immune responses for two model antigens. Pseudomonas exotoxin A domain III (PE-III) functions as an ADP-ribosyltransferase with significant cellular toxicity and has been incorporated into a recombinant immunotoxin for the treatment of cancer. The bacterial component of the PE-III immunotoxin is highly immunogenic and generates neutralizing antibodies that render subsequent treatments ineffective. A group of six single-amino-acid substitutions in PE-III that were predicted to disrupt CD4+ T-cell epitopes have been shown to reduce antibody responses in mice. Here we demonstrate that only one of the substitutions, R494A, exhibits reduced folding stability and proteolytic resistance through the removal of a hydrogen bond. This destabilization significantly reduces its antibody immunogenicity while generating CD4+ T-cell epitopes that are indistinguishable from those of wildtype PE-III. PE-III specific B cells isolated from R494A-immunized animals contained fewer somatic mutations, which are associated with affinity maturation, and exhibited a weaker germinal-center gene signature, compared to B cells from wildtype-immunized animals. Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possess a protease-sensitive reactive center loop that lies adjacent to the OT-II epitope. We took advantage of the previously described single-substitution-variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins to study how changes in loop size and protein stability influences CD4+ T-cell priming in vivo. We observed that OVA R339T loop-insertion increases overall stability and protease resistance and significantly shortens the reactive center loop. This results in reduced CD4+ T-cell priming of the OT-II epitope in SJL mice. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools. / 1 / Daniel Moss

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 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.

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 differentiate into multiple effector subsets that mediate pathogen clearance. ThCTL are anti-viral effectors with MHC-II restricted cytotoxicity. The factors regulating ThCTL generation are unclear, in part due to a lack of a signature marker. I show here that in mice, NKG2C/E identifies ThCTL that develop in the lung during influenza A virus (IAV) infection. ThCTL phenotype indicates they are highly activated effectors with high levels of binding to P-selectin, T-bet, IFNγ production, and degranulation. ThCTL express increased levels of granzymes and perforin and lower levels of genes associated with memory and recirculation compared to non-ThCTL lung effectors. ThCTL are also restricted to the site of infection, the lung in IAV and systemically in LCMV. ThCTL require Blimp-1 for their differentiation, suggesting a unique effector CD4 population. As ThCTL are highly activated, they also require antigen signaling post priming during IAV infection. Late antigen was necessary and sufficient for the differentiation of ThCTL. In the context of late antigen encounter, ThCTL surprisingly do not require CD80 and CD86 costimulation for their differentiation. Additionally ThCTL do not require late IL-2 for their differentiation and instead require late IL-15 signals for their efficient generation. Thus these data suggest ThCTL are marked by the expression of NKG2C/E and represent a unique CD4 effector population specialized for cytotoxicity.

<p>Understanding the mechanisms that control T cell function and differentiation is crucial to develop new strategies to modulate immune function and prevent autoimmune and inflammatory disease. The balance between effector (Teff; Th1, Th2 and Th17) and regulatory (Treg) T cells is critical to provide an appropriate, but not excessive, immune response and therapies to induce Treg or inhibit Teff are likely promising treatment strategies. It has recently become clear that T cell metabolism is important in both T cell activation and differentiation. T cells undergo a metabolic reprogramming upon activation and not all differentiated T cell subsets utilize the same metabolic fuels or programs.</p><p>These metabolic differences are not trivial, as T cell metabolism is tightly</p><p>regulated and dysregulation can lead to cell death or reduced immunity. An</p><p>understanding of the metabolic differences between Teff and Treg may lead to a new direction for treating inflammatory diseases by modulating the Teff:Treg balance through metabolic inhibition. Previous studies have shown that Teff express higher levels of the glucose transporter Glut1 than Treg, however the role of Glut1, and importantly, the cell-intrinsic role of glucose metabolism in T cell differentiation and inflammation was not previously examined. The work presented here examines the role of Glut1 in T cell differentiation. We show that effector CD4 T cells were dependent on Glut1 for proliferation and function both in vitro and in vivo. In contrast, Treg were Glut1-independent and capable of suppressing colitis in the absence of Glut1 expression.</p><p>Additionally, previous studies have shown broad metabolic differences between Teff and Treg, however the specific metabolic profiles of Teff and Treg are poorly understood. Here, Teff and Treg metabolism is examined to test if dependence on distinct metabolic pathways will allow selective targeting of different T cell populations. We show that pyruvate dehydrogenase kinase 1 (PDHK1) is differentially expressed in the T cell subsets and inhibition of PDHK1 selectively suppresses Th17 and promotes Treg differentiation and function. Because Teff and Treg have distinct metabolic profiles, we hypothesized that the Treg-­specific transcription factor FoxP3 may drive the Treg oxidative metabolic program. We therefore examined the role of FoxP3 in T cell metabolism and determined that FoxP3 promotes glucose and lipid oxidation and suppresses glycolytic metabolism. Importantly, we show that promoting glycolysis with transgenic expression of Glut1 inhibits Treg suppressive capacity. Together, these data suggest that FoxP3 drives an oxidative metabolic program that is critical to Treg function. Overall, this work examines the metabolic phenotypes and regulation of Teff and Treg and potential metabolic targets that could be used to treat autoimmune and inflammatory disease.</p> / Dissertation

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.

<p>Several variables have been found to affect the Th1/Th2 differentiation of newly activated CD4 T cells. This phenotype can be critical in determining effectiveness of immune responses. Experiments in this thesis were undertaken to better define the in-vivo cellular interactions involved in determining the Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>Lethally irradiated BALB/c mice reconstituted with a constant number of syngeneic, naive spleen cells were challenged with xenogeneic red blood cells (XRBC) conjugated to ovalbumin (OVA) and the Th1/Th2 phenotype of the anti-XRBC response assessed. Antigen-specific interferon-gamma (IFN-g) and interleukin-4 (IL-4) secreting cells obtained from spleens of immunized mice were enumerated by an ELISPOT assay; the relative number of IFN-g- and IL-4-producing cells is taken as a relative measure of Th1 and Th2 components of the response. When challenged with a standard dose of XRBC-OVA, predominant Th1 responses are generated; when challenged with a ten-fold lower dose, such reconstituted mice do not generate significant responses. This adoptive transfer system was employed to explore further the relationships between quantitative changes in the dose of immunizing antigen and the number of responding antigen-specific CD4 T cells, and the Th1/Th2 phenotype of immune responses generated. Unprimed transgenic CD4 T cells specific for OVA can modulate the Th1/Th2 phenotype of the anti-XRBC response upon immunization with XRBC-OVA. Addition of a small number of naive transgenic spleen cells to the standard reconstituting population of normal spleen cells results in the generation of significant numbers of SRBC-specific Th2 cells when mice are challenged with a standard dose, or can generate predominant Th1 responses when mice are challenged with a ten-fold lower dose. Transgenic cells only impact the Th1/Th2 phenotype of CD4 T cells specific for XRBC when OVA is linked to the XRBC. That CD4 T cells specific for different antigens cooperate only through the recognition of linked antigenic determinants has important implications for many aspects of immune regulation. Observations further show that thymocytes from transgenic mice can influence the XRBC-specific response phenotype in an identical manner as transgenic spleen cells, suggesting that previously polarized pro-Th1/Th2 cells are not required in the cooperative events influencing Th1/Th2 phenotype of newly activated CD4 T cells.</p><p>These observations lead to a quantitative description, whereby antigen-mediated CD4 T cell cooperation can affect the Th1/Th2 phenotype of a primary antigen-specific immune response, and provide a context for further analysis at the molecular level.

Trichuris trichiura is a gastrointestinal dwelling nematode that infects almost 500 million people worldwide. T. muris occurs naturally in mice and is very closely related the human whipworm, making it a suitable model to dissect the immune response against the parasite. Studies using the Trichuris muris system have identified CD4+ T cells as dictators of the outcome of infection. In wild type mice, infection with a high dose of T. muris eggs leads to resistance and worm expulsion, which are dependent on a Th2 response and the secretion of type 2 cytokines especially interleukin (IL) 13. Chronicity is dependent on a Th1 response and occurs when mice are infected with a low dose of T. muris eggs. It is well established that metabolic changes are essential to promoting T cell activation and effector function. Moreover, during chronic infection the host immune system is continuously exposed to parasite antigen, which represents a metabolic challenge. This thesis has investigated the importance of T cell metabolism during response against T. muris. Data presented here show that low and high dose T. muris infections promote upregulation of the glycolytic pathway in CD4+ T cells. During later stages of chronic infection, CD4+ T cells displayed supressed glycolysis and mitochondrial respiration, and may be due to metabolic modulation imposed by the parasite. Leucine uptake via the amino acid transporter Slc7a5 was previously shown to be required for mTORC1 activation and for T cell effector function. Data presented here show that in early stages following a high dose T. muris infection, mice that lack Slc7a5 in T cells have delayed worm expulsion, impaired production of antibodies, and lower levels of IL-13. Their CD4+ T cells present reduced glycolytic rates when compared to cells from cohoused infected wild type mice. However, at later stages of infection, antibody, IL-13 and glycolytic levels were restored together with worm expulsion. CD4+ T cells from the early stage of infection showed reduced phosphorylation of mTOR, which suggested that impairment of function was mTOR dependent. Indeed, mice lacking mTOR in T cells fail to expel a high dose of parasites. They showed abrogation of IL-13 production, impairment in antibody class switching and their CD4+ T cells failed to upregulate glycolysis. Thus, this thesis shows that mTOR is essential for the proper functioning of T cells during T. muris infection and efficient amino acid transport plays a significant role. Taken together, these data show that metabolic orchestration of T cell function influences the capacity to effectively control helminth infection and that even subtle changes in T cell metabolic control can have a major effect on response phenotype.

Humoral immunity provides protection against subsequent infections. Antigen-specific, high-affinity, class-switched antibodies are produced by B cells through rounds of proliferation, B cell receptor rearrangement and selection in the germinal centres (GC). T cells play an essential and indispensable role in this process and in the recent years the term T follicular helper cells (TFH) was coined to describe this cell subset. The aim of my thesis is to investigate whether there is more than one type of T cells within the TFH population and whether it has important functional consequences. Firstly, I use sheep red blood cell immunisation (SRBC) and Salmonella enterica infection to show phenotypical differences between TFH expressing high and low level of surface molecule PD-1. In order to investigate the relationship between different TFH populations gene profiling was carried out on the microarray platform. Detailed transcriptome analysis revealed the discrete nature of isolated TFH cell subsets and provided an overview of their genetic landscape. Secondly, I have investigated the dependence of TFH subsets on cognate interactions with B cell in SRBC model by generating BM chimeras. I have demonstrated that generation of PD-1HI TFH, but not of PD-1LO TFH, depends on antigen presentation by B cells. Furthermore, I have shown that provision of wild-type but not MHC II knock-out B cells rescues PD-1HI formation in BM chimeras after SRBC immunisation. Finally, I have explored plasticity within TFH subsets and showed that none of the populations is in a terminally differentiated state, as they can convert into one another. Thirdly, experiments with S. enterica model revealed that the absence of PD- 1HI TFH is independent of the splenic architecture disruption present within the first week of the response. Surprisingly, co-immunisation studies showed that PD-1HI population is not only present but even enhanced in the group which received both SRBC and S. enterica when compared to single immunisations. The work presented in the thesis documents that there is a significant and previously unappreciated heterogeneity within TFH subset. This knowledge is important for designing optimal vaccine strategies and treating autoimmune diseases, as in both processes the antibody production plays a crucial role and its manipulation (either enhancing or blocking antibody production, respectively) can significantly improve clinical interventions.