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The multi-functional FGL2 can be expressed as either a type II membrane-associated glycoprotein or a secreted tetrameric molecule. As an important effector of regulatory T cells, secreted FGL2 inhibits dendritic cell maturation and T cell proliferation. The mechanism of its immunomodulatory function remains unclear. The goals of this thesis are to identify receptor(s) of secreted FGL2, key biological functions and signaling pathways, and mechanism of FGL2 oligomerization. Soluble FGL2 was critical for all studies, and the production of recombinant FGL2 was compared in E. coli, insect cells and mammalian cells. Soluble and stable FGL2 was secreted only by mammalian cells, indicating the importance of post-translational modification. In flow cytometry and surface plasmon resonance assays, recombinant FcFGL2 and albumin tagged FGL2 fusion proteins bound to Fc gamma RIIB and Fc gamma RIII receptors expressed by antigen presenting cells (APCs), including lipopolysaccharide (LPS)-stimulated B lymphocytes, endothelial cells, thioglycollate-stimulated peritoneal macrophages, and bone marrow-derived dendritic cells (BM-DCs). The binding of recombinant FGL2 to Fc gamma RIIB and Fc gamma RIII was specific, dependent on receptor expression and blocked by anti-Fc gamma RIIB/III antibody. FcFGL2 inhibited the maturation of BM-DC derived from fc gamma riib wild type mice but not from fc gamma riib knock out mice. It also induced apoptosis of the A20 mouse B cell line (Fc gammaRIIB+), but not the A20IIA1.6 cell line (Fc gamma RIIB-). The activation of caspases induced by FcFGL2 binding to A20 cells was confirmed by flow cytometry, Western blotting and analysis of DNA fragmentation. The role of Fc gammaRIIB in FGL2-mediated immunosuppression was confirmed in vivo. Infusion of FcFGL2 into fc gamma riib+/+, but not fc gamma riib-/- C57BL/6J mice (H-2b) inhibited the rejection of fully mismatched BALB/cJ (H-2d) skin and heart allografts. Studies on the mechanism of FGL2 oligomerization employed site-directed mutagenesis and revealed that cysteines at positions 94, 97, 184, and 187 were critical. Mutation of these cysteines resulted in secretion of monomeric FGL2. Computer modeling of FGL2 tetramers predicted an asymmetric arrangement that was similar to the structure of multimeric ficolin. The data presented in this thesis provide mechanistic insights into the immunosuppressive activity of soluble FGL2, and a foundation for the development of a novel and potentially highly effective immunosuppressive therapy.

The multi-functional FGL2 can be expressed as either a type II membrane-associated glycoprotein or a secreted tetrameric molecule. As an important effector of regulatory T cells, secreted FGL2 inhibits dendritic cell maturation and T cell proliferation. The mechanism of its immunomodulatory function remains unclear. The goals of this thesis are to identify receptor(s) of secreted FGL2, key biological functions and signaling pathways, and mechanism of FGL2 oligomerization. Soluble FGL2 was critical for all studies, and the production of recombinant FGL2 was compared in E. coli, insect cells and mammalian cells. Soluble and stable FGL2 was secreted only by mammalian cells, indicating the importance of post-translational modification. In flow cytometry and surface plasmon resonance assays, recombinant FcFGL2 and albumin tagged FGL2 fusion proteins bound to Fc gamma RIIB and Fc gamma RIII receptors expressed by antigen presenting cells (APCs), including lipopolysaccharide (LPS)-stimulated B lymphocytes, endothelial cells, thioglycollate-stimulated peritoneal macrophages, and bone marrow-derived dendritic cells (BM-DCs). The binding of recombinant FGL2 to Fc gamma RIIB and Fc gamma RIII was specific, dependent on receptor expression and blocked by anti-Fc gamma RIIB/III antibody. FcFGL2 inhibited the maturation of BM-DC derived from fc gamma riib wild type mice but not from fc gamma riib knock out mice. It also induced apoptosis of the A20 mouse B cell line (Fc gammaRIIB+), but not the A20IIA1.6 cell line (Fc gamma RIIB-). The activation of caspases induced by FcFGL2 binding to A20 cells was confirmed by flow cytometry, Western blotting and analysis of DNA fragmentation. The role of Fc gammaRIIB in FGL2-mediated immunosuppression was confirmed in vivo. Infusion of FcFGL2 into fc gamma riib+/+, but not fc gamma riib-/- C57BL/6J mice (H-2b) inhibited the rejection of fully mismatched BALB/cJ (H-2d) skin and heart allografts. Studies on the mechanism of FGL2 oligomerization employed site-directed mutagenesis and revealed that cysteines at positions 94, 97, 184, and 187 were critical. Mutation of these cysteines resulted in secretion of monomeric FGL2. Computer modeling of FGL2 tetramers predicted an asymmetric arrangement that was similar to the structure of multimeric ficolin. The data presented in this thesis provide mechanistic insights into the immunosuppressive activity of soluble FGL2, and a foundation for the development of a novel and potentially highly effective immunosuppressive therapy.

The purpose of this study was to identify conditioning strategies for mesenchymal stromal cells (MSC) which optimize cellular immunosuppressive potency. By identifying new treatment strategies and previously unidentified small molecules capable of stimulating MSC we hope to pave the way tailoring licensed MSC phenotypes to be used in a specific disease state, rather than a one size fits all package. We sought to determine how MSC act in response to a changing immune response or environmental condition. MSC are exquisitely sensitive to changes in their environmental conditions and we show that cellular transcriptome and secretome changes are conditionally responsive to their inflammatory stimulus. One of the main subjects of analysis here is the observations of how these cellular profiles evolve over time in the presence of an inflammatory environment. Similarly, this study observes how MSC behavior changes after an inflammatory event has been resolved to address, in part, the plasticity of MSC licensing and the ability of MSC to rapidly recall a previous immunosuppressive state upon secondary challenge with an inflammatory stimulus. Data was obtained from in vitro experiments with human bone marrow derived MSC and donor human peripheral blood mononuclear cells (PBMC), while in vivo data was obtained using C57BL6/J mice. Overall this research demonstrated that MSC potency can be bolstered by small molecule and drug treatment conditioning, and that certain disease conditions may be more effectively paired with specific MSC conditioning strategies to improve their therapeutic effectiveness.

Natural Killer (NK) cells, lymphocytes of the innate immune response, play a vital role in controlling infections and in tumor surveillance. NK cells provide protection by direct cytolysis of infected cells and by the production of pro-inflammatory cytokines such as, IFN-γ and TNF-α. Notably, NK cells have recently been identified to regulate the immune response by producing the anti-inflammatory cytokine IL-10. Several other cells can produce IL-10 during infections, however NK cell derived IL-10 can be critical in regulating immune response during early phases of infection and therefore protecting the host from excessive immunopathology. Although the regulatory role of NK cells seems to be plausible, the physiological relevance of NK cell mediated immune regulation during infections has not been demonstrated in detail. To investigate the immunoregulatory function of NK cells, I used Murine Cytomegalovirus (MCMV) infection induced by a high dose challenge and demonstrated that NK cells are a major IL-10 producer during acute stage of the infection. To elucidate the role of NK cell derived IL-10 during infections, I generated NK cell specific IL-10 knockout, NKp46iCre  Il-10flox/flox mice (NK-Il-10-/-) by crossing Il-10flox/flox mice with mice expressing Cre recombinase exclusively under the NK cell specific promoter, NKp46 (NKp46iCre knock-in mice). My results indicated that Cre mediated Il-10 genomic deletion occurred predominantly in NK cells but not in NKT, T and B cells. Enriched NK cells from NK-Il-10-/- mice failed to produce IL-10 upon ex vivo IL-2/IL-12 stimulation. Furthermore, histological analysis of the colon indicated that NK-Il-10-/- mice are free from aberrant inflammation. During sustained MCMV infection, significantly higher production of IFN-γ by CD8+ T cells of NK-Il-10-/- mice in salivary glands indicates that NK cell derived IL-10 contributes to the establishment of the immune suppressive environment in the organ. NK-Il-10-/- mice also demonstrated increased susceptibility to acute Listeria monocytogenes (LM) infection based on enhanced body weight loss. Taken together, I have successfully generated NK-Il-10-/- mice that lack the Il-10 gene exclusively in NK cells. The NK-Il-10-/- mouse can be used as an ideal model to dissect the immunoregulatory role of NK cells during various microbial infections and tumorogenesis.

Torque-teno viruses (TTVs) are small ubiquitous non-enveloped single-stranded circular DNA viruses. Since their discovery in a post-transfusion hepatitis patient, they have been isolated in several vertebrate hosts with over 90% prevalence, including swine. They have been detected in the environment, water sources, human drugs, vaccine and blood product as contaminants. Intriguingly, the role of TTVs in human disease causation is still not fully understood. Several epidemiological studies have associated TTVs to human diseases, like cancers, hepatitis, and autoimmune diseases, but no clear link between infection and clinical disease has been demonstrated yet. In contrast, experimental studies done in pigs demonstrated that swine TTVs (TTSuVs) could an act as sole pathogens. Other studies also demonstrated that TTSuVs could exacerbate symptoms of other viral pathogens in coinfections. Here, we showed that TTSuV1 could be zoonotic, as we detected TTSuV1 DNA in human serum samples. We also showed that TTSuV1 could replicate in human immune cells, and consequently suppress their ability to respond to immune stimuli. Further in-vivo studies, to elucidate host immune regulation by TTSuVs, showed a delayed antibody response and minimal viremia. Also, we found that viral sensing could be limited to interferon-inducing sensors (DHX36), while upregulation of PD-1 could demonstrate how these viruses may establish chronic infections. In another study, we showed the use of our novel recombinant TTSuV1 culture system to study the synergistic interactions between TTSuV1 and porcine circovirus 1 (PCV1). When both viruses were cultured together in-vitro, their respective viral titers were increased, compared to the single virus infections. We also demonstrated that increased in-vitro replication of TTSuV1 could be relying on expression of PCV1 replicase. In addition, molecular mechanisms were used to explain this synergistic relationship; a strong promoter activity by the putative major promoter of TTSuV1 was shown to be blocked PCV1 and TTSuV1 replicase proteins, but protein-DNA interaction assays need further optimizations to demonstrate physical interaction between these viruses. In conclusion, our result showed new information about TTSuV1 transmission, pathogenesis, host innate immune regulation, and their role in coinfections.

[EN] Lung cancer is the leading cause of cancer-related death worldwide, and is the third most common cancer type; it can be classified into two subgroups based on histology: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). The 5-year survival still remains poor and despite the existence of several distinct tumour phenotypes, therapeutic decisions are mainly based on clinical features such as stage or performance status. This highlights the need for new diagnostic and prognostic biomarkers in different types of samples (such as blood, fresh-frozen tissue or formalin-fixed, paraffin-embedded samples). The field of tumour immunology has changed in the last decade, and it is now accepted that the immune system plays a pivotal role in cancer. Although the immune cells that infiltrate the tumour microenvironment are potentially capable of eliminating tumour cells, they cannot prevent tumour development and progression. Tumours acquire mechanisms to regulate their immune microenvironment such as the release of a series of factors to subvert normal reaction mechanisms, the modulation of co-stimulatory pathways, also known as immune checkpoints, and the induction and attraction of suppressor cells (myeloid-derived suppressor cells, tumour-associated macrophages, and regulatory T cells). The potential effect of the patient's immune system on clinical outcome is important for the identification of prognostic markers as well as markers that predict treatment responses. The study of immune-related markers, especially those implicated in immunoregulatory processes, could provide valuable prognostic information that could help in many applications in future clinical practice. Thus, the objective of this thesis is to characterise cancer immunoregulation biomarkers and to evaluate the possible correlation between these biomarkers and clinicopathological and prognostic variables in patients with NSCLC by the use of well-tested and accurate techniques such as quantitative PCR and immunohistochemistry. Furthermore, this study will provide information about the immunological features of the tumour microenvironment in NSCLCs. / [ES] El cáncer de pulmón es una de las principales causas de muerte relacionada con cáncer en el mundo, siendo el tercer tipo de cáncer más común. El cáncer de pulmón no microcítico (CPNM) representa casi el 85% de todos los cánceres de pulmón y la supervivencia a los 5 años va desde el 50% en estadios IA hasta el 15% en estadios IIIA. Hasta el momento, no se han descubierto biomarcadores capaces de predecir la progresión de la enfermedad en pacientes tanto en estadios resecables como en estadios avanzados, por lo que existe una clara necesidad de realizar estudios centrados en la búsqueda de biomarcadores pronósticos y diagnósticos en los diferentes tipos de muestra disponibles, como por ejemplo sangre, tejido fresco y tejido parafinado. El campo de la inmunología tumoral ha cambiado en la última década y actualmente se sabe que el sistema inmune juega un papel clave en cáncer. Las células inmunes que infiltran el tumor son un componente más del microambiente tumoral. Pese a que son potencialmente capaces de eliminar los antígenos tumorales, estas células no pueden evitar la formación y progresión tumoral. Esto es debido a que el tumor adquiere diversos mecanismos de regulación del microambiente tumoral con el objetivo de escapar del ataque del sistema inmune, como por ejemplo liberación de factores que impiden el correcto funcionamiento de los mecanismos de reacción inmune, modulación de vías co-estimuladoras y reclutamiento y activación de células inmunoreguladoras como las células T reguladoras, las células mieloides supresoras y los macrófagos asociados a tumores. El estudio de marcadores relacionados con la respuesta inmune y concretamente con los procesos de inmunoregulación puede proporcionarnos información pronóstica y predictiva relevante sobre los pacientes con cáncer. Por todo ello, el principal objetivo de esta tesis doctoral es analizar la presencia de marcadores relacionados con la inmunoregulación y evaluar su posible correlación con las variables clínico-patológicas y pronósticas en pacientes con CPNM mediante el uso de técnicas fiables y aplicables en la práctica clínica como la PCR cuantitativa y la inmunohistoquímica. Así mismo, esto nos permitirá conocer en mayor profundidad las características inmunológicas del microambiente tumoral en pacientes con CPNM. / [CA] El càncer de pulmó és una de les principals causes de mort relacionades amb càncer al món, sent a més a més el tercer tipus de càncer més comú. El càncer de pulmó no microcític (CPNM) representa el 85% de tots els casos de càncer de pulmó aproximadament i la supervivència als 5 anys continua sent molt baixa. Fins el moment, no s'han descobert biomarcadors capaços de predir la progressió de la malaltia tant en pacients en estadis inicials com en estadis avançats. Per aquest motiu, existeix una clara necessitat de realitzar estudis centrats en la recerca de biomarcadors pronòstics i predictius en els diferents tipus de mostres disponibles, com per exemple sang, teixit fresc i teixit parafinat. El camp de la immunologia tumoural ha canviat en l'última dècada i actualment se sap que el sistema immune exerceix un paper clau en el càncer. Les cèl¿lules immunològiques que infiltren el tumour són un component més del microambient tumoural. Malgrat que aquestes cèl¿lules són potencialment capaces d'eliminar el antígens tumourals, s'ha evidenciat que no poden previndre la formació i progressió tumoural. Una de les raons per les quals s'observa aquest fenomen és que el tumour adquireix diversos mecanismes de regulació del microambient tumoural. Aquests mecanismes es basen en l'alliberació de factors que impedeixen el correcte funcionament del sistema immune, la modulació de vies coestimuladores i el reclutament i activació de cèl¿lules immunoreguladores com poden ser les cèl¿lules T reguladores, les cèl¿lules mieloides supressores i els macròfags associats a tumour. L'estudi de marcadors relacionats amb la resposta immune i més concretament amb els processos d' immunoregulació pot proporcionar informació pronòstica i predictiva rellevant sobre els pacients amb càncer. Per tot això, el principal objectiu d'aquesta tesi doctoral és analitzar la presència de marcadors relacionats amb la immunoregulació i avaluar la seva possible correlació amb les variables clinicopatològiques i pronòstiques de pacients amb CPNM mitjançant l'ús de tècniques fiables i aplicables a la pràctica clínica com són la PCR quantitativa i la immunohistoquímica. Així mateix, aquestes anàlisis ens permetran conèixer amb major profunditat les característiques immunològiques del microambient tumoural de pacients amb CPNM. / Usó Marco, M. (2015). ANALYSIS OF IMMUNOREGULATORY BIOMARKERS IN NON-SMALL CELL LUNG CANCER [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/51283

Mesenchymal stem cells (MSCs) represent a heterogeneous population of stromal cells with a pluripotent differentiation potential. They can be isolated from multiple tissues of mesodermal origin, such as bone marrow, adipose tissue, umbilical cord blood and peripheral blood and afterwards externally expanded according their adherence to the plastic surfaces. These cells show remarkable immunomodulatory properties, suppressing T-, B- and NK-cell functions, and also modulating dendritic cell activities and influencing immune responses during tissue repair and recovery. MSCs have been shown to possess ability to migrate to sites of inflammation and tissue injury. All these properties make MSCs a promising tool for clinical application. Our primary goal was to identify processes that may influence immunoregulatory effects of MSCs. In order to promote immunossupressive qualities of MSCs we established the scheme comprising MSCs precultivated with various cytokines and Toll-like receptors (TLR) ligands in vitro, with the final aim to improve the therapeutic effect of MSCs on wound healing in vivo. We studied modulation of MSCs properties and consequently the effect of influenced MSCs on cells of the immune system. The immunosuppression is mainly mediated through secreted factors that MSCs produced after...