Dissertations / Theses on the topic 'Dendritic cell'

Thesis (M.S.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Mar. 26, 2008). "... in partial fulfillment of the requirements for the degree of Master of Science in the Department of Microbiology and Immunology, School of Medicine." Discipline: Microbiology and Immunology; Department/School: Medicine.

Les cèl·lules dendrítiques (DCs) són clau en la inducció de respostes immunitàries adaptatives gràcies a la seva capacitat de capturar, processar i presentar antígens derivats de patògens als limfòcits T. Tanmateix, aquestes cèl·lules podrien contribuir a la disseminació inicial del VIH-1 a través de la captura i transmissió viral a les cèl·lules T CD4+ diana, un procés conegut com a trans-infecció. Aquest mecanisme es basa en l’expressió del receptor Siglec-1 (CD169), que reconeix gangliòsids sialilats a la membrana viral. Els nivells de Siglec-1 augmenten en DCs estimulades amb interferó-alfa i lipopolisacàrid, factors immuno-activadors presents durant el decurs de la infecció per VIH-1. En aquesta tesi, hem demostrat que l’interferó-alfa secretat per DCs plasmacitoides (pDCs) infectades per VIH-1, així com l’inteferó-alfa autocrí secretat per cèl·lules mieloides en resposta a lipopolisacàrid, augmenten l’expressió de Siglec-1 en DCs. A més, les pDCs provinents de dones secreten quantitats superiors d’interferó-alfa que les d’homes, posant de manifest la rellevància d’estudiar la trans-infecció del VIH-1 en teixits clau per a l’adquisició del virus en dones. Així, també hem estudiat el paper de Siglec-1 en la transmissió del VIH-1 per part de DCs primàries aïllades directament de teixit cervical, identificant una població de cèl·lules mieloides cervicals que expressen Siglec-1 i capturen partícules de VIH-1 a través d’aquest receptor. Aquesta capacitat augmenta amb l’activació per interferó-alfa. A més, la transmissió del VIH-1 per cèl·lules mieloides del cèrvix es bloqueja de forma eficient amb un anticòs monoclonal dirigit contra Siglec-1. Per tant, hem generat una sèrie de nous anticossos monoclonals contra Siglec-1 amb la capacitat de bloquejar la tans-infecció del VIH-1 per DCs. S’han produït cinc nous clons, que han demostrat tenir una alta afinitat per diferents epítops localitzats a la regió N-terminal de Siglec-1. A més, aquests anticossos bloquegen la captura i trans-infecció del VIH-1 per DCs, de forma que podrien ser un component en estratègies microbicides dirigides contra aquest tipus de transmissió viral cèl·lula-cèl·lula. A banda del VIH-1, les cèl·lules dendrítiques participen en la patogènesi d’altres virus, com ara els filovirus d’Ebola i Marburg. A diferència del VIH-1, les DCs són permissives a la infecció per filovirus i són dianes primerenques en la patogènesi viral. Els factors cel·lulars implicats en l’entrada de filovirus en aquestes cèl·lules no han estat totalment caracteritzats, però tant Ebola com Marburg són virus embolcallats que incorporen gangliòsids sialilats durant el procés de gemmació viral. A més, els factors que activen l’expressió de Siglec-1 com ara interferó-alfa i lipopolisacàrid s’han trobat durant la infecció pel virus d’Ebola. Per tant, en aquesta tesi hem estudiat el paper de Siglec-1 en l’entrada de filovirus en DCs. Hem trobat que Siglec-1 està implicat en la captura de partícules no infeccioses d’Ebola per part d’aquestes cèl·lules, especialment després de l’activació per interferó-alfa i lipopolisacàrid. A més, les partícules capturades són acumulades en el mateix compartiment cel·lular en què prèviament s’havia detectat el VIH-1. Siglec-1 també facilita l’entrada citoplasmàtica del virus a les DCs, així que hem determinat la capacitat dels nous anticossos monoclonals contra Siglec-1 d’interferir amb aquest procés, i hem vist que aquests bloquegen tant la captura com l’entrada citoplasmàtica de partícules no infeccioses d’Ebola en cèl·lules mieloides activades. En general, l’activitat delsanticossos monoclonals contra Siglec-1 inhibeix l’accés de retrovirus i de filovirus a les cèl·lules mieloides, cosa que indica el seu potencial ús com a agents antivirals d’ampli espectre.
Las células dendríticas (DCs) son clave en la inducción de respestas inmunitarias adaptativas gracias a su capacidad de capturar, procesar y presentar antígenos derivados de patógenos a los linfocitos T. Sin embargo, estas células también podrían contribuir a la diseminación inicial del VIH-1 a través de la captura de partículas virales y de su transmisión a las células T CD4+ diana, un proceso conocido como trans-infección. Este mecanismo se basa en la expresión del receptor Siglec-1 (CD169), que reconoce gangliósidos sialilados en la membrana viral. Los niveles de Siglec-1 aumentan en DCs estimuladas con interferón-alfa (IFN-α) y lipopolisacárido (LPS), factores immuno-activadores presentes durante el curso de la infección por VIH-1. En este trabajo, hemos demostrado que el IFN-α secretado por DCs plasmacitoides (pDCs) infectadas por VIH-1, así como el IFN-α autocrino secretado por células mieloides en respuesta a LPS, aumentan la expresión de Siglec-1 en DCs. Además, las pDCs provenientes de mujeres secretan cantidades superiores de IFN-α que las derivadas de hombres, poniendo de manifiesto la relevancia de estudiar la trans-infección del VIH-1 en tejidos clave para la adquisición del virus en mujeres. Por lo tanto, también hemos estudiado el papel de Siglec-1 en la transmisión del VIH-1 por parte de DCs primarias aisladas directamente de tejido cervical, identificando una población de DCs cervicales que expresan Siglec-1 y capturan partículas de VIH-1 a través de este receptor. Esta capacidad aumenta con la activación por IFN-α. Además, la transmisión célula-célula del VIH-1 por células mieloides del cérvix se puede bloquear de forma eficiente con un anticuerpo monoclonal (mAb) dirigido contra Siglec-1. Se han producido cinco nuevos clones, que han demostrado tener una alta afinidad por diferentes epítopos localizados en la región N-terminal de Siglec-1. Además, bloquean de forma eficaç la captura y trans-infección del VIH-1 por DCs, de forma que podrían ser un posible componente en estrategias microbicidas dirigidas contra este tipo de transmisión viral célula-célula. Además del VIH-1, las DCs pueden jugar un papel importante en la patogénesis de otros virus, como los filovirus Ébola y Marburg. A diferencia del VIH-1, las DCs son permisivas a la infección por filovirus y son dianas tempranas en lapatogénesis viral. Los factores celulares implicados en la entrada de filovirus en DCs no han sido totalmente caracterizados, pero tanto Ébola como Marburg son virus envueltos que incorporan gangliósidos sialilados durante el proceso de budding. Además, los factores que activan la expresión de Siglec-1 como IFN-α y LPS se han encontrado durante la infección por el virus de Ébola. Por tanto, en esta tesis hemos estudiado el papel de Siglec-1 en la entrada de filovirus en DCs. Hemos encontrado que Siglec-1 está implicado en la captura de partículas no infecciosas de Ébola (VLPs) por parte de estas células, especialmente tras la activación por IFN-α y LPS. Además, las VLPs capturadas se acumulan en el mismo compartimento en el que previamente se había detectado VIH-1. Siglec-1 también facilita la entrada citoplásmica del virus en las DCs, así que hemos determinado la capacidad de los nuevos mAbs contra Siglec-1 para interferir en este proceso, y hemos visto que dichos mAbs bloquean tanto la captura como la entrada citoplásmica de VLPs de Ébola en células mieloides activadas. En general, la actividad de los mAbs contra Siglec-1 inhibe el acceso de retrovirus y filovirus en las células mieloides, cosa que indica su potencial uso como agentes antivirales de amplio espectro.
Dendritic cells are key inducers of specific adaptive immune responses due to their capacity to capture, process and present pathogen-derived antigens to T lymphocytes. However, they might also contribute to early HIV-1 dissemination by capturing HIV-1 particles and transmitting them to target CD4+ T cells, a process known as trans-infection. This mechanism relies on the expression of Siglec-1 receptor (CD169), which recognizes sialylated gangliosides on the viral membrane. Siglec-1 is potently up-regulated upon dendritic cell stimulation with interferon-alpha and lipopolysaccharide, which are both immune-activating factors present during the course of HIV-1 infection. Here, we demonstrated that interferon-alpha secreted by HIV-1-infected plasmacytoid dendritic cells and autocrine interferon-alpha secreted by myeloid cells in response to lipopolysaccharide up-regulate Siglec-1 on dendritic cells. Importantly, plasmacytoid dendritic cells derived from women secreted higher amounts of interferon-alpha than those derived from men, highlighting the relevance of studying HIV-1 trans-infection in key female tissues for HIV-1 acquisition. Thus, we next studied the role of Siglec-1 in HIV-1 transmission mediated by primary dendritic cells directly isolated from cervical tissues, identifying a subset of cervical myeloid cells that expressed Siglec-1 and captured HIV-1 particles in a Siglec-1-dependent manner. This capacity was enhanced upon activation with interferon-alpha. Moreover, HIV-1 cell-to-cell transmission mediated by these cells could be efficiently blocked using an anti-Siglec-1 monoclonal antibody, indicating the potential use of antibodies directed against Siglec-1 in prevention of sexually transmitted HIV-1 acquisition in women. Thus, we generated a set of new anti-Siglec-1 monoclonal antibodies with the capacity to block dendritic cell-mediated HIV-1 trans-infection. Five new clones were produced, demonstrating high affinity for different epitopes located in the N-terminal region of Siglec-1 receptor. Moreover, they efficiently blocked HIV-1 capture and trans-infection mediated by dendritic cells, indicating their potential use in microbicidal strategies targeting this type of viral cell-to-cell transmission. Aside from HIV-1, dendritic cells can play important roles in the pathogenesis of other viruses, including Ebola and Marburg filoviruses. In contrast to HIV-1, dendritic cells are permissive to filoviral infection and act as early targets in viral pathogenesis. The host factors governing filoviral entry into these cells are not fully characterized, but both Ebola and Marburg are enveloped viruses that incorporate sialylated gangliosides during the budding process. Moreover, Siglec-1-activating factors such as interferon-alpha and lipopolysaccharide have been found during Ebola virus disease. Thus, we investigated the role of Siglec-1 in filoviral entry into dendritic cells. We found that Siglec-1-mediated capture of non-infectious Ebola virus-like particles into these cells, especially upon interferon-alpha and lipopolysaccharide activation. Interestingly, captured Ebola virus-like particles accumulated in the same cellular compartment where HIV-1 was previously detected. Siglec-1 also facilitated Ebola cytoplasmic entry into dendritic cells, so we tested the capacity of novel anti-Siglec-1 monoclonal antibodies to interfere with this process. We found that capture and cytoplasmic entry of Ebola virus-like particles into activated myeloid cells was blocked by these novel antibodies. Overall, the activity of anti-Siglec-1 monoclonal antibodies inhibits the access of both retroviruses and filoviruses into myeloid cells and suggests their potential use as broad-spectrum antiviral agents.

Natural killer (NK) cells are innate lymphocytes with inherent ability to eliminate infected cells and produce several cytokines/chemokines. They express surface receptors to sense environment and interact with other immune cells including the Dendritic cells (DC). Reciprocally, DCs are also shown to activate NK-cells. NK/DC cross-talk is well-documented, yet the molecular interactions and the diverse NK-cell activities regulated by DC remain unclear. Several target proteins such as MHC-1, Qa-1 mediate NK-cell target recognition. One such antigen, Ocil/Clr-b functions as a cognate ligand of NKR-P1B/D, NK-inhibitory receptor. In first aim of my study, I documented that deficiency of Ocil/Clr-b expression not only augmented the sensitivity of DC towards NK-cell cytotoxicity but also regulated the development of mature NK-cells. Thus suggesting NKR-P1B/D:Ocil to be another receptor:ligand system, besides Ly49:MHC-1, that regulates NK-cell responsiveness. Src homology region 2-containing protein tyrosine phosphatase-1 (SHP-1) transmits inhibitory signals of the specific NK-inhibitory receptors, including NKRP-1B/D. SHP-1 silenced NK-cells showed unaffected target recognition towards prototypic target cells in this study. In addition, these cells also displayed an unexpected phenotype of self-killing in-vitro, thus implicated SHP-1 as an important regulator of some other unappreciated NK-cell functions. The data from my third study suggest that DCs are directly implicated in the induction of NK-cell migration. In summary, using a novel live-cell imaging microfluidic platform and conventional transwell migration assay this project established a clear molecular link between DC-derived soluble factors such as IP-10 and NK cell-chemokine receptor such as CXCR3. Previously, GM-CSF was shown as an inflammatory cytokine, involved in the development of DC as well as in mediating Th-1 immune responses. In this study I found that GM-CSF regulates NK-cell migration negatively. Lastly, the fourth aim of my thesis highlighted the critical role of immature-DC in the induction of maturation receptors (NK1.1 & Ly49) on differentiating NK-cells. I successfully established a multi-stage in-vitro NK-cell differentiation model and found that differentiating NK-cells required an active engagement with DCs, in addition to the soluble factors. I believe my PhD project findings would impact the existing knowledge to harness DC-based NK cell therapies in clinical settings.
October 2014

Renal Cell Carcinoma (RCC) has a very high rate of mortality since it does not respond to conventional therapies such as chemotherapy and radiation therapy. Furthermore, in the majority of cases, metastases are already present at the time of diagnosis. The objective of our study is to develop a noval treatment for RCC, using a dendritic cell (DC) vaccine. An animal model of RCC, RENCA, was used to develop the vaccine.

The cross-talk between DC and NK cells is relevant for immune responses to infectious agents and tumors. However, the molecular basis of such interaction was largely unknown. Here we defined the nature of the signals involved in DC-mediated NK cell activation, focusing on LPS stimulation, thereby mimicking the context of bacterial infections. NK cells were not able to directly sense LPS, but required TLR4-equipped accessory cells. We demonstrated that DC produce IL-2, IL-18 and IFN-β in response to LPS, and this is the minimal set of cytokines necessary and sufficient to activate NK cells in terms of IFN-γ release in vitro and in vivo. However, DC-derived IL-18 was released only in the presence of NK cells. Indeed, LPS stimulation alone was not able to trigger the secretion of IL-1 family members. In this respect, NK cells provided the inflammasome-activating signal required for full processing and secretion of IL-18. NK cell stimulatory capability seemed to be restricted to the CD8-negative DC subset. Differently from IFN-γ release, cytotoxic responses were enhanced only by IFN-β but not by IL-2 or IL-18.

Dendritic cells (DC) have a fundamental role in priming naive T-cell responses and a suspected importance in the regulation of central and peripheral tolerance. Before DC can be responsibly used in human clinical trials, the generation of both immature and mature subsets must be standardised and their in vivo migratory and functional characteristics explored. The generation of human blood monocyte-derived DC in either fetal calf serum (PCS) or autologous plasma (HP) were compared. Phenotypic and functional assays demonstrated that DC derived from either system were similarly immature and underwent comparable maturation in response to LPS, TNF-α and MCM (decreasing potencies.) Furthermore, we demonstrated that adherent cells from HP cultures were likely DC but failed to react with the anti-rat CD8 antibody OX-8 which labels nonadherent DC. DC grown from mouse bone marrow (BMDC) using GM-CSF (GM) plus (IL4) were capable of undergoing further maturation with TNF-α or LPS. In contrast, the growth of BMDC in GM alone, gave rise to N418+ immature DC which could not be matured subsequently using TNF-α, LPS or IL1-β under our conditions. The migration of immature and mature BMDC was compared after IV injection. Fluorochrome labelled cells were found in splenic T-cell areas 24 hours after injection of all DC subsets into either syngeneic or allogeneic hosts. Furthermore, DC could be re-isolated from spleen and characterised by FACS analysis at various times after administration. Tc-^99m radiolabel studies demonstrated similar quantitative migration of BMDC subsets and primary splenic DC (LODACS) to peripheral tissues (spleen, liver and lung) by 24 hours after injection. Emerging evidence suggests that inhibition of costimulatory signalling during antigen presentation may lead to specific unresponsiveness. The ability of immature versus mature donor strain DC pre-treatment to alter cardiac allograft survival was investigated. Only GM-DC (immature) but not more mature GM/IL4-DC subsets were capable of inducing significant graft survival prolongation (MST>100d). Furthermore, the effect was dependant upon pre-treatment 7d before transplantation and was strain specific. The CD4+ T-cell priming patterns of immature versus mature BMDC were investigated using TCR transgenic mice recognising OVA plus MHCII. Mature OVApulsed DC were able to induce antigen-specific T-cell proliferation, activation marker upregulation and intracellular IL2, IL4 and TNF-α production, while immature GMDC proliferation was less, activation marker expression limited, and no IL4 seen. Our findings represent the first demonstration that cytokine cultured DC migrate similarly to primary DC after IV injection. Furthermore, the comparable migratory patterns of immature and mature BMDC subsets contrasts with differences in CD4+ T-cell priming responses in spleen and the unique ability of immature GM-DC to selectively induce cardiac allograft prolongation.

Most currently defined hematopoietic progenitor pools are heterogeneous, contributing to uncertainty regarding the development of certain blood cells. The origins of plasmacytoid dendritic cells, for instance, have long been controversial and progenitors exclusively committed to this lineage have never been described. We show here that the fate of hematopoietic progenitors is determined in part by their surface levels of 9-O-acetyl sialic acid. Pro-plasmacytoid dendritic cells were identified as lineage negative 9-O-acetyl sialic acid low progenitors that lack myeloid and lymphoid potential but differentiate into pre-plasmacytoid dendritic cells. The latter cells are also lineage negative, 9-O-acetyl sialic acid low cells but are exclusively committed to the plasmacytoid dendritic cell lineage. Levels of 9-O-acetyl sialic acid provide a distinct way to define progenitors and thus facilitate the study of hematopoietic differentiation.

Human immunodeficiency virus (HIV) infection is a worldwide epidemic where infected individuals usually develop acquired immunodeficiency syndrome (AIDS). HIV is primarily spread by sexual transmission across mucosal tissue where dendritic cells (DC) reside. DC regulate immune responses through their unique ability to capture antigen, migrate to lymphoid tissue, and activate naive T cells. In this Thesis, we have investigated whether HIV influences the migration of DC, thereby influencing their capacity to regulate immune function and facilitate transport of HIV to T cell rich lymphoid tissue. Transmigration assays demonstrated that the predominant HIV strain during primary infection, R5 HIV-1, was chemotactic for immature DC (iDC). Addition of soluble CD4 enhanced iDC migration to R5 HIV, presumably by binding to R5 HIV and altering the conformation to enhance binding to CCR5. Our results suggested that iDC migrated specifically to R5 and not X4 HIV gp120, through interactions between the extracellular loop-2 (ECL-2) domain of CCR5 with the V3 loop region of R5 gp120. iDC prepared from HIV-infected subjects were shown to have impaired chemotaxis to inflammatory chemokines compared with iDC from healthy individuals. Furthermore, the level of inhibition appeared to be proportional to the severity of disease progression in HIV infected subjects. Interestingly, chemotaxis of iDC from long-term non-progressor individuals was similar to normal individuals, whereas migration of iDC from typical progressors was greatly impaired. These differences did not appear to be related to the level of CCR5 expression or patient viral load. The protease inhibitor Indinavir used in antiretroviral therapy, limited DC trans-endothelial migration to chemokines, reduced DC-SIGN expression and increased CD83 on iDC. The results suggested that Indinavir inhibited proteases necessary for DC migration by adversely affecting interactions between DC-SIGN, VLA-4 and VLA-5 and ligands on the endothelium and underlying fibronectin matrix. A novel method has been successfully developed for amplifying rare HIV-specific CDS cells using DC transfected with HLA antigens matching HIV-infected subjects. This has enabled us to amplify HIV-specific CDS T cells by 10- to 60-fold. This may help us to clone and characterise HIV-specific CDS T cells from highly exposed persistently seronegative (HEPS) individuals. In summary, the results in this Thesis demonstrate that R5 HIV mimics chemokines to subvert the natural trafficking of DC. Indeed, we have shown that DC from typical progressors have severely impaired migration. This may have serious consequences on DC immunoregulation, compromising the immune function of these infected individuals.

The focus of this project is a human-specific C-type lectin with potential roles in cell signalling: blood dendritic cell antigen 2 (BDCA-2). BDCA-2, a plasmacytoid dendritic cell-specific molecular marker, has been evaluated as a therapeutic target against auto-immune disorders, because antibodies to BDCA-2 inhibit the production of type I interferon. Accordingly, key goals of the project were to identify endogenous ligands for BDCA-2, to characterise the mechanism of ligand binding and ultimately to determine how ligands stimulate signalling pathways. A combination of BDCA-2 affinity chromatography column and mass spectrometry revealed that α2 macroglobulin and immunoglobulins, IgA, IgM and IgG are potential endogenous ligands in human serum. Competition binding studies conducted to characterise the binding affinity for each glycoprotein demonstrated that IgA has the highest affinity. Strategies for biochemical development of defined glycoforms of IgG Fc domain were established. The Chinese hamster ovary cell system for expression of Fc domain and the activity of enzymes necessary for chemoenzymatic glycoengineering have been tested. BDCA2 organisation in the cell membrane was studied by development of a transfected cell system which was analysed by affinity purification of BDCA-2 followed by analysis of protein-protein interactions. The results demonstrate that it is likely that BDCA-2 assembles with Fc receptor γ-chain in a 2:2 complex.

Apoptotic cells are an important source of many autoantigens and the realization that dendritic cells (DCs), the main antigen presenting cell of the adaptive immune system, not only internalise such drying cells but present antigen derived from them had important implications for our understanding of autoimmunity, tumour immunology and anti-viral responses. The aim of this thesis was to explore the likely consequences of clearance of cells dying by constitutive apoptosis by myeloid phagocytes, with particular emphasis on the mechanism and outcome of DC clearance and the implications for autoimmunity. Firstly it will show that DCs generated from murine bone marrow demonstrated many characteristics attributed to DCs in vivo including endocytosis and phagocytosis and mature upon receipt of danger signals such as endotoxin. However, internalisation of apoptotic cells does not augment DC maturation but rather inhibits subsequent responses to LPS, rendering these DCs less efficient than their neighbours at stimulating naïve T cells. These effects do not appear to be due to secretion of inhibitory cytokines such as TGFβ or IL10 and are not dependent on CD36 or β3/5 integrins, receptors thought to be involved in DC internalisation of apoptotic cells. In addition apoptotic cells inhibit LPS driven IL12 production by ex vivo DCs and in mice immunised with apoptotic cells in vivo. Furthermore, macrophages, likely to be found in high numbers in the inflammatory site also inhibit DCs and these inhibitory effects could be further augmented by the presence of apoptotic cells. Taken together these data demonstrate that internalisation of apoptotic cells by myeloid phagocytes modulates the adaptive immune response and suggests that the likely outcome of internalisation of cells dying by constitutive apoptosis will be tolerance rather than autoimmunity.

Langerhans’ cells (LC) are the dendritic cells (DC) of the epidermis and, as sentinels of the immune system, act as a bridge between the innate and adaptive immune responses. When LC, and other DC, recognise an antigen or pathogen they mature and are stimulated to migrate to the lymph nodes, where they orchestrate immune responses. Pathogen derived toll-like receptor (TLR) ligands, and chemical allergens, are recognised as being potentially harmful and stimulate LC to mobilise and mature. Cytokine signals, including tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-18, all induce LC migration and are required for initiating LC mobilisation in response to certain contact allergens. Subsequently, chemokines promote the migration and localisation of LC within the draining lymph nodes. Chemokines are also involved in shaping the adaptive immune response by promoting differential T cell activation, such as T helper (Th)1 or Th2 responses, which are involved in immunity against different pathogens, and also in the development of different types of chemical allergy. The hypothesis is that LC phenotype (activation, migration and chemokine production), is dependent on the nature of the challenge ligand. The murine LC-like cell line XS106 was used to investigate the response of LC following stimulation with TLR ligands and chemical allergens. In addition, LC migration in response to these stimuli was investigated in vivo and the role of TNF-α was examined using mice deficient in either one of the two TNF-α receptors; TNF-R1 or TNF-R2.XS106 cells and freshly isolated LC were associated with a selective type 2 immune response, as determined by preferential expression of type 2 associated chemokines. Furthermore, XS106 cells responded to type 2, but not to type 1, associated TLR ligands. In contrast, all of the TLR ligands tested induced the migration of LC from the epidermis in vivo. Similarly, chemical allergens failed to induce a maximal response of XS106 cells, but did induce the migration of LC in vivo. There were differences in LC migration between the two mouse strains tested, with C57/BL6 strain mice being less responsive to administration of TNF-α and the contact allergen oxazolone compared with BALB/c strain mice. However, C57/BL6 and BALB/c strain mice responded similarly after exposure to the contact allergen 2,4-dinitrochlorobenzene (DNCB). Furthermore, DNCB was able to induce LC migration in mice deficient in TNF-R2, the TNF-α receptor expressed by LC.Collectively, these data suggest a paradigm in which keratinocytes and LC in the epidermis have distinct roles in promoting type 1 and type 2 immune responses, respectively. Therefore, LC may not be activated directly by certain TLR ligands or chemical allergens that are associated with type 1 responses. Consequently the migration of LC in vivo after encounter with these stimuli may be secondary to interaction with keratinocytes, or with other skin resident cells. Together, LC and keratinocytes allow the epidermis to respond to a range of pathogens, in addition to developing the necessary type 1 and type 2 responses. Chemical allergens may have divergent cytokine signalling requirements for the induction of LC migration as, unlike other contact allergens (and other stimuli such as irritant and ultraviolet [UV]B exposure), DNCB may induce LC migration independently of TNF-α.

The human immunodeficiency virus (HIV)-1 is the causative agent of acquired immune deficiency syndrome (AIDS) worldwide. Till date there are no vaccines or cure for this infection as the virus has adapted myriad ways to remain persistent in the host where it causes severe damage to the immune system. Both humoral and cellular immune responses are mounted against HIV-1 during the initial phase of infection but fail to control viral replication as these responses are severely depleted during disease progression. Of great importance in HIV-1 research today is the in depth understanding of the types of immune responses elicited, the mechanisms behind their decline and how these responses can be  maintained overtime. The focus of this thesis was to examine the possibility of priming HIV-1 specific T cell responses in vitro from whole viral particles and in detail, scrutinize the type of T cell responses and epitope specificities generated. Next was to investigate in vitro the factors responsible for impaired immune responses in HIV-1 infected individuals. We were also interested in understanding the underlying mechanisms through which HIV-1 initiate suppression of T cell functionality. Results showed that using HIV-1 pulsed monocyte derived dendritic cells (DCs), we were able to prime HIV-1 specific CD4+ and CD8+ T cells from naïve T cells in vitro. The epitopes primed in vitro were located within the HIV-1 envelope, gag, and pol proteins and were confirmed ex vivo to exist in acute and chronically infected individuals. We established that many of the novel CD4+ T cell epitopes primed in vitro also existed in vivo in HIV-1 infected individuals during acute infection. These responses declined/disappeared early on, which is in line with HIV-1 preferential infection of HIV-1 specific CD4+ T cells. Besides declining HIV-1 specific T cell responses, many HIV-1 infected individuals also have impaired T cell functionality. We established that one reason behind the decline and impairment in immune responses was the increased expression of inhibitory molecules PD-1, CTLA-4, and TRAIL on HIV-1 primed T cells. These T cells had the capacity to suppress new responses in a cell-cell contact dependent manner. The ability of the HIV-1 primed T cells to proliferate was severely impaired and this condition was reversed after a combined blockade of PD-1, CTLA-4 and TRAIL. Furthermore, more inhibitory molecules TIM-3, LAG-3, CD160, BLIMP-1, and FOXP3 were also found increased at both gene and protein levels on HIV-1 primed T cells. Additionally, we showed decreased levels of functional cytokines IL-2, IFN-γ and TNF-α, and the cytolytic proteins perforin and granzyme in DC T cell priming cocultures containing HIV-1. This could be as a result of the decreased T cell activation or impaired production by T cells. The mechanisms responsible for the elevated levels of inhibitory molecules emanated mainly from the P38MAPK/STAT3 pathways. Blockade of these pathways in both allogeneic and autologous DC-T cell assays significantly suppressed expression of inhibitory molecules and subsequently rescued T cell proliferation. In conclusion, HIV-1 pulsed DCs have the capacity to prime HIV-1 specific responses in vitro that do exist in HIV-1 infected individuals and we found evidence that many of these responses were eliminated rapidly in HIV-1 infected individuals. HIV-1 triggers through P38MAPK/STAT3 pathway the synthesis of inhibitory molecules, namely CTLA-4, PD-1, TRAIL, TIM-3, LAG-3, CD160, and suppression associated transcription factors FOXP3, BLIMP-1 and DTX1. This is followed by decreased T cell proliferation and functionality which are much needed to control viral replication.

L’esclerosi múltiple (EM) és considerada una malaltia autoimmune crònica que afecta el sistema nerviós central. Els tractaments actuals pels pacients amb EM remitent-recurrent (EM-RR) redueixen la freqüència dels brots i l’activitat inflamatòria general, però el seu efecte en la progressió de la malaltia encara no està clar. Per això és necessari desenvolupar noves aproximacions terapèutiques més específiques per tal de modificar el curs de la malaltia. En aquest sentit, una estratègia terapèutica interessant és la inhibició o supressió específica de les cèl·lules T autoreactives amb la finalitat de restablir la tolerància en malalties autoimmunitàries com l’EM. L’objectiu principal d’aquest treball ha estat la generació i caracterització de cèl·lules dendrítiques tolerogèniques (tolDCs) de pacients amb EM-RR, carregades amb pèptids de la mielina, com a eina terapèutica per restablir la tolerància als antígens de la mielina en aquests pacients. Els resultats obtinguts mostren que la utilització de diferents agents immunosupressors i estímuls de maduració permet generar tolDCs de grau clínic amb diferències rellevants per a la seva aplicació terapèutica. S’ha valorat la viabilitat, el fenotip, el perfil de les citocines produïdes, l’estabilitat i la funcionalitat d’aquestes tolDCs. La comparació de diferents agents tolerogènics de grau clínic (dexametasona, rapamicina i vitamina-D3) ha permès observar que les tolDCs tractades amb dexametasona mostren un fenotip semi-madur i produeixen grans quantitats de IL-10; que les tolDCs tractades amb rapamicina redueixen la secreció d’interferó-γ dels limfòcits T co-cultivats i expandeixen cèl·lules T reguladores; i finalment que les tolDCs obtingudes amb vitamina-D3 presenten un fenotip semi-madur, produeixen IL-10, i disminueixen la producció d’interferó-γ dels limfòcits T. Aquestes característiques, juntament amb la reproductibilitat dels resultats en les diferents mostres, fan que la vitamina-D3 hagi estat considerat l’agent més adient per generar tolDCs com a eina terapèutica per a l’EM. Tenint en compte l’estímul de maduració, el còctel de citocines pro-inflamatòries −composat per TNF-α, IL-1β i PGE-2− és considerat com a estímul maduratiu òptim per a la generació de tolDCs (obtingudes amb vitamina-D3), ja que aquestes tolDCs han estat les úniques en mostrar una estabilitat funcional i una capacitat de suprimir la resposta immunitària in vitro. La generació de tolDCs de pacients amb EM-RR (generades amb vitamina-D3 i madurades amb el còctel de citocines pro-inflamatòries) no ha mostrat diferències significatives en comparació amb tolDCs obtingudes de controls sans, ambdues presentant un perfil tolerogenic. Les tolDCs de pacients amb EM-RR carregades amb pèptids de la mielina són capaces d’induir un estat d’hipo-resposta/anergia, estable i antigen-específica, als limfòcits T autòlegs i reactius a la mielina in vitro. En resum, aquest treball ha conduit al desenvolupament d’un protocol per generar tolDCs de grau clínic i ha establert les bases per al seu ús com a eina terapèutica amb la finalitat de restablir la tolerància en pacients amb EM-RR.
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system. Current treatments for relapsing-remitting multiple sclerosis (RR-MS) patients decrease the frequency of relapses and reduce inflammatory activity in a nonspecific manner, but their effect on disease progression is still unclear. Therefore, in order to modify the course of MS, new and more specific therapeutic approaches are necessary. Specific inhibition or deletion of autoreactive T cells represents an interesting goal for restoring peripheral tolerance in autoimmune diseases such as MS. The main goal of this work has been to generate and characterize tolerogenic dendritic cells (tolDCs) from RR-MS patients, loaded with myelin peptides as specific antigen, as a therapeutic tool to re-establish tolerance to myelin-antigens in these patients. Our results show that using different immunosuppressive drugs and different maturation stimulus led to the generation of clinical-grade tolDCs products with differences that are relevant to therapeutic applicability. We evaluated the viability, phenotype, cytokine profile, stability and functionality of these tolDCs. The comparison of different pharmacological grade tolerogenic agents (dexamethasone, rapamycin and vitamin-D3) led to the observation that dexamethasone-treated DCs showed a semi-mature phenotype and high IL-10 secretion; that rapamycin-treated DCs impaired IFN-γ in co-cultured T cells and expanded T regulatory cells; and finally that vitamin-D3-treated DCs presented a semi-mature phenotype, produced IL-10, and reduced IFN-γ in T cells. These features, along with their reproducibility among different samples, made consider vitamin-D3 as the most convenient of the three compared agents to generate tolDCs for MS therapy. Regarding the maturation of tolDCs, the cytokine cocktail (composed by TNF-α, IL-1β and PGE-2) was determined as the optimal maturation stimulus to generate tolDCs (induced by vitamin-D3 treatment), since these tolDCs were the unique exhibiting functional stability and capability to suppress an immune response in vitro. The generation and characterization of tolDCs from RR-MS patients (generated with vitD3 and maturated with the pro-inflammatory cytokine cocktail), showed no significant differences compared with tolDCs generated from healthy controls, both presenting a tolerogenic profile. Importantly, myelin peptide-loaded tolDCs from RR-MS patients induced antigen-specific and stable hyporesponsiveness in autologous myelin-reactive T cells in vitro. Altogether this work has conducted to the development of a protocol to generate clinical-grade tolDCs and set up the bases for their use as a therapeutic tool to re-establish tolerance in RR-MS patients.

The intention of the work described in this thesis was to identify whether the Notch signalling pathway is utilized by antigen presenting cells in order to influence CD4+ adaptive immune responses. The notion that Notch proteins may be involved in polarising CD4+ T cells is relatively recent and most of the work that had been done in this area so far has concentrated on the consequences of Notch signalling within T cells. In contrast, the work that I have done has focussed on Notch ligand expression by antigen presenting cells and addresses the question whether Notch signalling is a redundant, necessary or irrelevant tool in the arsenal of antigen presentation.

This is a comprehensive study of human kidney proximal tubular epithelial cells (PTEC) which are known to respond to and mediate the pathological process of a range of kidney diseases. It identifies various molecules expressed by PTEC and how these molecules participate in down-regulating the inflammatory process, thereby highlighting the clinical potential of these molecules to treat various kidney diseases. In the disease state, PTEC gain the ability to regulate the immune cell responses present within the interstitium. This down-regulation is a complex interaction of contact dependent/independent mechanisms involving various immuno-regulatory molecules including PD-L1, sHLA-G and IDO. The overall outcome of this down-regulation is suppressed DC maturation, decreased number of antibody producing B cells and low T cell responses. These manifestations within a clinical setting are expected to dampen the ongoing inflammation, preventing the damage caused to the kidney tissue.

Dendritic cells play an essential role in activating immune responses upon recognition of pathogens. This results in maturation and migration to the lymph nodes, where T cells are stimulated by upregulated antigen presentation, co-stimulation and cytokine secretion. DCs are also considered important in inhibiting inappropriate immune responses against self-peptides which could lead to the development of autoimmunity. This has been attributed to DCs that demonstrate inhibited co-stimulation and cytokine secretion. It has been previously shown that the continuous ligation of an immunomodulatory receptor, LILRB1, during DC differentiation results in such a DC population that demonstrates an immature phenotype even after exposure to bacterial components and resulted in inhibiting primary T cell responses. The mechanisms by which LILRB1-DCs promote tolerance are, therefore, here investigated. Previous studies revealed significantly altered expression for a large number of gene targets which varied from immune to cytoskeletal and bone-related functions. One of these includes DcR3, a soluble protein with a poorly defined role in immune regulation. It is here demonstrated that DcR3 has a positive role in the induction of IL-17, a cytokine implicated in autoimmunity. However, DcR3 was not secreted by LILRB1-DCs, possibly accounting for some of their tolerogenic functions. In addition, the expression of several cytoskeletal proteins was significantly changed in response to LILRB1 ligation and was associated with decreased ability for phagocytosis and migration. Lastly, it has been recently identified that DCs are able to trans-differentiate into osteoclasts, the main cell type linked with inflammatory bone disorders, such as rheumatoid arthritis. It is here shown for the first time that ligation of LILRB1 inhibits this process and results in decreased bone resorption. Overall, these data provides evidence that ligation of LILRB1 on DCs affects normal inflammatory functions and suggests its potential for the development of new treatments against several autoimmune diseases.

Embryonic stem cells (ESC) possess immune privileged properties and have the capacity to modulate immune activation. ESCs can persist across allogeneic immunological barriers, prevent lymphocyte proliferation in mixed lymphocyte reaction (MLR) assays and can promote graft acceptance. However, clinical application of live ESC to treat immunological disorders is not feasible as live ESC can form teratoma in-vivo. In order to harness these properties of ESCs without adverse risk to patients, we hypothesized that ESC derived extracts may retain immune modulatory properties of whole cells and therefore could be used to abrogate allo-immune responses. We found addition of ESC-extracts from human lines H1 and H9, significantly prevented T cell proliferation in allogeneic MLRs. These results were confirmed using murine J1 ESC line. In-vitro studies showed human ESC EXT were able to modulate maturation of human monocyte derived dendritic cells (DC) by suppressing up-regulation of important co-stimulatory and maturation markers CD80, HLA-DR and CD83. In addition, DCs educated in the presence of human ESC extracts significantly lost their ability to stimulate purified allogeneic T cells compared to control extract treated DCs. We also determined that ESC extracts have an independent effect on T cells. ESC extracts prevented T cell proliferation in response to anti CD3/CD28 stimulation. In MLRs, ESC derived factors significantly down-regulated IL-2 and IFN-γ expression, while up-regulating TGF-β and Foxp3 expression. Furthermore, lymphocytes and purified T cells activated with anti-CD3/CD28, ConA and PMA proliferated poorly in the presence of ESC derived factors, while proliferation in response to ionomycin was not affected. Western blot analysis indicated that ESC derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. Therefore, ESC extracts have potent immune suppressive properties and may have clinical applications in ameliorating transplant rejection and autoimmune conditions.

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-cell Chronic Lymphocytic Leukaemia were generated by culture in Interleukin-4 and Granulocyte Macrophage-Colony Stimulating Factor. Lysate-pulsed autologous dendritic cells were used as antigen presenting cells in co-culture with autologous B-cell Chronic Lymphocytic Leukaemia T-cells. B-cell Chronic Lymphocytic Leukaemia T-cells stimulated with B-cell Chronic Lymphocytic Leukaemia lysate-pulsed autologous dendritic cells showed a significant increase in cell surface expression of Interleukin-2 Receptor (CD25), Interferongamma secretion and cytotoxicity against autologous B-cell Chronic Lymphocytic Leukaemia B-cell targets hut not against targets from healthy volunteers. Responses were only stimulated by the B-cell Chronic Lymphocytic Leukaemia B cell lysate. Cytotoxicity was Major Histocompatibility Complex Class II restricted. The addition of maturation agents such as Lipopolysaccharide, Tumour Necrosis Factor-alpha and Polyriboinosinic Polyribocytidylic Acid to monocyte derived dendritic cells was unsuccessful at increasing anti-tumour responses. Pre-treatment of T cells with Interleukin-15 before stimulation by lysate pulsed autologous dendritic cells increased numbers of activated cells, cytokine secretion and specific cytotoxicity to B-cell Chronic Lymphocytic Leukaemia 8-cells. Fusion of monocyte derived dendritic cells and B-cell Chronic Lymphocytic Leukaemia B-cells generated both Major Histocompatibility Complex Class I and Class II restricted cytotoxicity to B-cell Chronic Lymphocytic Leukaemia B-cell targets. When B-cell lysates were analysed using reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, a B-cell Chronic Lymphocytic Leukaemia specific hand at 42,000 Dalton and other patient specific bands were observed. Only the 65,000 Dalton and 42,000 Dalton hands were capable of stimulating comparable T cell responses as the whole lysate. The 65,000 Dalton band from normal healthy volunteers showed a dominant peptide that closely matched Human Serum Albumin. The 42,000 Dalton band from B-cell Chronic Lymphocytic Leukaemia patients showed a possible match with Human Actin.

The quality of life of a significant proportion of IBD patients is poor as a result of persistent disease activity and repeated surgery, among others. Current treatments for Crohn’s disease are not able to neither prevent this serious impact nor improve the long term prognosis of a significant proportion of patients. Therefore, new therapeutic approaches are needed in order to modify the immune response of these patients. We hypothesize that administration of ex-vivo generated autologous tol-DCs to Crohn’s disease patients may arrest Th1 lymphocyte proliferation and therefore may restore specific tolerance against non-pathogenic antigens in the gut. The overall objective of this thesis was to generate and characterize tol-DCs for the purpose of implementing an autologous immunotherapy treatment for Crohn’s disease patients. In the first study, we described the generation of tol-DCs from healthy donors and Crohn’s disease patients by use of clinical-grade reagents in combination with dexamethasone as immunosuppressive agent and characterized their functional properties. Our main findings demonstrated that the combination of dexamethasone with a specific cytokine cocktail yields clinical-grade DCs with the following characteristics: a semi-mature phenotype, a pronounced shift towards anti-inflammatory versus inflammatory cytokine production and low T-cell stimulatory properties. This characteristic tolerogenic profile is maintained when tol-DCs are activated using heat-inactivated Gram-negative bacteria as maturative stimulus. Whole microorganisms contain multiple PAMPs capable of stimulating DCs by different pathways. Our results clearly showed a strong inhibitory effect on DC phenotype, a robust inhibition of pro-inflammatory cytokines, increased IL-10 secretion, and inhibition of T-cell proliferation and Th1 induction. Interestingly, we showed that tol-DCs have reduced immunogenic capacity in autologous, allogeneic and antigen-specific T-cell responses. We further evaluated the ability of tol-DCs to induce CD4+ T-cell hypo-responsiveness. Our results demonstrated that T-cells or antigen-specific T-cells previously cultured with tol-DCs are anergic exhibiting a reduced capacity to proliferate as well as reduced IFN-gamma secretion when rechallenged with fully competent mDCs. With regard to tol-DCs clinical application, we importantly found that their tolerogenic properties remain stable after washing out dexamethasone and subsequent restimulation with LPS, CD40L or different Gram-negative enterobacteria strains. All these properties led us to conclude that this cell product is suitable to be tested in clinical trials of immune-based diseases such as Crohn’s disease. We further identified a positive biomarker for tol-DCs, MERTK receptor is highly expressed on clinical grade dexamethasone-induced human tol-DCs and contributes in their tolerogenic properties. Our results demonstrated that MERTK expression in human DCs is regulated by glucocorticoids and described a new function of this receptor in directly regulating T-cell response. Interestingly, our findings showed that neutralization of MERTK with monoclonal antibodies in allogeneic MLR cultures leads to increased T-cell proliferation and IFN-gamma production. The direct regulation of T-cell response was confirmed by the use of recombinant MERTK-Fc protein, used to mimic MERTK on DCs. Our results remarkably showed that MERTK-Fc suppresses naïve and antigen-specific memory Tcell proliferation and activation. These findings identified a new non-cell autonomous regulatory function of MERTK expressed on DCs. Additionally, we described that this regulation is mediated by the neutralization of MERTK soluble ligand PROS1. We also found that MERTK is expressed on T-cell surface and that PROS1 drives an autocrine pro-proliferative effect on these cells. In summary, the results of this work demonstrated that MERTK on DCs regulates T-cell activation and expansion through the competition for PROS1 interaction with MERTK in the T-cells. We showed that MERTK expression in human DCs has a key role in instructing adaptive immunity and identified MERTK as a potent suppressor of T-cell response. Therefore targeting MERTK may provide an interesting approach to effectively increase or suppress tolerance for the purpose of immunotherapy.
Esta tesis doctoral estudia el proceso de generación de células dendríticas tolerogénicas en grado clínico, con el objetivo de establecer un protocolo destinado al tratamiento de la enfermedad de Crohn. El estudio realizado ha permitido la caracterización de dichas células y sus propiedades tolerogénicas, incluyendo la descripción novedosa de un marcador de células tolerogénicas y el estudio de sus propiedades funcionales relacionadas con la inducción de tolerancia.

Dendritic cells (DC) play a major role in the detection of antigens, initiation of immunity and induction and regulation of tolerance. DCs are Bone Marrow (BM) derived and their development may be influenced by haematological malignancy in several ways. Firstly, myelodysplastic, myeloproliferative or leukaemic transformation of bone marrow progenitors may involve DC precursors directly, when they become part of a malignant clone, or indirectly when neoplastic expansion of other lineages compromises the development of DCs. Secondly, neoplasia of the dendritic cell lineage itself may occur in a heterogeneous group of histiocytic disorders including Langerhans cell histiocytosis (LCH) and Erdheim Chester disease (ECD). The first part of this thesis concerns the effect of haematological malignancies on the generation of DCs; in particular the relationship between DC, monocyte, B and NK lymphoid cell (DCML) deficiency, caused by GATA2 gene mutations, sporadic myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Comprehensive flow cytometric methods for profiling of the DCs and monocytes in the peripheral blood (PB) and peripheral tissues, and the CD34+ progenitors of the BM have been developed. Using these methods, it was possible to see that patients with the three diseases differed on several grounds. DCML deficiencies associated with a younger age of presentation, better preserved haemoglobin, neutrophils and platelets and much more severe defects of DCs, monocytes, and lymphoid cells. In patients with MDS, deficiencies are more moderate and mononuclear cell generation in AML patients is surprisingly preserved. Serum levels of Fms-like tyrosine kinase 3 ligand (FLT3L) also differed, with massively increased levels in DCML deficiency, stable levels in MDS and deficiencies in AML. Finally, strategies of grouping of AML patients have proven to be possible by using progenitor cell or DC related phenotypic markers which may correlate to known cytogenetic abnormalities. The second part of this thesis explores the origin of the cells of LCH and ECD, both neoplasia involving DC-like cells. RT-PCR assays were developed to detect the BRAFV600E mutation, which is present in ~60% of LCH and ECD viii cases. Surprisingly, it was seen that LCH and ECD express BRAFV600E in differing peripheral blood fractions. In LCH, 78-94% of all BRAFV600E alleles were found in HLA-DR+Lineage- cells, localised to the monocytes and CD1c+ myeloid DCs (mDCs). However in ECD, 80-82% of mutated alleles were recovered from the HLA-DR-Lineage- quadrant, localised to CD33+ early myeloid cells, and no mutation was found in the monocytes or mDCs. The final part of the thesis examines whether the circulating cells which carry the BRAFV600E mutation in LCH and ECD can actually develop into LCH-like cells. In order to do this, culture systems were developed with a variety of cytokines and conditions. These experiments showed that CD14+ and CD16+ monocytes and CD1c+ mDCs could all induce the Langerhans Cell (LC) markers CD1a and Langerin in response to GM-CSF, TGFβ and BMP7, however only the CD1c+ mDCs could express Langerin at the high levels seen in LCs, along with EpCam and Birbeck granules. Several techniques have been developed to study DCs in haematological malignancies. Throughout this thesis, these techniques have provided valuable data on the development and homeostasis of human DCs.