Thèses sur le sujet « Immuno evasione »

Il numero di pazienti affetti da leucemia mieloide acuta (LMA) trattati con trapianto di cellule ematopoietiche allogeniche (TCE-allo) è in costante aumento. L'efficacia terapeutica di questa procedura si basa principalmente sul trasferimento dal donatore al paziente di cellule immunitarie, in grado di riconoscere ed eliminare le cellule tumorali residue. Tuttavia, fino al 50% dei pazienti trapiantati con LMA va incontro a recidiva e la prognosi di questi pazienti rimane estremamente negativa. Pertanto, lo scopo del mio lavoro di tesi era quello di migliorare la comprensione attuale dell'immunobiologia della recidiva post-trapianto, studiando i) come le terapie e la leucemia stessa influenzano la ricostituzione immunitaria, ii) come perfezionare il rilevamento della ricomparsa della leucemia nella fase di malattia minima residua (MMR) e iii) come scoprire i meccanismi molecolari alla base dell’evasione della leucemia dal sistema immune. In particolare, presenterò per la prima volta i risultati di uno studio prospettico volto a valutare l'utilità clinica di monitorare il chimerismo specifico del paziente sul sangue periferico, anziché sul midollo osseo attualmente utilizzato, impiegando la PCR quantitativa (PCRq) per la diagnosi precoce delle recidive di leucemia dopo trapianto. In seguito, saranno presentati i risultati di due studi sulla dinamica della ricostituzione delle cellule NK e T dopo il TCE-allo. Entrambi gli studi mirano a comprendere i determinanti dell'insufficienza del sistema immunitario del donatore nel controllo della recidiva della malattia LMA con la possibilità di utilizzare le caratteristiche identificate come biomarcatori per prevedere la recidiva post-trapianto. Nelle ultime sezioni presenterò dati sia pubblicati che non pubblicati sui meccanismi biologici della recidiva della malattia post-trapianto, riferendo in che modo questa conoscenza possa essere facilmente tradotta in nuove opzioni terapeutiche per combattere la recidiva della malattia. In queste sezioni sono incluse due recensioni che ho scritto di recente, focalizzate, rispettivamente, sull'immunobiologia della recidiva post-trapianto e sulle attuali terapie epigenetiche all’avanguardia per la LMA e i loro effetti sul sistema immunitario.
The number of acute myeloid leukemia (AML) patients cured through allogeneic hematopoietic cell transplantation (allo-HCT) is constantly increasing. The therapeutic effectiveness of this procedure mainly relies on the transfer from the donor to the patient of immune cells, capable of recognizing and eliminating residual tumor cells. Still, up to 50% of transplanted AML patients will eventually relapse, and the prognosis of these patients remains extremely poor. Thus, aim of my thesis work was to improve current understanding on the immunobiology of post-transplantation relapse, by investigating i) how therapies and leukemia itself affect immune reconstitution, ii) how to refine detection of leukemia reappearance at the stage minimal residual disease (MRD), and iii) how to uncover the molecular mechanisms at the basis of leukemia immune evasion. In particular, I will first present the results of a prospective study aiming at evaluating the clinical utility of monitoring patient-specific chimerism on peripheral blood, instead of the currently used bone marrow specimens, employing quantitative PCR (qPCR) for the early detection of leukemia relapses after transplantation. Will next present the results of two studies on the dynamics of recovery of NK and T cells after allo-HCT. Both studies aim at understanding the determinants of donor immune system failure in controlling AML disease recurrence with the potential implications of using the identified features as biomarkers to predict post-transplantation relapse. In the last sections I will present both published and unpublished data on the biological mechanisms underlying post-transplantation disease relapse, reporting how this knowledge can be easily translated in novel therapeutic rationales to combat disease recurrence. Included in these sections are two recent reviews I authored, focused, respectively, on the immunobiology of post-transplantation relapse, and on current state-of-the art epigenetic therapies for AML and their effects on the immune system.

Glioblastoma multiforme (GBM) is the most common form of primary brain cancer and the current prognosis for patients is poor. New therapies are required that target the invasive cells that are characteristic of GBM. GBM is infiltrated by immune cells but, as with other cancers, immune evasion pathways minimise productive anti-tumour immunity. Natural killer (NK) cells are able to recognise and kill tumour cells and are being developed for the immunotherapy of other cancers. The aim of this work was to analyse the interaction between human NK cells and GBM cells in vivo and in vitro, as a prerequisite to future NK cell based immunotherapy of GBM. Analysis of the cell surface phenotype for GBM infiltrating NK and T cells revealed that the tumour microenvironment exerts localised immune evasion mechanisms which downregulate activation receptors and upregulate inhibitory receptors. The interaction of NK cells with patient-derived GBM stem cells, which are thought to be responsible for recurrent disease, was investigated in vitro. A high-throughput, multiplex flow cytometry-based screen of tumour cells revealed the expression of a number of cell surface molecules that regulate NK cell activation. Furthermore, GBM cells were more susceptible to NK cell lysis in vitro compared to a non-cancerous neural progenitor cell line, revealing specificity in the NK cell response. Furthermore, this screen identified potential mechanisms by which GBM might evade immune surveillance in vivo. Targeting these pathways and restoring functional immune surveillance provides a potential route for future immunotherapy of this disease. However, GBM patients often experience cerebral oedema and are treated with immunosuppressive corticosteroids, such as dexamethasone; this induces a similar immunosuppressed phenotype to that observed with the GBM infiltrating NK cells, and inhibits their lytic function. Gene expression profiling identified the transcription factor c-Myc as a key regulator of NK cell activation and as a hub for the immunosuppressive action of steroids and the immunosuppressive cytokine TGF-β. The demonstration that therapeutic steroids target the same pathway as TGF-β and induce immunosuppression has important implications for the use of steroids in patients undergoing immunotherapy.

Induction of the innate immune response by viral pathogens is characterized by a rapid production of Type I interferons (IFNβ/α). Recognition of viral components by Toll-like receptors (TLRs) or RIG-I-like receptors (RLRs), initiates multiple intracellular signalling cascades that culminate in the activation of the transcription factor NF-B and the interferon regulatory factors-3 and 7 (IRF-3 and IRF-7). As a consequence, these events lead to the production of immunoregulatory molecules, including Type I IFNs, pro-inflammatory cytokines and IFN-stimulated genes (ISGs), resulting in the inhibition of virus replication. Throughout evolution, viruses have developed strategies to subvert the innate immune response for their own benefit. Human immunodeficiency virus type-1 (HIV-1), the etiological agent of the acquired immunodeficiency syndrome (AIDS), has been shown to evade the innate immune response, resulting in disease progression. Thus, understanding the distinct mechanisms by which HIV-1 modulates TLRs and RLRs signaling pathways will ultimately lead to the development of novel strategies to inhibit HIV-1 replication and propagation. Studies have indicated that TLRs that signal via NF-B enhance HIV-1 replication. However, TLR4 stimulation triggers both NF-B and the IFN pathway and thus may have inhibitory effects on HIV-1 replication. Our first study was aimed at better understanding the role of TLR4 in HIV-1 replication. Therefore, we first characterized IRF-3 and IRF-7 activation following TLR4 stimulation. Our results demonstrate that the non-canonical TBK1 and IKKε are activated with distinct kinetics resulting in the activation of IRF-3 and subsequent induction of Type I IFNs. Thus, activation of the IFN pathway via TLR4 stimulation can provide a novel strategy to inhibit HIV-1 replication. Our second study sought to further delineate the different signaling pathways activated by HIV-1. Accordingly, transcriptional changes induced by distinct HIV-1 subtypes in immature dendritic cells were examined by microarray analysis. Our findings demonstrate that during the late phase of HIV-1 infection, a subset of genes is differentially regulated by the subtypes. In addition, this study highlights the important role of immature dendritic cells in HIV-1 replication and dissemination. Finally, given the importance of RLRs in the recognition of RNA viruses, the objective of the final study was aimed at investigating the evasion mechanisms employed by HIV-1 to counteract the initial antiviral response. Our results reveal that HIV-1 RNA is detected by the cytosolic receptor RIG-I. However, the HIV-1 viral protease sequesters RIG-I to the lysosomes and thus inhibits RIG-I-mediated antiviral response. Overall, the research presented in this thesis provides new avenues for developing novel therapeutic and preventive strategies to combat HIV-1/AIDS.
L'induction de la réponse immunitaire innée par des pathogènes viraux est caractérisée par une production rapide des interférons de Type I (IFNβ/α). Les Toll-like (TLR) ou RIG-like (RLR) récepteurs détectent divers composants viraux induisant multiples voies de signalisation intracellulaire impliquées dans l'activation du factor de transcription-NF-B- ainsi que des facteurs de régulation de l'interféron-3 et -7 (IRF-3 et IRF-7). Ces évènements mènent à la synthèse de molécules immunorégulatrices, tel que les interférons (IFN) de Type I, les cytokines pro-inflammatoires et les gènes stimulés par l'IFN (ISG), qui jouent un rôle important dans l'inhibition de la réplication virale. Au cours de l'évolution, les virus ont développé des stratégies pour contrer la réponse immunitaire innée afin de se répliquer. Le virus de l'immunodéficience humaine de type 1(VIH-1), l'agent infectieux du syndrome de l'immunodéficience acquise (SIDA), échappe à la réponse immunitaire innée, ce qui favorise la progression de la maladie. Par conséquent, une meilleure compréhension des mécanismes par lesquels le VIH-1 module les voies de signalisation des TLR et des RLR pourrait mener au développement de nouvelles stratégies thérapeutiques pour empêcher la réplication et donc la propagation du VIH-1. Des études ont démontré que les TLR qui signalent par l'intermédiaire de NF-B augmentent la réplication du VIH-1. Cependant, la stimulation du TLR4 déclenche à la fois la voie de signalisation de NF-B et celle des IFN, pouvant avoir ainsi des effets inhibiteurs sur la réplication du VIH-1. L'objectif de notre première étude était de comprendre le rôle du TLR4 dans la réplication du VIH-1. Par conséquent, nous avons caractérisé la voie d'activation des IRF-3 et IRF-7 suite à la stimulation du TLR4. Nos résultats démontrent que les kinases non-canoniques TBK1et IKKε sont activées avec une cinétique distincte ayant pour conséquence l'activation de l'IRF-3 et l'induction subséquente des IFN de type I. Par conséquent, l'activation de la voie de signalisation des IFN par la stimulation du TLR4 pourrait offrir une nouvelle stratégie pour inhiber la réplication du VIH-1. Notre deuxième étude a eu pour but de définir les différentes voies de signalisation activées par le VIH-1. Les changements transcriptionels induits par les différents sous-types du VIH-1 dans les cellules dendritiques immatures ont été examinés par analyse de microréseaux. Nos résultats démontrent que pendant la phase tardive de l'infection VIH-1, un ensemble de gènes est différemment régulé par les différents sous-types du VIH-1. En plus, cette étude accentue le rôle important des cellules dendritiques immatures dans la réplication et la dissémination du VIH-1. En conclusion, étant donné l'importance des RLR dans la reconnaissance des virus à ARN, l'objectif de la dernière étude a été d'étudier les mécanismes d'évasion utilisés par VIH-1 pour contrer la réponse antivirale innée. Nos résultats démontrent que l'ARN du VIH-1 est détecté par le récepteur cytosolique RIG-I. Cependant, une protéine du VIH-1 -la protéase- séquestre le récepteur RIG-I dans les lysosomes et empêche l'activation de la réponse antivirale initié par le récepteur RIG-I. De façon générale, la recherche présentée dans cette thèse propose de nouvelles avenues pour développer des stratégies préventives et thérapeutiques afin de combattre le VIH-1/SIDA.

HIV is a master of immune evasion, utilising a range of different techniques to not only survive the human immune system, but also mediate its eventual catastrophic decline. Understanding how HIV evades the adaptive immune response is paramount to developing effective treatments and vaccines. This thesis aimed to investigate three key ways in which HIV-1 and HIV-2 mediate immune evasion in the context of clinically latent infection. Chapter three summarises a study into selection pressure and mutation in the gp120 envelope gene in a narrow-source HIV-1 cohort of former plasma donors (FPDs) from China. This study further characterised the cohort, and identified specific mutations in the gene consistent with antibody and CTL-driven selection pressure. Chapter four describes an investigation into the downregulation of HLA-I mediated by primary isolates of HIV-1 and HIV-2 Nef. Nef-mediated HLA-I downregulation contributes to the evasion of CTL responses. In stark contrast to previous reports, no evidence for differential downregulation of HLA-A and HLA-B was detected, but primary isolates invariably showed reduced activity relative to laboratory-adapted and consensus variants. In performing this study, a number of limitations came to light regarding how bifurcate analyses are used to interpret flow cytometric data collected in studies of receptor modulation. A novel technique - SWARM - was therefore developed to address these limitations, and is described in chapter five. Chapter six aimed to address why CTL responses against HIV-2 Nef are rare, despite HIV-1 Nef being highly immunogenic. A series of in vitro (immuno)proteasomal processing assays revealed HIV-2 Nef is more extensively digested than HIV-1 Nef, but further experimentation is required to explain the difference in response. Finally, chapter seven briefly summarises a successful collaborative attempt to resolve the first ever crystal structure of HIV-2 Nef.

Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions.

Cytotoxic T lymphocytes (CTLs) are activated to kill virally infected cells by the recognition of viral peptides presented on the cell surface by major histocompatibility (MHC) class I molecules. Peptides are generated by proteasOIpal degradation of viral proteins in the cytosol, and then translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Here, peptides are loaded onto newly synthesised MHC class I molecules, before trafficking ') to the cell surface. Clearly it is advantageous' for viruses to inhibit cell- surface expression of MHC class I, as this enables them to avoid a CTL response. Human cytomegalovirus (HCMV) encodes US6, an ER resident glycoprotein that inhibits peptide translocation by TAP. HCMV US6 prevents ATP binding by TAP, effectively starving the transporter of its energy source, and cutting off the supply of peptides for loading onto MHC class I molecules. The mechanism through which HCMV US6 inhibits ATP binding by TAP is poorly understood, nor is it clear how HCMV US6 localises to the ER. Thus the aims of this study were three-fold.' Firstly, to further investigate HCMV US6 inhibition of MHC class I cell surface expression, in particular its interactions with TAP. Secondly, to characterise a chimpanzee cytomegalovirus (CCMV) homologue of HCMV US6, in order to identify conserved residues required for TAP inhibition. Thirdly, to ~nvestigate interactions that may mediate HCMV US6 localisation to the ER. A flow cytometric· assay for TAP activity was developed in which cell surface expression of the TAP-depe.ndent allele HLA-B2705 was measured. Using a series of HCMV US6 truncations and site directed mutants in this assay it was demonstrated that that residues 89-104 ofHCMV US6 are required for TAP inhibition. Additionally, two separate regions of HCMV US6 stabilise the interaction with TAP; one at the Nterminus comprising residues 81-89, and another at the C-terminus ofHCMV US6. In contrast CCMV US6 had no effect on HLA-B2705 expression and could not inhibit ATP binding by human TAP. Sequence alignments indicated that CCMV US6 differs from HCMV US6 in seven of the sixteen residues required for TAP inhibition, corresponding to residues 89-104 ofHCMV US6. Site directed mutagenesis was used to create a CCMV US6 molecule that was identical to HCMV US6 in fifteen of the sixteen residues required for TAP inhibition. Significantly this chimeric US6 protein reduced cell surface expression of HLA-B2705, and inhibited ATP binding by TAP. Overall, these data provide further insight into the molecular interactions between HCMV US6 and TAP, from which a possible mechanism ofinhibition can be proposed. Previously published work revealed that HCMV US6 binds both TAP and the ER chaperone calnexin. To investigate if either of these interactions was required for HCMV US6 localisation to the ER, TAP-deficient, and calnexin-deficient cell lines were utilised. In both of these cell types HCMV US6 localised to the ER, suggesting that neither TAP nor calnexin was, by itself, required to retain HCMV US6 in the ER. Additionally, the intracellular localisation of a HCMV US6 truncation series was assessed to map the sequence requirements for ER localisation; however this could not be conclusively defined.

Ross River virus (RRV) is an Alphavirus distributed throughout Australia. It is transmitted by mosquitoes and is known to cause moderate to severe disease symptoms in humans. Along with other alphaviruses such as Sindbis virus and Chikungunya virus, RRV is known to cause arthritic symptoms, characterised by muscle and joint inflammation. Several investigations have established the role of macrophage cells and pro-inflammatory host factors in the development of RRV-induced disease. In this study, we attempted to determine differences between RRV passaged in mammalian and mosquito cells. There is strong evidence that arthropod-borne viruses are able to display enhanced infectivity when passaged into arthropod cell line. We showed that mosquito cell-derived RRV (mos-RRV) was able to replicate to higher titres than mammalian cell-derived RRV. We also showed that mos-RRV failed to induce Type I IFN-associated antiviral responses. The second aim of this study was to investigate the role of TNF-ᬠa pro-inflammatory cytokine implicated in arthritic diseases, in the development of RRV disease. We treated RRV-infected C57BL/6J mice with a commercially available TNF-ᠩnhibitor drug and monitored disease signs. We found that the TNF-ᠩnhibitor does not ameliorate RRV disease (RRVD) symptoms, and that it does not prevent muscle and joint inflammation. We analysed histological sections of muscle and joint tissue of Enbrel-treated and untreated, RRV-infected cells. We also determined and compared host cytokine expression profiles. Finally, we sought to determine the requirement for natural killer (NK) cells in RRV disease. NK cells have been detected in the synovium of RRV-infected patients since early studies, but their role in disease pathogenesis remains unclear. Using a NK-dysfunctional mouse (C57BL/6J-Lystbg), we showed that mice lacking a functional NK system are more susceptible to RRV disease than wildtype, C57BL/6J mice. We monitored disease symptoms following RRV infection and assessed muscle and joint inflammation in Lystbg and C57BL/6J mice. This thesis examines mechanisms of viral infection and immune evasion employed by RRV, as well as into the role of host cells and cytokines in RRVD pathogenesis disease mechanisms. We showed that a functional NK cell system is required for the regulation of RRV-induced muscle and joint inflammation. Our characterisation of the use of a commercial TNF-ᠩnhibitor in RRV-induced disease in mice may provide information on the role of TNF-ᠩn viral arthritis, and may help towards developing safe and effective treatment.

Staphylococcus aureus is a common commensal organism and a clinically important pathogen. S. aureus is able to establish infection in a range of tissues owing to its production of a wide variety of proteins which aid its invasion, attachment and immune evasion. Characterisation of S. aureus proteins is required to deepen understanding of infection and immunity and for continued development of antibacterial therapy. Extracellular fibrinogen binding protein, Efb, binds both fibrinogen and complement components. The C-terminal complement modulating domain of Efb, Efb-C, is well characterised. NMR data analysis suggests that the N-terminal fibrinogen binding region of Efb is natively unstructured. Previously characterised fibrinogen binding domains of S. aureus proteins are typically structured and so Efb may show a novel mechanism of fibrinogen binding. Staphylococcal binder of immunoglobulin, Sbi, confers host immune evasion through binding to both immunoglobulins and complement components. The solution structure of the complement binding domain of Sbi, Sbi IV, has been determined and reveals a conformation seen in other complement modulating proteins of S. aureus. The effect of Sbi IV and Efb on the complement activation pathways has been investigated and reveals key differences in the action of each of these proteins. The crystal structure of Sbi IV in complex with C3d has been determined and reveals that the interaction of Sbi IV with C3d is similar to that of other complement modulating proteins with C3d. NMR and X-ray diffraction data also reveal a potential second physiologically relevant mode of Sbi IV – C3d binding. Immunodominant staphylococcal antigen B, IsaB, is an uncharacterised protein whose expression may be correlated with immune evasion. NMR data indicate that IsaB adopts a folded conformation in solution but results of preliminary functional analyses of IsaB are currently unclear.

Varicella zoster virus (VZV) is an alpha herpes virus that causes primary infection with varicella (chicken pox), establishes latency in ganglia and may later reactivate as herpes zoster (shingles). Innate immune effectors are thought to control initial viral replication, but it is the adaptive immune system, involving T cells that mediates eventual control of viraemia and the associated clinical disease. Although both CD4⁺ and CD8⁺ T cells mediate viral clearance during acute illness, memory responses are dominated by CD4⁺ T cells. We tested the hypothesis that the paucity in memory CD8⁺ T cell effectors is partly attributed to immune evasion mechanisms that are mounted by VZV. We confirmed that VZV readily down regulates cell surface HLA-A and HLA-C but spares HLA-B onVZV infected keratinocytes and VZV infected Mewo cells. Analysis of intracellular HLA protein expression and gene transcription showed global down regulation of all HLA subtypes. Further analysis showed that VZV inhibits IFN-γ mediated up regulation of HLA expression and augments IFN-γ mediated up regulation of HLA-E and CD71 expression. Furthermore, we show that acute VZV infection lowers the frequency of circulating peripheral blood myeloid dendritic cells (mDC), reduces the expression of the DC activation marker HLA-DR and impairs inflammatory cytokine secretion in blood DC populations. Inhibition of DC cytokine secretion was found to be dependent on viral replication as irradiated virus resulted only in mild inhibition of IFN-α and TNF-α secretion. Lastly, we observed that VZV infection results in increased expression of host peptides, including MHC derived leader sequences that potentially bind to HLA-E. Cell surface HLA-E is known to be a ligand for the natural killer (NK) cell inhibitory receptor CD94/ NKG2A identifying a novel mechanism of viral immune escape from NK cell surveillance. In conclusion, our data reiterates the fact that VZV targets different aspects of antigen presentation to evade the immune system with implications for pathogenesis and approaches to improved vaccination and treatment.

Pseudomonas aeruginosa est l'une des bactéries multirésistantes les plus répandues. Bien que le rôle du facteur de virulence LecB, une protéine se liant au fucose, ait été montré comme important pour la fixation aux cellules hôtes, son interaction avec le système immunitaire reste à élucider. Nous montrons ici que LecB cible les cellules endothéliales (CE) dans les ganglions lymphatiques (GL) drainant après injection cutanée chez la souris. Vingt-quatre heures après l'injection, LecB provoque une accumulation de lymphocytes dans les GL drainant la peau. Bien que l'injection de lymphocytes traçables ait révélé que LecB n'accélère pas le recrutement des lymphocytes dans le GL, nous montrons plutôt dans les expériences de blocage de l'entrée des lymphocytes que LecB empêche la sortie de ces derniers. Nous démontrons que LecB modifie les CE in vivo et in vitro, ce qui suggère une fonction de barrière endothéliale renforcée et un rôle possible pour LecB dans la perturbation de la circulation des lymphocytes entre le sang et le GL ce qui est essentiel pour l’immunosurveillance et l’établissement des réponses immunitaires adaptatives protectrices
Pseudomonas aeruginosa is one of the most common multidrug-resistant bacteria. The role of its virulence factor LecB, a fucose-binding protein, in the attachment to host cells has been shown. Its interaction with the immune system, however, is still to be elucidated. Here, we show that LecB targets endothelial cells within the draining lymph nodes (LNs) after cutaneous injection in mice. Twenty-four hours after injection, LecB causes an accumulation of lymphocytes within the skin-draining LNs. While injection of traceable lymphocytes revealed that LecB does not enhance the recruitment of lymphocytes into the LN, we show instead in lymphocyte entry-blocking experiments that LecB impedes lymphocyte egress. We demonstrate that LecB modifies endothelial cells in vivo and in vitro, suggesting a reinforced endothelial barrier function and a possible role for LecB in disturbing lymphocyte circulation between the blood and the LN, which is essential for immunosurveillance and the establishment of protective adaptive immune responses

Thesis (Ph.D.)--Indiana University, 2005.
Source: Dissertation Abstracts International, Volume: 66-08, Section: B, page: 4071. Chair: Randy R. Brutkiewicz. Title from dissertation home page (viewed Oct. 10, 2006).

Staphylococcus aureus is a major human pathogen, leading cause of soft tissue and blood stream infections. One of the causes of its success as a pathogen is the peculiar array of immune evasion factors that enables the bacterium to avoid host defenses, in which the staphylococcal protein A (SpA) plays a major role thanks to its IgG binding activities. Moreover, SpA has been recently proposed as a promising vaccine antigen. In this study, we evaluated the expression of SpA in a collection of staphylococcal strains, among which about 7% did not express SpA (SpA- strains), despite the presence of the gene. By a comparative genomic analysis, we identified that a mutation in the spa 5’ UTR sequence affecting the RBS is responsible for the loss of SpA in a subset of SpA- strains. Using a high-throughput qRT-PCR approach on a selected panel of virulence-related genes, we identified that the SpA- phenotype is associated with lower spa transcript levels and increased expression and production of capsule as well as other changes in the transcription of several key virulence factors. Our data suggest that the SpA- phenotype has occurred in geographically distinct strains through different molecular mechanisms including both transcription and translation alterations. Furthermore, we provide evidence that the SpA- strains are highly susceptible to phagocytic uptake mediated by anti-capsule antibodies. These data suggest that S. aureus may alter its virulence factor expression pattern as an adaptation to the host or environment. Vaccination strategies targeting both SpA and capsule could therefore result in broader coverage against staphylococcal isolates.

IIn the first study, we investigated the functional implications of prolonged TLR signalling on IFN-γ mediated killing of mycobacteria by murine macrophages in vitro. TLR2, but not TLR4 ligation interfered with IFN-γ mediated killing of mycobacteria in macrophages. In terms of mechanisms, neither TNF nor nitric oxide (NO) production was significantly affected, and the refractoriness induced could be reversed with increasing amounts of IFN-γ In the second study, we aimed to identify immunological markers of diagnostic importance in both the respiratory tract and serum during pulmonary mycobacterial infection in mice. We found that increased levels of immunological markers in the respiratory tract, but not serum, correlated better with active mycobacterial infection in the lungs, suggesting that the immune response in the respiratory tract is more reflective of the infection status and pathology than the systemic response. Finally, we investigated the level and nature of immune responses to pulmonary mycobacterial infection in BALB/c and C57BL/6 mice, two mouse strains known to exhibit different susceptibilities to infection with several intracellular pathogens, including mycobacteria. We showed that increased susceptibility of BALB/c mice to early mycobacterial infection was associated with reduced Th1 immune responses, and increased sTNFR secretion in the lung. Moreover, BALB/c mice recruited fewer monocytes/macrophages to the lung, and although IFN-γ stimulation of infected bone marrow derived macrophages in both mouse strains resulted in induction of antimycobacterial activity, BALB/c mice had a reduced capacity to kill ingested bacteria. The work presented in this thesis provide further insight into the mechanisms involved in the host-pathogen interaction; from persistence, to the immunological processes induced by the pathogen, to susceptibility of the host to infection.

Equine herpesvirus-1 (EHV-1) is one of the major viral pathogens causing respiratory disease, abortion, perinatal mortality and neurologic disease among horses resulting in significant economic losses to the equine industry. The virus can also remain latent in the horses and recrudesce at any time. Type-I interferons (IFNs) act as a first line of defense against many viral infections. In this study we investigated the type-I IFN response against the neuropathogenic T953 strain of EHV-1 in equine endothelial cells (EECs). The results showed that after a transient induction of IFN-β mRNA as well as protein at an early time (3h) post infection (p.i.), T953 strain of EHV-1 suppressed further induction of IFN-β at later times (12h onwards). Studies were done to confirm that the suppression of type-I IFN induction at later time points was not due to the normal IFN-β induction kinetics, it was rather because of the active interference by the virus. Investigation of the mechanisms by which T953 interferes with IFN-β production revealed that the virus degraded the endogenous level of the transcription factor, interferon regulatory factor 3 (IRF-3) and also down-regulated the activation of IRF-3 followed by its accumulation in the nucleus. However, T953 infection caused degradation of nuclear factor κB (NF-κB) inhibitory protein IκBα and also induced p50 subunit to translocate into nucleus from cytoplasm suggesting activation of NF-κB signaling. This also indicated that inhibition in the type-I IFN production was probably not due to the inhibition of NF-κB. The results of these studies also indicated that T953 virus was resistant to the biological effect of the recombinant equine IFN-α in vitro. Investigation of the reason of this resistance showed that T953 virus interfered with the cellular JAK-STAT signaling mechanism by which type-I IFN exerts its antiviral effect. Moreover, the studies revealed that downstream of the JAK-STAT signaling, T953 virus also inhibited the expression of cellular antiviral proteins including interferon stimulated gene 56 (ISG56) and viperin. Altogether, these data indicate that the T953 strain of EHV-1 interfered with the host cell innate immune responses by modulating type-I IFN mediated immune responses at multiple levels in vitro.

During lytic cycle replication EBV expresses at least three genes; BNLF2a, BILF1 and BGLF5, which individually act to inhibit efficient processing and presentation of CD8+ T cell epitopes. This thesis sets out to assess the relative contribution of these potential immune-modulating proteins to the evasion from CD8+ T cells at different stages of EBV lytic cycle. Lentiviral vectors for shRNAs were used to silence expression of these individual viral genes in EBV-transformed B-cells, which were then probed with CD8+ T cell effector clones of specificities for epitopes derived from the three phases of the EBV lytic cycle; allowing us to determine the contribution each immune evasion gene makes towards the inhibition of antigen presentation during lytic cycle. Cells replicating viruses lacking BNLF2a were more efficiently recognised by CD8+ T cells specific for immediate early and early expressed antigens relative to those lacking BGLF5 and BILF1. Conversely, cells lacking the expression of BILF1 were better recognised by CD8+ T cells specific for early and late lytic antigens. These data suggest that whilst the role BNLF2a plays in interfering with antigen presentation diminishes as lytic cycle progresses (IE>E>>L), BILF1 plays a more active role with the progression of lytic cycle (IE