published_or_final_version / Paediatrics / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Chlamydia is an obligate intracellular bacterial pathogen that causes a wide spectrum of diseases worldwide. At present, there are no vaccines to prevent chlamydial infections due to poor understanding of how anti-chlamydial immunity ensues. In this study, we employed a variety of in vitro and in vivo systems, including knockout (KO) mice and adoptive transfer, to investigate the role of lung dendritic cells (LDCs) and their relationship with innate lymphocytes, natural killer (NK) and invariant NKT (iNKT) cells, in host defense against chlamydial lung infections in mice. We found that iNKT cells altered the phenotype and cytokine production pattern of LDCs following C. pneumoniae infection. Adoptive transfer of LDCs from infected Jα18-KO mice, which lack iNKT cells, into naïve wild-type (WT) mice promoted Th2 (IL-4) immunity following infection challenge, whereas the transfer of LDCs from the infected WT mice induced protective Th1/Tc1 (IFN-γ) immunity. On the other hand, upon adoptive transfer, LDCs from C. muridarum-infected NK-cell-depleted mice (NK-LDCs) conferred reduced protection after chlamydial challenge than the recipients of LDCs from infected sham-treated mice (NK+LDCs). NK+LDC recipients exhibited an enhanced Th1/Th17, in contrast to Th2, response compared to the NK-LDC recipients. In coculture experiments, NK cells isolated from the infected mice promoted IL-12p70, IL-6, and IL-23 production by LDCs through NKG2D receptor signaling. These findings indicate that iNKT and NK cells condition LDCs to confer protective Th1/Tc1/Th17 immunity against chlamydial lung infection. We also analyzed the contribution of major LDC subsets, CD103+ and CD11bhi LDCs, in host defense against C. muridarum infection. We found that CD103+ and CD11bhi LDC subsets expanded following chlamydial infection. CD103+ LDCs showed higher expression of costimulatory molecules and greater production of Th1- and Th17-inducing cytokines (IL-12, IL-6 and IL-23) than CD11bhi LDCs. Coculture of Chlamydia-specific CD4+ T cells with LDC subsets revealed that the T cells cultured with CD103+ LDCs produced larger amounts of IFN-γ and IL-17 compared to those with CD11bhi LDCs. To test their function in vivo, we isolated CD103+ and CD11bhi LDC subsets from infected mice and transferred them into naïve syngeneic mice that received chlamydial challenge. CD103+ LDC-recipients showed better protection, as evidenced by their reduced body weight loss, bacterial burden and lung pathology, than CD11bhi LDC recipients. Mice that received CD103+, compared to CD11bhi, LDCs produced enhanced Th1/Th17 cytokines (IFN-γ and IL-17) in the lung and the MLNs. In conclusion, these findings demonstrate that CD103+ LDCs are more efficient in inducing Th1/Th17 immunity to chlamydial infection than CD11bhi LDCs. Taken together, our findings have provided direct in vivo evidence on the role of LDCs and their conditioning by iNKT and NK cells in generating mucosal T-cell immunity against a bacterial lung infection. The findings have added new knowledge to the field of lung immunology, which have implications for developing prophylactic and/or therapeutic strategies against respiratory diseases. / October 2015

Dendritic cells (DCs) comprise a heterogeneous population of mononuclear phagocytes that play a critical role in both innate and adaptive immunity. DCs in mice can be divided into two main types. Plasmacytoid DCs (pDCs) secrete type I interferons (IFN-α/β) in response to viruses. Classical or conventional dendritic cells (cDCs) are highly adept at Ag presentation. There are two main subsets of cDCs; the CD11b+ cDC subset presents exogenous Ag to CD4+ T cells on major histocompatibility complex class II (MHCII) and the CD8α+/CD103+ cDCs uniquely capable of cross-presenting exogenous Ag to CD8+ T cells on MHCI. Functional equivalents of both subsets have been identified in humans and have been designated cDC2 and cDC1, respectively. All DCs develop from progenitors found in the bone marrow (BM) by a process directed primarily by the cytokine Fms-like tyrosine kinase 3 ligand (FL). The specification of DC types is driven by several transcription factors such as IRF8, while terminal cDC differentiation is guided by tissue-specific signals mediated through signaling pathways such as Notch and lymphotoxin-β. Notch is an evolutionarily conserved pathway of cell-cell communication that plays an essential role in the development of immune cell types, including T and B lymphocytes. DC-specific gene targeting, has been used to establish the role of Notch2 receptor signaling in the differentiation of cDC2 subset in the spleen and intestine and splenic cDC1. Because primary cDCs, particularly cDC1, are rare in vivo their study and use in translational applications require methods to generate functional cDC subsets in vitro. Commonly used cultures of primary BM with the cytokines FL or granulocyte-monocyte colony stimulating factor (GM-CSF) produce a mixture of pDC, cDC2 and cDC1-like cells, or cDC2-like cells and macrophages, respectively. Thus, new approaches are needed to yield high numbers of fully differentiated cDCs, particularly of mature cDC1. Given the critical role of Notch signaling in cDC differentiation in vivo, I hypothesized that it would facilitate cDC differentiation in vitro. Indeed, coculture of murine primary BM cells with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) facilitated the generation of bona fide, IRF8-dependent CD8α+ CD103+ Dec205+ cDC1 with an expression profile resembling ex vivo cDC1. Critically, the resulting cDC1 showed improved Ccr7-dependent migration, superior T cell cross-priming capacity and antitumor vaccination in vivo. Further, OP9-DL1 cocultures of immortalized progenitors allowed for the de novo generation CD8α+ cDC1. This discovery can help further our understanding of the mechanisms of DC differentiation while providing a tool to allow for the generation of unlimited numbers of cDCs for functional studies. Further, as cDC1 are essential for the cross-priming of cytotoxic T cells against tumors, they hold great promise as cellular vaccines. However, the use of DCs in clinical applications has been hampered by inadequate methods to generate large quantities of functionally mature cDC1 in vitro. As such, these findings should help to advance the use of cDCs in translational and therapeutic applications, such as antitumor vaccination and immunotherapy.

Viral protein R (Vpr) is an evolutionarily conserved but poorly understood protein encoded by all primate lentiviruses, including the lineages that gave rise to both human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), the causative agents of AIDS in humans. In this work, I sought to define the contribution of primate lentiviral Vpr to viral replication and evasion from cell-intrinsic antiviral defenses. I found that HIV-1 infection of human dendritic cells (MDDCs) is substantially attenuated upon infection with Vpr-deficient (HIV-1/ΔVpr) virus compared to wild-type (WT) infection. This replication defect to HIV-1/ΔVpr is evident in a single round of infection, results in reduced levels of viral transcription, and is relieved upon complementation by virion-associated Vpr. The block to transcription is alleviated through Vpr-engagement with the Cul4A/DCAF/DDB1 (DCAFCRL4) ubiquitin ligase complex and a yet-to-be identified host factor, hypothesized to induce the DNA damage response (DDR) in infected cells. MDDCs are critical immune cells that are poised to detect invading viruses through a variety of cell-intrinsic antiviral sensors, resulting in the production of type I interferon (IFN) and restriction of virus replication. Surprisingly, infection of MDDCs with Vpr-deficient lentiviruses (HIV-2 or SIVmac) resulted in production of type I IFN indicating that this pathway is targeted by Vpr. I determined that signaling cascades that induce NF-κB-dependent type I IFN production are triggered in response to lentiviral integration, an obligatory process in lentivirus life cycle that results in host DNA lesions and subsequent repair by cellular DNA repair machinery. I also demonstrated that mutations in SIVmac Vpr that ablate the ability to initiate DDR are unable to counteract the antiviral type I IFN response. Together, our work suggests the existence of a novel host factor that detects lentiviral integration in MDDCs to trigger an innate immune response that blocks virus dissemination. I hypothesize that Vpr by overcoming this cell intrinsic block to integration would be a critical viral adaptation to facilitate cross-species transmission that resulted in the HIV pandemic. / 2018-11-01T00:00:00Z

L’objectif de cette thèse est de définir des biomarqueurs relatifs à l’immunothérapie allergénique (ITA). Il peut s’agir de biomarqueurs prédictifs d’une réponse au traitement qui vont permettre aux cliniciens d’adapter les schémas thérapeutiques ou de biomarqueurs d’efficacité facilitant le suivi clinique des patients au cours du traitement. La stratégie de recherche est basée sur une hypothèse qui consiste à dire que les cellules dendritiques (DCs) sont impliquées dans le succès de l’immunothérapie. En particulier, nous supposons que le traitement induit une baisse des DCs effectrices et une augmentation des DCs tolérogènes.Dans une première partie, un criblage de molécules biologiques et pharmacologiques a été entrepris sur les DCs dérivées des monocytes afin de générer in vitro des DCs effectrices de type DC1 etDC17 et des DCs régulatrices. Quatre molécules ont ainsi été identifiées pour leurs propriétés polarisantes. En particulier, les protéases d’Aspergillus oryzae se sont révélées être des inducteurs forts de tolérance. Le phénotype des DCs régulatrices obtenu a été étudié en détail ainsi que la polarisation et la fonctionnalité des lymphocytes T générés après cocultures.Dans une deuxième partie, deux approches de protéomique quantitative (la 2D-DIGE et la LCMS/MS sans marquage) ont été utilisées pour comparer les protéomes des DCs régulatrices et desDCs effectrices. Le différentiel d’expression des protéines les plus pertinentes a été validé au niveau transcriptionnel et protéique dans différents modèles. Le suivi des marqueurs dans des cellules du sang de patients traités ou non par ITA lors d’une étude clinique randomisée, contrôlée, en double aveugle, a permis de définir deux nouveaux biomarqueurs d’efficacité précoce de l’immunothérapie. Ces marqueurs pourront être suivis lors des traitements de désensibilisation pour distinguer les patients répondeurs des non-répondeurs. Par ailleurs, le suivi de ces biomarqueurs pourrait être essentiel dans d’autres pathologies comme les maladies auto-immunes ou encore la transplantation. / The aim of this thesis is to define biomarkers of allergen-specific immunotherapy (SIT).These biomarkers can be predictive of a clinical response or could be efficacy biomarkersable to discriminate responders versus non responder patients. The research strategy is based on the following hypothesis: if immunotherapy works, effector DCs are decreased where as regulatory DCs are increased locally or in the peripheral blood.First, we screened several biological or pharmacological agents to identify effector orregulatory DCs polarization agents. Four distinct molecules lead to the generation of eitherDC1, DC17 or regulatory DCs. In particular, proteases from Aspergillus Oryzae were clearinducer of tolerogenic DCs. The phenotype of those cells and the CD4+ T cell polarization induced after coculture were characterized extensively.In a second part, two proteomic approaches were used to compare the whole cell proteome of generated DCs. Most pertinent markers of polarization were validated in several cellular models. Markers were also followed in a randomized, double blind, placebo controlled clinical trial testing the efficacy of grass pollen tablets. Two markers were up regulated in patients who responded to the treatment pointing to a potential role of these proteins as early efficacy biomarkers. These markers are of crucial interest in the follow up of patients after SIT and could also be used in other diseases like autoimmune diseases or transplantation.

by Tang Mei Kuen. / "May 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 134-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Infection with the helminth parasite Schistosoma mansoni leads to chronic inflammation and Th2 mediated fibrosis, which result in severe pathology characterised by hepatosplenomegaly. Dendritic cells (DCs) are adept initiators of CD4+ T cell responses, but their fundamental importance in this regard in Th2 settings remains to be demonstrated. Indeed, the role of DCs at different stages of infection with S. mansoni is also yet to be determined. In addition, the importance of the interaction of DCs with tissue factors in the tissue microenvironment on the development of Th2 response to S. mansoni antigens is an area of active research and debate. This thesis is comprises of four studies. The first study tackles the involvement and importance of DCs in the induction and development of Th2 responses against S. mansoni using CD11c–diphtheria toxin receptor mice to deplete CD11c+ cells during the priming stage of the CD4+ Th2 response against S. mansoni. Diphtheria toxin treatment significantly depleted CD11c+ DCs from all tissues tested, with 70-80% efficacy. Even this incomplete depletion resulted in dramatically impaired CD4+ T cell production of Th2 cytokines, altering the balance of the immune response and causing a shift towards IFN-γ production. In contrast, basophil depletion using Mar-1 antibody had no measurable effect on Th2 induction in this system. These data underline the vital role that CD11c+ antigen presenting cells can play in orchestrating Th2 development against helminth infection in vivo, a response that is ordinarily balanced so as to prevent the potentially damaging production of inflammatory cytokines. The second study addresses whether the exposure of DCs to the cercarial stage of the parasite is critical for either parasite survival or the subsequent development of the Th2 immune response against later stages of infection. It was found that CD11c depletion prior to infection resulted in increased parasite survival, but did not impair the development of CD4+ T cell Th2 response later in infection. The third study asked whether DCs continue to be necessary for the maintenance of the chronic immune response during infection with S. mansoni. In contrast, depletion of CD11c+ cells during the initiation (4 to 6 weeks) or maintenance (6 to 8 weeks or 12 to 14 weeks) of Th2 response to eggs, resulted in severely impaired Th2 cytokine production. Interestingly, depletion during the later stages of infection led to dramatic weight loss and mortality, coincident with impaired CD4+ T cell responses. These data suggest that CD11c+ antigen presenting cells, in addition to being important in the early priming phase, also play a vital role in the maintenance and homeostasis of chronic CD4+ T cell responses in a Th2 infection setting, the disruption of which can have lethal consequences. The final study in this thesis aimed to establish whether the tissue factor thymic stromal lymphopoietin (TSLP) is able to enhance or modulate the Th2 responses initiated by DCs stimulated with SEA. Contrary to previous studies, it was found that BMDCs do not become phenotypically activated by TSLP, in particular, they do not up-regulate the costimulatory molecule OX40L, nor does TSLP suppress the production of IL-12p40 or IL-12p70 in response to LPS or CpG. Further, exposure to TSLP had no impact on DC cytokine production or survival. Irrespective of this unaltered profile in vitro, TSLP exposed DCs transferred in vivo induced the production of significantly more Th1 and Th2 cytokines from polyclonally restimulated splenocytes than DCs exposed to medium alone. In addition to this, TSLP altered the kinetic of the immune response induced by DCs stimulated with the soluble egg antigen (SEA) of S. mansoni. This was characterised by the antigen specific production of T cell cytokines starting more rapidly than with non-TSLP treated control DCs. The alteration in the kinetics of the immune response was not restricted to Th2 antigens and was also seen to some extent in Propionibacterium acnes stimulated DCs. This suggests a possible role for TSLP in either inducing faster DC migration or greater production of T cell chemoattractants and thus, enhancing the rate of DC interaction with T cells.

Langerhan’s cells (LCs) are the immune sentinels of the skin, sampling the cutaneous microenvironment and presenting captured antigen to T cells. A sheet-like proliferation of LCs is termed Langerhan’s cell histiocytosis (LCH), an enigmatic and poorly understood disorder with a widely varied clinical spectrum and disease course. In non-pulmonary LCH all cases reported to date have been monoclonal. Clonality argues for LCH as a neoplastic rather than reactive disorder. After initial investigation of the limitations of formalin fixed paraffin embedded tissues for downstream analysis, lesions of LCH were collected from 4 sites across Scotland. To further define the spectrum of LCH, clonality was assessed using an X inactivation assay based on the polymorphous region of the Human Androgen Receptor. To improve understanding of the assay, a study on post-mortem material was undertaken. This demonstrated a unique insight into patterns of X inactivation across different tissues of the same individual and highlighted potential pitfalls in interpretation. An important question was whether lesions of LCH associated with haematopoietic neoplasms were polyclonal or monoclonal proliferations? For the first time, associations of LCH with B-cutaneous lymphoid hyperplasia (B-CLH), lymphomatoid papulosis (LyP) and mycosis fungoides (MF) are reported. In two female cases, the LCs were polyclonal providing some reassurance that such lesions are reactive in nature and should not be regarded as potential second neoplasms. In a more expanded study a wide variety of primary LCH lesions were assessed for clonality. Significant limitations were posed by the quality of the material available; in 2 cases the lesions were found to be polyclonal. This is the first time such a result has been reported. Monoclonality was identified in 2 other cases including one of pulmonary LCH. The findings reported herein suggest that clonality and hence neoplasia cannot be assumed in all cases of primary non-pulmonary LCH. The possible functions of LCs in cutaneous lymphoma were explored. In T-cell lymphoma 2 cases reported here suggest a role for LCs in disease progression. In contrast, LCs play no significant part in the development or progression of cutaneous B-cell proliferations although other types of dendritic cells probably have an important role. By studying proliferations of LCs in a variety of settings, this work has extended knowledge of the spectrum of LCH. Displaying similar histopathological appearances, lesions of LCH may be best defined by clonality as well as cytokine expression and level of maturation. In future, such markers may be employed as prognostic indicators allowing individualised and targeted management.