No description available.

No description available.

Experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell mediated autoimmune disease of the central nervous system and shares many characteristics with multiple sclerosis (MS). Induction of EAE is mediated by myelin reactive CD4+ T helper (Th) cells, particularly Th1 and Th17 cells, which can be provoked by the immunization with myelin derived protein (or peptide) and Toll-like receptor (TLR) stimulus (eg, complete Freund¡s adjuvant, CFA). If given an injection of soluble peptide before immunization, mice do not develop EAE (they are tolerant). This approach has been widely applied, evoking tolerance in primary responses (i.e., in naive T cells). Therefore the first hypothesis of this thesis is that peptide induced protection from EAE is a result from T cell deletion or / and anergy. As MS patients have ongoing disease and over 85% of MS patients develop a relapsing-remitting course, memory T cells are key targets when considering peptide-induced tolerance as a therapeutic strategy. Thus, a model for ¡memory EAE¡ was established to test a second hypothesis that the myelin reactive memory T cells can be controlled by the administration of soluble peptide. Here, adoptive transfer of T cells from T cell receptor transgenic mice (2D2) recognizing myelin oligodendrocyte glycoprotein 35-55 (pMOG) was used to investigate the pMOG-reactive memory responses. Soluble pMOG administration could induce a transient expansion of 2D2 T cells followed by their loss through apoptosis. A model using double immunization was established by immunizing mice first with pMOG together with unmethylated CpG oligonucleotide (CpG) as an adjuvant, and subsequently immunizing with pMOG in CFA. This produced EAE with early onset and high incidence compared to mice which received pMOG/CFA only. Cells from mice that received the double immunization protocol produced high levels of IFN-γ, suggesting that memory T cell responses have been triggered in the mice. Administration of soluble peptide before secondary immunization could ameliorate EAE, indicating that memory T cells are susceptible to tolerance induction. pMOG-reactive memory T cells were further assessed by isolating CD4+ CD25- CD44high CD62Llow cells from pMOG-experienced 2D2 mice. These cells showed early and high production of IFN-γ, and early but transient production of IL-2, compared with naive population. These data provide basic information relevant to translating peptide-induced T cell tolerance from mice to humans.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.

The Tumor necrosis factor receptor (TNFR) family member 4-1BB and its TNF family ligand, 4-1BBL, are important in modulating multiple stages of the CD8 T cell response. Here I show that during a mild influenza infection, 4-1BBL is completely dispensable for initial T cell responses, viral clearance and mouse survival. In contrast, during severe influenza infection with prolonged viral load, 4-1BB expression is sustained on lung T cells and 4-1BBL is upregulated in the lung compared to mild influenza infection. Under these conditions, 4-1BBL-deficiency results in a decreased CD8 T cell response in the lungs, higher viral load, impaired lung function and increased mortality. These findings suggest that the sustained expression of 4-1BB and its ligand as a function of viral load fine-tunes the CD8 T cell response to a level appropriate for the severity of infection. 4-1BBL is also important for maintaining CD8 memory T cell survival following the clearance of an infection. I found that 4-1BB is selectively expressed on a subset of memory CD8 T cells in the bone marrow. I further showed that the TNFR family member GITR is intrinsically required on CD8 memory T cells for 4-1BB expression in vivo, and that 4-1BB on CD8 T cells interacting with 4-1BBL on a radio-resistant cell in the bone marrow contributes to CD8 memory T cell survival. Immunotherapy with 4-1BB agonists has shown efficacy in eradication of tumors in several mouse models. These effects have been attributed to 4-1BB on multiple cell types. I found that 4-1BB either on transferred T cells or on host T cells was necessary and sufficient for inducing regression of established tumors when anti-4-1BB is combined with adoptive T cell therapy. This thesis highlights the importance of the CD8 T cell intrinsic role of 4-1BB in the immune system.

Les maladies inflammatoires chroniques sont associées à un maintien de la réponse immunitaire et notamment la présence de cellules T CD4+ mémoires. Les maladies inflammatoires chroniques de l’intestin ont pour conséquence une perte osseuse due à une augmentation du nombre et de l'activité des ostéoclastes, les cellules responsables de la résorption osseuse. Les cellules CD4+ ont été décrites comme participant à la différenciation des ostéoclastes mais la nature exacte de ces cellules reste encore indéterminée. Des études in vivo et in vitro chez la souris et chez l’Homme nous ont permis de décrire une population inflammatoire Th17 TNFα CD4+ présente dans la moelle osseuse participant à la différenciation des ostéoclastes. Ces cellules Th17 participent au recrutement et à la différenciation des monocytes en ostéoclastes en agissant sur les cellules stromales mésenchymateuses. De plus, les maladies inflammatoires chroniques de l’intestin sont associées à une augmentation de la prolifération et de la différenciation des cellules souches hématopoïétiques en cellules myéloïdes. Suite à un stress chimique ou mécanique, les ostéoclastes participent à la mobilisation des cellules souches hématopoïétiques depuis la moelle osseuse. Nous avons montré qu’au cours des maladies inflammatoires de l’intestin, l’augmentation de l’activité des ostéoclastes participe à la prolifération et la différenciation des cellules souches en cellules myéloïdes. Ainsi, les ostéoclastes sont devenus une nouvelle cible thérapeutique intéressante dans la lutte des maladies inflammatoires chroniques de l’intestin / Chronic inflammatory diseases are associated to maintain of memory response and the presence of memory CD4+ T cells. Inflammatory bowel diseases are associated with bone loss due to an increase of the number and activity of osteoclasts, the bone-resorbing cells. CD4+ T cells participated to osteoclasts differentiation, but their nature is still undetermined. In vivo and in vivo studies in mice and human allow us to describe an inflammatory Th17 TNFα CD4+ T cells in the bone marrow that participated to osteoclast differentiation and link inflammation and bone loss. Th17 T cells induce the recruitment and the differentiation of monocytes into osteoclasts acting on mesenchymal stromal cells. Moreover, inflammatory bowel diseases are associated in an increase of hematopoietic stem cell (HSC) mobilization. In stress condition, osteoclasts act on HSC niches and induce their mobilization. We have shown that in inflammatory bowel disease, increase osteoclast activity is involved in the proliferation and differentiation of stem cells into myeloid cells. These myeloid cells are responsible for the intestinal inflammation. Thus, osteoclasts have become an exciting new therapeutic target in the fight of inflammatory bowel diseases

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Das Knochenmark (BM) beherbergt wesentliche Komponenten des adaptiven Immunsystems, die einen langfristigen Schutz gegen wiederkehrende Pathogene vermitteln können, sodass es sich als Reservoir für ein immunologisches Gedächtnis qualifiziert. Neben langlebiger Antikörper-produzierender Plasmazellen bleiben auch Antigen (Ag)-spezifische CD8+ und CD4+ T-Gedächtniszellen dauerhaft im Knochenmark erhalten, auch wenn sie in den sekundären lymphoiden Organen (SLOs) und im Blut abwesend sind. Es wird angenommen, dass diese T-Gedächtniszellen bei erneutem Kontakt mit den gleichen systemischen Pathogenen schnell reagieren können. Allerdings sind die biologischen Mechanismen für ihre langfristige Aufrechterhaltung immer noch umstritten und demnach ungeklärt. Unklar ist auch, wie die T-Gedächtniszellen des Knochenmarks bei erneuter Konfrontation mit demselben Antigen reagieren. Hier wird dieser Frage begegnet, indem durch klassiche Immunisierung mit definieren Antigenen eine stabile Population Ag-spezifischer CD8+ und CD4+ T-Gedächtniszellen im Knochenmark erzeugt wird. / The bone marrow (BM) harbors critical components of the adaptive immune system being able to provide long-lasting protection against previously encountered pathogens, thus qualifying as a reservoir of immunological memory. In addition to long-lived antibody producing plasma cells, antigen (Ag)-specific CD8+ and CD4+ memory T lymphocytes are maintained long-term in the BM even when they are absent from secondary lymphoid organs (SLOs) and blood. Those memory T cells are thought to respond fast upon re-encounter of systemic pathogens. However, the biological mechanisms behind their long-term maintenance in the BM are still a matter of debate and thus remain unclear. Similarly, it is also unclear how the memory T cells of the BM react to antigenic re-challenge. Here we address these issues by generating a stable pool of Ag-specific CD8+ and CD4+ memory T lymphocytes in the BM by classical immunizations with defined antigens.

Virtual memory (VM) CD8+ T cells represent a population of antigen-inexperienced T cells with an apparent memory phenotype. In lymphoreplete germ-free mice VM CD8+ T cells represent 10-20% of all peripheral CD8+ T cells. Their origin correlates with the levels of self-reactivity where the main factor that determinates the T-cell fate decision is the strength of homeostatic signals. In the first part of this thesis, we demonstrated that VM CD8+ T cells and naïve CD8+ T cells had distinct TCR repertoire and T-cell subsets contained different clonotypes. Moreover, 'VM clones' were enriched among VM T cells and were also present in naïve T cells. In contrast, 'naïve clones' were almost exclusively detected in naïve T cells. Next, we characterized the signaling of particular OVA-reactive TCRs from both naïve and VM subsets. We confirmed that 6 out of 8 tested TCRs were responsive to Kb-OVA. In the last part of the thesis, we developed and optimized a qPCR-based method for the relative quantification of specific T-cell clonotypes prior to and during the immune response. This method will serve as a tool for studying the biology of particular VM and naïve T-cell subsets and their role during the immune response. Keywords: T-cell receptor, homeostatic signaling, self-reactivity, virtual memory cells, T cells

Verstärkte Anstrengungen zur Förderung der T-Zell-basierten Immunität haben eine zwingende Notwendigkeit für unser Verständnis der menschlichen T-Zell-Funktion und –Erhaltung geschaffen. Das Paradigma, dass Gedächtnis-T-Lymphozyten kontinuierlich durch den Körper zirkulieren wurde vor kurzem durch die Entdeckung der Gedächtnis-T-Zellen, die in einer Vielzahl von Geweben, einschließlich des Knochenmarks angesiedelt sind, herausgefordert. Allerdings bleibt der Unterschied zwischen Funktionsweise von zirkulierenden und gewebeansässigen Gedächtnis-T-Zellen nur unzulänglich verstanden. Die Knochenmark ist die Heimat für eine große Anzahl Gedächtnis-T-Zellen. CD4+ Gedächtnis-T-Zellen aus dem Knochenmark beinhalten ein breites Spektrum an Antigenspezifitäten. Interessanterweise wurden CD4+ Gedächtnis-T-Zellen spezifisch für systemische Kindheitsantigene im Knochenmark von älteren Menschen gefunden, auch wenn sie nicht mehr in der Blutzirkulation nachgewiesen werden konnten. Gedächtnis-T-Zellen aus dem Knochenmark sind sesshaft und ruhend und Langzeitgedächtnis gegen systemische Antigene erhalten. Sowohl der Überlebensmechanismus von Gedächtnis-T-Zellen, als auch die Kapazität von gewebsansässigen Gedächtnis-T-Zellen nach einer systemischen Herausforderung mobilisiert zu werden, sind bisher nur unzureichend geklärt. Ich habe gezeigt, dass Gedächtnis-T-Zellen aus dem peripheren Blut und Knochenmark unterschiedliche Überlebensfähigkeiten haben. Weiterhin habe ich die Rolle von Überleben Faktoren in ihrer Erhaltung identifiziert. Zudem habe ich bestimmt, dass Gedächtnis-T-Zellen aus dem Blut und Knochenmark unterschiedliche Zellpopulationen sind, mit unterschiedliche TCRβ Repertoires. Schließlich konnte ich zeigen, dass sesshafte Gedächtnis-T-Zellen, die spezifisch für systemische Antigene sind, schnell in die Blutzirkulation mobilisiert werden. Zusammenfassend bieten diese Studien ein umfassenderes Verständnis der Funktion und des Erhalts des immunologischen Gedächtnisses. / Intensified efforts to promote protective T cell-based immunity in vaccines and immunotherapies have created a compelling need to expand our understanding of human T cell function and maintenance. The paradigm that memory T lymphocytes are continuously circulating through the body in search of their cognate antigen has been recently challenged by the discovery of memory T cells residing in a variety of tissues, including the bone marrow (BM). However, the division of labor and lifestyle of circulating versus tissue resident memory T cells remains poorly understood. The human BM is home to a great number of memory T cells. BM memory CD4+ T cells contain a wide array of antigen specificities. Interestingly, memory CD4+ T cells specific for systemic childhood antigens have been found in the BM of elderly humans, even when they were no longer detectable in peripheral blood (PB) circulation. BM memory T cells are resident, resting and maintain long-term memory to systemic antigens. The survival mechanisms of circulating and BM resident memory T cells; as well as the capacities of tissue resident memory T cells to be mobilized into blood circulation after systemic antigen re-challenge to confer us with immune protection remains to be elucidated. I have shown that PB and BM memory T cells have different survival capacities, as well as identified the role of survival factors in their maintenance. Moreover, using sequencing analysis of the TCRβ repertoire, I have determined that PB and BM memory T cells are separated cell populations. Finally, by tracking the dynamics of antigen-specific memory CD4+ T cells after systemic MMR re-vaccination I could show that TRM CD4+ T cells specific for systemic antigens can be rapidly mobilized into blood circulation and contribute to the immune response. These studies provide a more comprehensive understanding of the function and maintenance of immunological memory in humans.