Microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC) is a common cause of chronic watery diarrhea. The diagnosis relies on typical histopathological changes observed upon microscopic examination. The studies in this thesis investigated innate and adaptive immune responses in the colonic mucosa of MC patients, also comparing patients with active disease (CC and LC) and histopathologically in remission (CC/LC-HR). We first analyzed expression of interleukin-1/Toll-like receptor (IL-1/TLR) signaling regulators in MC patients (Paper I). Our results showed enhanced IRAK-M, microRNA-146a, -155 and -21 expressions, whereas IL-37 gene expression was reduced in CC and LC patients as compared to non-inflamed controls. These results suggest different pathophysiological mechanisms in MC patients. The mixed inflammatory cell infiltrations seen in the lamina propria of MC patients might be a result of dysregulated expression of chemotactic mediators. In Paper II, we showed that MC patients display mainly an increased expression of chemokines and chemokine receptors in active disease as compared to noninflamed controls. In Paper III, we examined if the decreased IL-37 expression seen in Paper I could mediate the upregulation of chemokines seen in Paper II. We showed that a relatively small reduction in the ability of epithelial cells to produce IL-37 results in mainly increased chemokine expressions in a pattern similar to the findings in Paper II. In order to understand the nature of infiltrating T cells commonly observed in MC patients, we analyzed the T cell receptor (TCR) β chains in colonic biopsies of MC patients (Paper IV). Our results showed significant differences in TCRβ repertoire, which suggests selectively expanded T cell clones in active MC and histopathologically in remission patients. Altogether, these results i) increase the knowledge of MC pathogenesis by showing changes in TLR signaling regulators, enhanced chemokine and their receptor expressions involved in a mixed immune cell infiltrations and selectively expanded T cell clones in CC and LC patients, as well as in histopathological remission ii) might potentially increase the possibility of more target-specific therapies based on IL-37 induction, chemokines or chemokine receptor inhibitions, or hindering T cell infiltration according to TCR clonality.

No description available.

Les rythmes circadiens contrôlent de nombreux aspects de la physiologie chez les mammifères. Parmi ces processus physiologiques, les horloges circadiennes contrôlent entre autres la réponse immunitaire innée et adaptative. Depuis des décennies, de nombreuses études ont commencé à couvrir ce sujet. Cependant, le contrôle circadien de la réponse adaptative reste peu étudié. Dans le cadre de ce projet de recherche de doctorat, nous avons exploré le rôle des rythmes circadiens dans la réponse des lymphocytes T CD8 à la présentation antigénique par des cellules dendritiques. Des travaux du laboratoire publiés par Erin E. Fortier et al. ont mis en évidence une différence jour/nuit dans l’expansion des lymphocytes T CD8 dont le récepteur T (TCR) est spécifique au complexe KbOVA exprimé par les cellules dendritiques ainsi que dans le nombre de lymphocytes T CD8 CD44hi IFN+ spécifiques pour l’antigène de l’ovalbumine (OVA)1. En effet, la réponse des lymphocytes T CD8 est plus importante après une vaccination faite en milieu de jour (zeitgeber time (ZT) 6) par rapport à une vaccination faite en milieu de nuit (ZT18). Cependant, ces travaux de recherche n’ont pas démontré le rôle des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Mes travaux de recherche de doctorat ont dans un premier temps confirmé l’implication des horloges circadiennes dans le rythme de réponse des lymphocytes T CD8 à la présentation antigénique. Nous avons ensuite démontré la contribution des horloges circadiennes des cellules dendritiques ainsi que le rôle essentiel des horloges circadiennes des lymphocytes T CD8 dans ce rythme de réponse. De plus, nous avons montré que ce rythme avait un impact sur la capacité des lymphocytes T CD8 à contrôler une infection bactérienne (Listeria monocytogenes). En effet, les variations jour/nuit de la charge bactérienne dans la rate et le foie des souris de type sauvage étaient abolies dans les souris déficientes pour le gène des horloges circadiennes Bmal1 dans les lymphocytes T CD8. Dans un second temps, nous avons mis en évidence suite à l’analyse du transcriptome des lymphocytes T CD8 de souris naïves collectés toutes les 4 heures sur 48 heures que ces cellules sont plus enclines à être activées le jour et à l’opposé plus enclines à être inhibées la nuit. Ces résultats corrèlent avec le rythme de réponse des lymphocytes T CD8 à la vaccination. Dans un dernier temps, nous avons confirmé que le rythme de réponse des lymphocytes T CD8 à la présentation antigénique agissait de manière précoce dans l’activation de ces cellules. Pour cela nous avons irradié des souris de type sauvage et nous les avons ensuite reconstituées avec une moelle osseuse contenant 1% de précurseurs de cellules OT-I (lymphocytes T CD8 spécifiques au complexe KbOVA, exprimant la chaîne du TCR V5. Après vaccination de ces souris en milieu de jour subjectif (circadian time (CT) 6) ou en milieu de nuit subjective (CT18), nous avons observé un rythme de la réponse des lymphocytes CD8 pour différents marqueurs impliqués dans la réponse précoce des lymphocytes T CD8, telles que CD69, CD5, IRF4, et la phosphorylation de S6 (marqueur de l’activité de mTOR) et de AKT. L’ensemble des recherches de mon doctorat ont permis de mettre en évidence un tout nouveau mécanisme impliquant les horloges circadiennes dans la réponse des lymphocytes T CD8 en réponse à une vaccination. Une meilleure compréhension du fonctionnement des horloges circadiennes dans la réponse immunitaire permettra de mettre en place des nouveaux traitements personnalisés en fonction du type d’infection et du type de maladie, délivré à un certain moment de la journée dans le but d’améliorer l’efficacité tout en réduisant les effets secondaires. / Circadian rhythms control various aspects of the physiology in mammals. Among these processes, circadian clocks control the innate and the adaptive immune responses. Since few decades, numerous studies started to uncover the role of the circadian system in the immune response. However, the circadian control of the adaptive immune response remains poorly studied. My PhD work focused on the circadian control of the CD8 T cell response to vaccination by dendritic cells. Erin E. Fortier et al. published in The Journal of Immunology that wild type mice vaccinated with antigen presenting cells loaded with the OVA peptide present a day/night variation of the CD8 T cell response after a vaccination done during the middle of the day (zeitgeber time (ZT) 6) compared to a vaccination done during the middle of the night (ZT18)1. Indeed, the proportion of CD8 KbOVA+ cells and the proportion of CD8 CD44hi IFN+ T cells were higher during the middle of the day than the middle of the night. However, this work showed a diurnal but not a circadian rhythm, that remained to be confirmed. The first part of my PhD research confirmed a role of the circadian system in the rhythm of the CD8 T cell response to vaccination. We showed a contribution of the dendritic cell clock as well as an essential role of the CD8 T cell clock. Moreover, this rhythm impacts the ability to control an infectious challenge as shown by a circadian variation in bacterial load (Listeria monocytogenes) in wild type but not in mice lacking clock in mature CD8 T cells. The second part of my research focused on the analysis of the transcriptome of CD8 T cells from naive mice collected every 4 hours over 48 hours. We showed that CD8 T cells are more prone to be activated during the day and at the opposite are more prone to be inhibited during the night. These results are correlated with the rhythm of the CD8 T cell response to vaccination. Finally, during the third part of my PhD, we confirmed that the rhythm of the CD8 T cell response to antigen presentation was acting at the early stage of the CD8 T cell activation. We used wild type mice reconstituted with bone marrow cells containing 1% of OT-I precursor cells (CD8 T cells restricted for the KbOVA complex, expressing the receptor chain of the TCR for the OVA peptide, V5) and vaccinate these mice during the middle of the subjective day (CT6) or during the middle of the subjective night (CT18). At the early stage of the CD8 T cell response to vaccination, we showed a higher expression of several activation markers after a vaccination done during the middle of the day than during the middle of the night, such as CD5, CD69, IRF4 and the phosphorylation of S6 (marker of the mTOR activity) and AKT. Altogether, my PhD work highlights a new mechanism involving the circadian system in the control of the immune response. A better understanding of how circadian clocks act on the immune response will allow implementing new treatment strategies in order to increase their efficacy as well as to decrease side effects.

No description available.

Antigen presentation and recognition are key processes of the immune system necessary to initiate the adaptive immune response. Longstanding goals of these fields have been to understand the molecular mechanism of MHC II-peptide binding, the way in which dysregulation of this process can lead to disease, and determining how γδTCRs recognize their ligands. To examine some of these outstanding questions, I designed photocleavable peptides that could bind HLA-DR1 and could be used to facilitate peptide exchange. I also performed studies to understand whether peptide exchange on HLA-DR1 can be affected by glycation modifications, which occur in hyperglycemic conditions such as diabetes. I observed that while glycation modifications on HLA-DR1 did not affect peptide exchange, these modifications decreased the catalytic effect of HLA-DM on this reaction, which could affect antigen presentation in diabetic patients. For studies on antigen recognition by γδTCRs, I focused on γδNKT cells, a subset of γδT cells known to play a role during Listeria infection. I used four different variants of the γδNKT TCR to study the restrictions on Vγ junctional region usage by this TCR for ligand recognition. I found that all the TCR variants I examined could recognize cells infected with Listeria, indicating that this TCR is not restricted by γ-chain usage in order to recognize ligand. My research generated reagents that could serve in future studies of HLA-DR1 peptide binding and contributed to understanding the effect of hyperglycemic conditions on antigen presentation, as well as provided greater understanding of γδTCR restriction for ligand recognition.

T cells use their highly variable T cell receptor (TCR) to engage major histocompatibility molecules (MHC) presenting peptides on the surface of antigen presenting cells during an immune response. The TCR repertoire of developing T cells is shaped by thymic selection, resulting in a self-tolerant and foreign peptide specific naïve T cell population. However, naive T cells are alloreactive and generate immune responses towards foreign MHC alleles in clinical settings involving transplantation. While T cell immune responses towards foreign pathogens are peptide specific, the overall specificity of allo-responses is still debated. Under normal circumstances, immune system homeostasis and self-tolerance is maintained by specialized natural T regulatory cells (nTregs) that develop in the thymus. nTregs respond to self-peptide MHC they encountered in peripheral tissues with immune-suppressive activities. However, the identify of self-peptides that stimulate nTregs, specificity towards these self-peptides, and the method nTreg TCRs engage self-peptide MHC molecules is not clear. Here, we built a library of defined MHC-linked self-peptides eluted from the I-Ab MHC molecule to screen alloreactive T cells and self-reactive nTregs for activating self-peptides. We used this library to show that negative selection shapes the TCR repertoire’s specificity to self-peptides. We also provide evidence that alloreactive T cells have degenerate self and foreign peptide recognition if the foreign MHC allele is largely different from the host’s MHC allele. Finally, we identified a self-peptide that activates an nTreg, and present protein crystal structures that reveal its TCR engages self and foreign peptide MHC complexes via fairly conventional mechanisms.

T cell receptors (TCR) bind to peptides from various sources on MHC (Major Histocompatibility Complex) molecules. A long-standing goal in the field is to understand the mechanisms of MHC-peptide exchange and MHC-TCR interactions. Here, I present work from three uniquely different systems that address the following: HLA-DR1 conformational stability, self-tolerant mechanisms of TCRs isolated from self-reactive TCR transgenic mice, and TCR cross-reactivity mechanisms between LCMV and VV. First, I present a crystal structure of HLA-DR1 in complex with A1L9 peptide, a peptide with two amino acid substitutions from the parental peptide. The singly substituted A1 peptide, which has a pocket 1 alanine substitution, decreases intrinsic half-life between MHC-peptide and increases susceptibility to HLA-DM mediated peptide exchange. This data agrees with previous models of HLA-DM-mediated peptide exchange in which the major determinant is located at the HLA-DR1 pocket 1. However, the L9 substituted peptide, which has a pocket 9 leucine substitution, displays the opposite phenotype: increased intrinsic half-life and decreased HLA-DM susceptibility. The crystal structure presented here shows that HLA-DR1 in complex with a doubly substituted peptide, A1L9, is in the same conformation as HLA-DR1 with the wild-type peptide, demonstrating that pocket 9 residues can rescue pocket 1 residue binding deficiencies and that HLA-DR1 stability is determined by amino acids along the peptide, not only at pocket 1. Next, I present crystal structures of two self-tolerant TCRs in complex with IAb-3K pMHC. To elucidate molecular mechanism for self-reactivity and self-tolerance, the TCRs J809.B5 and 14.C6 are compared to each other and its parental self-reactive TCR, YAe-62.8. In comparison to YAe-62.8, J809.B5 interacts with the same pMHC, but utilizes more peptide specific interactions, a mechanism that may distinguish self-reactive receptors from self-tolerant receptors. Additionally, the crystal structure of 14.C6 TCR, which bears a different CDR3α sequence from J809.B5, demonstrates that CDR3 sequences can modulate interactions of germline encoded CDR1 and CDR2 loops. Together, these results highlight that in addition to CDR3 VDJ recombination, diversity is generated in the mature TCR repertoire by differential chain pairing, either of which can affect the interactions of germline encoded CDR loops. Next, I present a detailed analysis of cross-reactive TCRs between Kb-GP34 and Kb-A11R. The mature LCMV-immune repertoire was analyzed by DNA deep sequencing of TCRβ CDR3 sequences, which led to the identification of new cross-reactive sequence motifs. Cross-reactive sequence motifs varied by each Vβ gene, suggesting a role of CDR1, CDR2, and CDR3 loop interplay in cross-reactivity. Lastly, I present the crystal structures of a GP34/A11R cross-reactive TCR in complex with both Kb-GP34 and Kb-A11R. Analysis of the crystal structures revealed that the two complexes are largely the same, despite differences in peptide sequences. Surprisingly, the TCR to peptide interactions were dominated by three out of eight peptide side-chains. Cross-reactivity between these two complexes is likely due to a large amount of interactions from TCR to MHC compared to interactions of TCR to peptide. We note two unique MHC-peptide interactions that may allow Kb to be an allele prone to cross-reactivity. The first is an interaction at the C-terminus of the A11R peptide which pulls A11R P7 asparagine away from TCR interactions. The second interaction is from an arginine at position 155, which sits at the interface between TCRα and TCRβ , and contributes the most buried surface area in the interaction interface. Because Kb’s arginine 155 is a long side chain that hydrogen bonds with the peptide backbone, and is also at the center of the TCR-peptide interface, GP34 and A11R peptide sequence differences may be occluded from TCR discrimination by Kb presentation. The data presented in this dissertation demonstrate that interactions between MHC-peptide and MHC-TCR act harmoniously and coopertively, whereby proximal interactions are affected by interactions elsewhere. While previous models of HLA-DR/HLA-DM interactions demonstrate the importance of interactions at HLA-DR1 pocket 1, I showed that pocket 9 also contributes to HLA-DR stability and therefore, HLA-DM susceptibility. I also showed that TCR CDR3 loop sequences affect germline CDR1/CDR2 loop interactions and vice versa. Lastly, I showed that allele specific MHC side chain interactions with the bound peptide influence TCR ligand binding and hence, TCR cross-reactivity.

No description available.

According to the centers for disease control and prevention (CDC) and the world healthorganization (WHO), heart disease and immune related diseases such as diabetes and cancer areamong the leading causes of death around the world. Thus, the regulation of the function ofimmune cell plays a key role in health and disease. Calcium (Ca2+) ions play a critical role inimmune cell activation, function and in a robust immune response. Defects in Ca2+ signalinginfluences the development of cardiac disease, Alzheimer disease, immune cell metabolism,muscle dysfunction, and cancer. Each immune cell is unique in its activation and function,making it relevant to understand how activation of each type of immune cell is regulated. Herewe describe the role of the nBMP2 protein in macrophage activation and function and the role ofthe CD5 co-receptor in helper T cell activation and function.The nuclear bone morphogenetic protein 2 (nBMP2) is the nuclear variant of the bonemorphogenetic protein 2 (BMP2), a growth factor important in heart development, neurogenesis,bone, cartilage and muscle development. To better understand the function of nBMP2, transgenicnBMP2 mutant mice were generated. These mice have a slow muscle relaxation and cognitivedeficit caused in part by abnormal Ca2+ mobilization. Mutant nBMP2 mice also have an impairedsecondary immune response to systemic bacterial challenge. Here we have further characterizedmacrophage activation and function from mutant nBMP2 mice before and after bacterialinfection. We describe how nBMP2 influences the Ca2+ mobilization response and phagocytosisin macrophages, revealing a novel role of the nBMP2 protein in immune cell regulation.CD5 is a surface marker on T cells, thymocytes, and the B1 subset of B cells. CD5 isknown to play an important role during thymic development of T cells. CD5 functions as anegative regulator of T cell receptor (TCR) signaling and fine tunes the TCR signaling response.Here we describe our characterization of CD5 regulation of Ca2+ signaling in naïve helper Tcells. We also outline our findings examining how CD5-induced changes in helper T cellactivation influence other biological processes such as immune cell metabolism, the diversity ofthe gut microbiome, and cognitive function and behavior. Thus, this work elucidates theinfluence of the CD5 co-receptor on the functional outcomes in multiple systems when CD5 isaltered.

(ULSI) causes an increase of the resistance of the wiring system by increased scattering of electrons at side walls and grain boundaries in the state of the art Cu technology, which increases the RC delay of the interconnect system and thus degrades the performance of the device. The outstanding properties of carbon nanotubes (CNT) such as a large mean free path, a high thermal conductance and a large resistance against electromigration make them an ideal candidate to replace Cu in future feature nodes. The present thesis contributes to the preparation and properties of CNT based vertical interconnections (vias). In addition, all processes applied during the fabrication are compatible to ULSI and an interface between CNT based vias and a Cu metallization is studied. The methodology for the evaluation of CNT based vias is improved; it is highlighted that by measuring the resistance of one multiwall CNT and taking into account the CNT density, the performance of the CNT based vias can be predicted accurately. This provides the means for a systematic evaluation of different integration procedures and materials. The lowest contact resistance is obtained for carbide forming metals, as long as oxidation during the integration is avoided. Even though metal-nitrides exhibit an enhanced contact resistance, they are recommended to be used at the bottom metallization in order to minimize the oxidation of the metal-CNT contact during subsequent processing steps. Overall a ranking for the materials from the lowest to the highest contact resistance is obtained: Ta < Ti < TaN < TiN « TiO2 « Ta2O5 Furthermore the impact of post CNT growth procedures as chemical mechanical planarization, HF treatment and annealing procedures after the CNT based via fabrication are evaluated. The conductance of the incorporated CNTs and the applicable electrical transport regime relative to the CNT quality and the CNT length is discussed. In addition, a strong correlation between the temperature coefficient of resistance and the initial resistance of the CNT based vias at room temperature has been observed. / Die kontinuierliche Miniaturisierung der charakteristischen Abmessungen in hochintegrierten Schaltungen (ULSI) verursacht einen Anstieg des Widerstandes im Zuleitungssystem aufgrund der erhöhten Streuung von Elektronen an Seitenwänden und Korngrenzen in der Cu-Technologie, wodurch die Verzögerungszeit des Zuleitungssystems ansteigt. Die herausragenden Eigenschaften von Kohlenstoffnanoröhren (CNT), wie eine große mittlere freie Weglänge, hohe thermische Leitfähigkeit und eine starke Resistenz gegenüber Elektromigration machen diese zu einem idealen Kandidaten, um Cu in zukünftigen Technologiegenerationen zu ersetzen. Die vorliegende Arbeit beschreibt die Herstellung und daraus resultierenden Eigenschaften von Zwischenebenenkontakten (Vias) basierend auf CNTs. Alle verwendeten Prozessierungsschritte sind kompatibel mit der Herstellung von hochintegrierten Schaltkreisen und eine Schnittstelle zwischen den CNT Vias und einer Cu-Metallisierung ist vorhanden. Insbesondere das Verfahren zur Evaluierung von CNT Vias wurde durch den Einsatz verschiedener Methoden verbessert. Insbesondere soll hervorgehoben werden, dass durch die Messung des Widerstandes eines einzelnen CNTs, bei bekannter CNT Dichte, der Via Widerstand sehr genau vorausgesagt werden kann. Dies ermöglicht eine systematische Untersuchung des Einflusses der verschiedenen Prozessschritte und der darin verwendeten Materialien auf den Via Widerstand. Der niedrigste Kontaktwiderstand wird für Karbidformierende Metalle erreicht, solange Oxidationsprozesse ausgeschlossen werden können. Obwohl Metallnitride einen höheren Kontaktwiderstand aufweisen, sind diese für die Unterseitenmetallisierung zu empfehlen, da dadurch die Oxidation der leitfähigen Schicht minimiert wird. Insgesamt kann eine Reihenfolge beginnend mit dem niedrigsten zum höchsten Kontaktwiderstand aufgestellt werden: Ta < Ti < TaN < TiN « TiO2 « Ta2O5 Desweiteren wurde der Einfluss von Verfahren nach dem CNTWachstum wie die chemischmechanische Planarisierung, eine HF Behandlung und einer Temperaturbehandlung evaluiert, sowie deren Einfluss auf die elektrischen Parameter des Vias untersucht. Die Leitfähigkeit der integrierten CNTs und die daraus resultierenden elektrischen Transporteigenschaften in Abhängigkeit der CNT Qualität und Länge werden besprochen. Ebenso wird die starke Korrelation zwischen dem Temperaturkoeffizienten des elektrischen Widerstandes und des Ausgangswiderstandes der CNT basierten Vias bei Raumtemperatur diskutiert.