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

The contributions of peptide antigen affinity for TCR in driving T cell memory is unclear. Effector CD4 T cells must recognize cognate antigen again at an effector checkpoint, 5-8 days post-infection, to generate an optimal memory population. In this thesis, we examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We used an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP- peptide variants that bind FluNP TCR with a broad range of avidity. Varying peptide avidity in vivo at the effector checkpoint revealed that higher affinity interactions yielded greater numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN. The major impact of avidity was on memory cell number, not cytokine production, and was already apparent within several days of transfer. These memory cells demonstrated enhanced protection against lethal IAV infection with a robust early day 5 secondary effector response in the lung. We previously showed that autocrine IL-2 production during the effector checkpoint prevented default effector apoptosis and supported memory formation. Here, peptide avidity determined the level of IL-2 produced by effectors while IL-2R expression was unaffected. However, IL-2Ra expression by APC drove more memory cell formation, suggesting that transpresentation of IL-2 by APC at this checkpoint enhanced CD4 memory generation. Secondary memory generation was also avidity-dependent. We propose this pathway selects CD4 effectors of highest affinity to progress to memory and can instruct future vaccine design.

<p>Understanding the mechanisms that control T cell function and differentiation is crucial to develop new strategies to modulate immune function and prevent autoimmune and inflammatory disease. The balance between effector (Teff; Th1, Th2 and Th17) and regulatory (Treg) T cells is critical to provide an appropriate, but not excessive, immune response and therapies to induce Treg or inhibit Teff are likely promising treatment strategies. It has recently become clear that T cell metabolism is important in both T cell activation and differentiation. T cells undergo a metabolic reprogramming upon activation and not all differentiated T cell subsets utilize the same metabolic fuels or programs.</p><p>These metabolic differences are not trivial, as T cell metabolism is tightly</p><p>regulated and dysregulation can lead to cell death or reduced immunity. An</p><p>understanding of the metabolic differences between Teff and Treg may lead to a new direction for treating inflammatory diseases by modulating the Teff:Treg balance through metabolic inhibition. Previous studies have shown that Teff express higher levels of the glucose transporter Glut1 than Treg, however the role of Glut1, and importantly, the cell-intrinsic role of glucose metabolism in T cell differentiation and inflammation was not previously examined. The work presented here examines the role of Glut1 in T cell differentiation. We show that effector CD4 T cells were dependent on Glut1 for proliferation and function both in vitro and in vivo. In contrast, Treg were Glut1-independent and capable of suppressing colitis in the absence of Glut1 expression.</p><p>Additionally, previous studies have shown broad metabolic differences between Teff and Treg, however the specific metabolic profiles of Teff and Treg are poorly understood. Here, Teff and Treg metabolism is examined to test if dependence on distinct metabolic pathways will allow selective targeting of different T cell populations. We show that pyruvate dehydrogenase kinase 1 (PDHK1) is differentially expressed in the T cell subsets and inhibition of PDHK1 selectively suppresses Th17 and promotes Treg differentiation and function. Because Teff and Treg have distinct metabolic profiles, we hypothesized that the Treg-­specific transcription factor FoxP3 may drive the Treg oxidative metabolic program. We therefore examined the role of FoxP3 in T cell metabolism and determined that FoxP3 promotes glucose and lipid oxidation and suppresses glycolytic metabolism. Importantly, we show that promoting glycolysis with transgenic expression of Glut1 inhibits Treg suppressive capacity. Together, these data suggest that FoxP3 drives an oxidative metabolic program that is critical to Treg function. Overall, this work examines the metabolic phenotypes and regulation of Teff and Treg and potential metabolic targets that could be used to treat autoimmune and inflammatory disease.</p> / Dissertation

Objetivo: Determinar la principal causa de mortalidad en pacientes con VIH/SIDA en el Hospital Guillermo Almenara Irigoyen. Materiales y Méto-dos: Se realizó un estudio observacional, descriptivo, retrospectivo, median-te la revisión manual de 38 historias clínicas de todos los pacientes fallecidos en el Hospital Nacional Guillermo Almenara Irigoyen con diagnóstico de infección por VIH/SIDA durante el periodo 2012-2014. Se incluyeron todos los pacientes fallecidos con VIH positivos y se recolectó datos como edad, género, estado civil, opción sexual, recuento CD4, cantidad de carga viral, estadio clínico, causa frecuente de muerte, tiempo de enfermedad, entre otros. Resultados: Se encontró que el porcentaje de fallecimientos durante el año 2012 el 1.44% (15 pacientes) falleció por VIH, en el 2013 fue el 1.27% (14 pacientes), mientras que en el 2014 sólo fue el 0.77% (9 pacientes); el 89.5% fueron hombres y el 10.5% mujeres, la edad promedio fue 47.2 años (45-59); las enfermedades infecciosas fueron la causa más frecuente de muerte con un 81.5% (31 pacientes); el recuento promedio de linfocitos T CD4 fue menor de 200 células/ ul, con una carga viral mínima de 999 copias. La presentación clínica fue de curso subagudo, asociadas a anemia severa y diarrea crónica. Entre los síntomas más frecuentes al ingreso fueron nauseas, vómitos, deposiciones liquidas, transtorno hidroelectrolítico, tos productiva, disnea, transtorno del sensorio, hipertensión endocraneana. El 62.5% (20 pacientes) recibía tratamiento antirretroviral y el 57% (22 pacientes) tenían un tiempo de enfermedad menor de 5 años. Conclusiones: Las enfermedades infecciosas fueron una de las principales causas de muerte en pacientes con VIH, las cuales tuvieron como punto de partida el abdominal y el respiratorio en mayor proporción.

Introducción: En la actual epidemia por VIH/SIDA que vivimos se está presentando un aumento del número de pacientes con enfermedad no definitoria de SIDA, sin embargo aún no se establecen las causas de este aumento, más aún estas enfermedades presentan un comportamiento distinto en cada cohorte de estudio. En nuestro país no se ha realizado un estudio que las evalúe como tal. Objetivo: Determinar las proporciones de enfermedades no definitorias de SIDA presentes en los pacientes infectados por el VIH del Hospital Edgardo Rebagliati Martins durante el año 2014. Diseño: Estudio observacional, exploratorio, retrospectivo, de corte transversal. Mediante un muestreo aleatorio sistemático se seleccionaron 137 pacientes infectados con VIH, de entre todos aquellos diagnosticados con infección por VIH. Se recolectó información de las fichas de registro y de las historias clínicas y se registraron en fichas electrónicas codificadas, luego, con la base de datos se utilizó Microsoft Excel 2013 para el análisis exploratorio de los datos. Resultados: Se encontraron 5 pacientes (3.65%) infectaos con enfermedad vascular periférica (EVP), 2 (3.65%) con ictus, 1 (0.73%) con cirrosis hepática, 1 (0.73%) con falla renal, 1 (0.73%) con linfoma de Hodgkin y 127 (92.7%) sin enfermedad (NE). Las medianas para los valores de CD4 más bajos fueron Ic: 294.5, EHC: 282, LH: 216, EVP: 178, ERC: 78 y NE: 143.5 cel/uL. De los pacientes con SIDA y enfermedad los pacientes con EHC fueron el 100%, ERC el 100%, HTA el 92.86%, EVP el 80%, NE el 77.17% respecto de sus pares con enfermedad. Conclusiones: Entre las enfermedades no definitorias de SIDA la enfermedad vascular periférica se determinó como la más frecuente, seguida luego de ictus. Comparativamente se encontró mayor presencia de enfermedad en pacientes con mayores niveles de CD4, sin embargo hubo mayor proporción de enfermedades en pacientes con SIDA. El estudio cumplió con su objetivo de estudiar las proporciones de enfermedad, además contribuye a la generación de hipótesis y señala sus limitaciones y sugerencias para futuros trabajos. / Introduction: In the actual HIV/AIDS epidemic we live, it is being presenting an increase of the number of patients with non-aids defining illnesses, however there are not yet stablished the causes of this increase, also, these diseases have a different presentation in each study cohort. In our country it has not been performed a study that evaluate these diseases as such. bjective: To determine the frecuencies of non-aids defining illnesses in HIV patients of the Edgardo Rebagliati Martins Hospital in the 2014 year. Design: Observational, exploratory, retrospective, transversal study. Throught ramdom systematic sampling it has been selected 137 HIV patients, among all HIV diagnosed patients. It has been collected information from registration cards and medical records of HIV patients. The information was registered in codified electronical cards, then, with the database it has been used Microsoft Excel 2013 for the exploratory analysis of data. Results: It has been found 5 patients(3.65%) with peripheral vascular disease (EVP), 2 (3.65%) with stroke, 1 (0.73%) with hepatic cirrhosis, 1 (0.73%) with kidney failure, 1 (0.73%) with Hodgkin lymphoma and 127 (92.7%) without disease(NE). The medians for the lowest CD4 values were: Ic: 294.5, EHC: 282, LH: 216, EVP: 178, ERC: 78 y NE: 143.5 cel/uL. The patients with AIDS and illness who had EHC were the 100%, ERC the 100%, EVP the 80%, NE the 77.17%, IC the 50% respect of its pairs with illness. Conclusions: Among non-aids defining illnesses the vascular peripheral disease was the most frequent, followed by stroke. It has been found, comparatively, a greater presence of disease in patients with higher CD4 cells levels, however, ther was more proportion of diseases in AIDS patients. The study acomplished its objective of study the diseases proportions, moreover it contributes to the generation of hypotheses and mentions its limitations and gives suggerences for future studies. Non-aids defining illnesses; CD4-Positive T-Lymphocytes; Anti-Retroviral Agents

Les cellules dendritiques plasmacytoïdes (pDCs) font le lien entre l'immunité innée et l'immunité adaptative en produisant de l'interféron de type 1 en grandes quantités ainsi qu'en induisant l'activation et la prolifération des cellules T naïves de manière antigène-spécifique. Les pDCs expriment à haut niveau les récepteurs TLR7 et TLR9 et détectent ainsi les acides nucléiques d'origine virale. Les TLRs 7 et 9, localisés dans le réticulum endoplasmique (RE) à l'état basal, sont relocalisés vers les endosomes tardifs, lors de l'activation, pour initier la signalisation. Ce processus est dépendant de la protéine chaperon UNC93B1. Au cours de ma thèse, j'ai caractérisé une nouvelle molécule de la famille des protéines membranaires associées aux lysomes (LAMP), nommée BAD-LAMP. Cette protéine est, au sein du système immunitaire humain, exprimée spécifiquement dans les pDCs. Contrairement aux autres membres de la famille, BAD-LAMP n'est pas détectée dans les lysosomes mais est retenue dans le RE. J'ai également démontré que BAD-LAMP est régulée négativement lors de l'activation des pDCs induite par un ligand de TLR9. L'utilisation d'un système de cellules HeLa tranfectées et de différents mutants de BAD-LAMP avec des défauts de localisation m'ont permis d'établir que BAD-LAMP et UNC93B1 sont capables d'influencer mutuellement les adressage, par un mécanisme restant à identifier. BAD-LAMP pourrait aussi remplir un rôle de chaperon du complexe UNC93B1-TLR9 et moduler la réponse TLR9. L'étude d'un tel mécanisme de contrôle permettrait de mieux comprendre la régulation fine de la réponse immunitaire. / Plasmacytoid dendritic cells (pDCs) link innate and adaptative immunity by producing large amounts of type-1 interferon and inducing naive T cell activation and proliferation in an antigen-specific manner. pDCs express high levels of TLR7 and TLR9 and thereby sense viral nucleic acids. TLRs 7 and 9, which rest in the endoplamic reticulum (ER) at steady-state, are re-localized to the late endosomal compartment upon activation for signaling. this process is dependent of the interaction between TLRs and the chaperone UNC93B1. During my thesis, I characterized a new molecule of the lysosome-associated membrane protein (LAMP) family, named BAD-LAMP. In the human immune system, this protein is exclusively expressed in pDCs. BAD-LAMP is not detected in lysosomes, as opposed to the other LAMP family members, but is retained in the ER compartment. I also demonstrated that BAD-LAMP is down-regulated after pDCs activation by a TLR9 ligand. Using trnasfered HeLa cells and several mutant forms of BAD-LAMP with localization defects, I etablished that BAD-LAMP and UNC93B1 can influence reciprocally their intercellular trafficking by a yet uncharacterize mechanism. BAD-LAMP could therefore act as a chaperone of UNC93B1-TLR9 complex and moduate the TLR9 response. The study of such a regulatory mechanism could help to understand better the fine tuning of the immune response.

LAG-3 is an immune inhibitory marker. These immune inhibitory markers function to reduce the capability of immune cells to elicit a proper immune response and to help maintain tolerance. During an acute infection, these markers assist in controlling the immune system, however, during a chronic infection these markers prevent the immune response from persisting to effectively fight the disease. Contrary to other immune inhibitory markers, LAG-3 is not highly expressed on T cells during chronic viral infections, such as HIV. The majority of information available on LAG-3 has been gained from murine models and cell lines. This thesis uses primary human T cells in order to observe rapid expression of surface LAG-3 from a pre-formed intracellular store and cleavage of these surface molecules into soluble variants by matrix metalloprotease cleavage. LAG-3 and sLAG-3 expression is compared with CD69 and cytokine expression to help understand the early immune response. / October 2015

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

MSc (Med) Molecular Medicine and Haematology, Faculty of Health Sciences, University of the Witwatersrand / In recent years there has been much progress in understanding and defining the key protein structure-function relationships that mediate Human Immunodeficiency Virus (HIV-1) entry into host CD4+ cells. This process involves fusion of the virus and host cell membranes, following engagement of corresponding viral (gp120) and target (CD4) receptor proteins. Binding of gp120 to CD4 triggers extensive conformational changes in gp120, exposing binding sites for the co-receptor proteins (CCR5 or CXCR4), and facilitating insertion of gp41, the viral fusion protein, into the target cell membrane. Following insertion of gp41, oligomerisation of fusogenic domains on gp41 is thought to drive the juxtaposition of the virus and host cell and fusion of their membranes. Recent reports suggest that detergent-resistant membrane domains, known as lipid rafts, play a crucial role in orientating the receptor molecules during this step of HIV-1 infection. Lipid rafts are typically rich in cholesterol, sphingolipids and GPI-anchored proteins, and are biophysically distinct from the glycerophosolipid bilayer, which constitutes the bulk of mammalian cell membranes. The role of lipid rafts in virus entry, however, is still controversial, and further experimentation is needed to define their importance in this regard. To provide insight into the role of lipid rafts during HIV-1 entry, we evaluated the natural distribution of the host receptor proteins in HIV-1 target cells (U87.CD4.CCR5/CXCR4). CD4 was detected in membrane samples fractionated by sucrose density gradient centrifugation using immunoblotting techniques, while CCR5 and CXCR4 were detected on whole cells by fluorescence microscopy. We then used a primary CCR5-utilising subtype C HIV-1 isolate (FV5) to characterise dynamic changes in the distribution of these receptors and gp41 during viral entry in real-time. Viral fusion assays were set up by inoculating v target cells with FV5 at 23 ºC, a temperature that allows HIV-1 attachment, but is nonpermissive for advancement of the fusion reaction. This prefusogenic form of the virus-host receptor complex is defined as the temperature-arrested state (TAS). We found that, under normal, uninfected conditions, CD4, CCR5 and CXCR4 are distributed throughout both raft and non-raft microdomains on the U87 cell surface, and there is little evidence for any significant redistribution of these receptors into lipid rafts during the HIV-mediated fusion reaction. Interestingly, we observed a change in the structure of CD4 during the fusion process, which could describe a functionally important event in HIV-1 entry, or be related to compromises in the integrity of the virally-infected membranes. Moreover, we discovered that gp41 is capable of membrane insertion and oligomerisation at TAS, in contrast to previous reports that suggest the fusion peptide is not capable of breaching the membrane at this temperature. Our results provide valuable novel insights into the HIV-1 subtype C entry process, and the involvement of lipid rafts in this stage of the viral lifecycle, that may be relevant to novel therapy and immunogen design.

Monoklonale Antikörper (mAk) werden bereits erfolgreich zur therapeutischen Depletion verschiedener Zellpopulationen in vivo verwendet, die Mechanismen der Depletion sind jedoch unklar geblieben. In dieser Arbeit wurden im Mausmodell die molekularen Grundlagen der CD4+ T-Zelldepletion (CD4 TZD) nach einmaliger Gabe (i.p.) von 100 µg des anti-CD4-mAk YTS191.1 untersucht. Dabei konnte eine starke Korrelation zwischen Depletion und der Modulation des CD4-Moleküls von der Oberfläche beobachtet werden. Gleichzeitig zeigten sich organabhängige Unterschiede, sowohl im zeitlichen Verlauf, als auch in der Effizienz der Depletion. Im Thymus konnten weder Depletion noch Modulation detektiert werden, in Milz und Lymphknoten (Lk) war die CD4 TZD nach starker CD4-Modulation bereits nach 48 h mit 80-90 % maximal, in den Peyer-Plaques jedoch niedriger und verzögert (50-60 % nach 72 h). Anhand C3-defizienter Mäuse konnte ferner kein wesentlicher Beitrag von Komplement an der CD4 TZD beobachtet werden. Im Gegensatz dazu konnte durch die Verwendung verschiedener FcGamma-Rezeptor (FcGammaR)-defizienter Mäuse (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III und FcRGamma) wie auch durch die Blockade des FcGammaRIV eine starke, zudem organabhängige Beteiligung von FcGammaR an der CD4 TZD gezeigt werden. Während in der Milz die CD4 TZD von FcGammaRIV vermittelt wurde, waren in den Lk und Peyer-Plaques FcGammaRI/III involviert. Diese Befunde korrelierten mit der starken Expression von FcGammaRIV in Milz, Lunge, Darm, Niere und Leber, während in den Lk nur eine schwache und in Thymus und Peyer-Plaques keine Expression detektiert werden konnte. Innerhalb der Milz konnten erstmalig F4/80hoch Makrophagen als FcGammaRIV+ identifiziert und somit als potenzielle Effektorzellen der CD4 TZD bestimmt werden. Der direkte Vergleich der Depletion von CD4+ T-Zellen mit der Depletion von ICOS+ T-Zellen verdeutlichte darüber hinaus, dass die Effizienz der Zelldepletion nicht nur von den Eigenschaften des verwendeten mAk, sondern auch von denen des Zielmoleküls abhängig ist. / Monoclonal antibodies (mAb) are efficiently used for the therapeutic depletion of various cells in vivo yet the mechanisms of depletion are still unclear. In this work, the molecular principles of CD4+ T cell depletion (CD4 Tcd) by a single application of 100 µg of the anti-CD4 mAb YTS191.1.1 were investigated in the mouse. A strong correlation between the depletion and the surface modulation of the CD4 molecule could be observed. At the same time, organ-dependent differences in the kinetics as well as in the efficiency of depletion could be detected. In the thymus, neither modulation nor depletion were detectable. In the spleen and the lymph nodes (Ln), the modulation was strong and the depletion was maximal (80-90%) 48 h after mAb treatment. Interestingly, both modulation and depletion were decreased and delayed (50-60% after 72 h) in the Peyer`s patches. By using C3-deficient mice, no major contribution of complement to the CD4 Tcd was seen. On the contrary, with the help of different FcGamma-receptor (FcGammaR)-deficient mice (FcGammaRI, FcGammaRII, FcGammaRIII, FcGammaRI/III, and FcRGamma) and through the blockade of FcGammaRIV, a strong organ dependent involvement of FcGammaR could be shown. While the depletion in the spleen was clearly dependent on FcGammaRIV, in the Ln and the Peyer`s patches, FcGammaRI/III were involved. These findings correlated with the strong expression of FcGammaRIV in the spleen, the lung, the colon, the kidney, and the liver, while in the Ln the expression was weak and undetectable in the thymus and the Peyer`s patches. For the first time, F4/80high macrophages in the spleen could be identified as also being FcGammaRIV+, and are therfore considered as the potential effector cells of the CD4 Tcd. The direct comparison of the depletion of T cells via CD4 or ICOS pointed out that the target cell depletion is not only dependent on the properties of the mAb used, but also on those of the target molecule.