Introduction : Le glioblastome (GBM) est la tumeur cérébrale primitive la plus fréquente et la plus grave de l’adulte. Des études épidémiologiques ont mis en évidence que les antécédents d’allergie sont un facteur protecteur, soulignant le possible impact de l’immunité sur le GBM. Plusieurs études transcriptomiques ont également mis en évidence des signatures immunes plus ou moins associées à la survie. Matériel et méthodes : Pour clarifier ce lien et déterminer quels gènes immuns étaient les plus impliqués dans le GBM, nous avons étudié l’expression de 791 gènes immuns dans des échantillons de GBM et de cerveaux normaux. Les interactions entre les gènes immuns ont été étudiées par une analyse de co-expression. Nous avons ensuite recherché une association entre les gènes immuns et la survie selon 3 méthodes statistiques, avant d’établir un modèle de risque mathématique validé sur plusieurs jeux de données. Enfin, nous avons étudié les cellules immunes infiltrantes sur des échantillons de gliomes dont 73 GBM par cytométrie de flux. Résultats : Un profil d’expression génique différent significativement entre le cerveau normal et le GBM a été établi de manière robuste, mais pas au sein des GBM. L’analyse de co-expression a mis en évidence 6 modules dont 5 sont enrichis en gènes ayant un lien avec la survie. Cent huit gènes immuns ont une association significative avec la survie et un prédicteur de risque à 6 gènes immuns a permis de distinguer deux groupes de patients en fonction de leur survie, y compris chez les patients dont la tumeur a un promoteur MGMT méthylé et dans le sous-groupe de GBM proneuraux. Enfin, nous avons mis en évidence, dans tous les échantillons de GBM analysés, une infiltration leucocytaire par des cellules macrophagiques/microgliales et parfois par des cellules lymphocytaires ou granulocytaires. L’infiltration de lymphocytes uniquement est associée significativement avec la survie dans notre cohorte. Conclusion : Des gènes, impliqués dans diverses fonctions immunes, sont différentiellement exprimés entre le cerveau normal et le GBM et au sein des GBM. Un prédicteur à 6 gènes robuste a été établi, il sépare les patients en 2 groupes bas et haut risque y compris ceux ayant un bon pronostic. Nous avons enfin mis en évidence dans une série de GBM une infiltration de cellules immunes, dont une infiltration lymphocytaire associée positivement à la survie. / Background: Glioblastoma is the most common and lethal primary brain tumor in adults. Epidemiological studies have revealed that a history of allergies is a protective factor, thereby underlining the likely impact of the immune system on GBM. A number of transcriptomic studies have also identified immune signatures more or less associated with patient survival. Methods: In order to clarify and identify which immune-associated (IA) genes were the most involved in GBM, we studied the expression of 791 immune genes in GBM and normal brains samples. Interactions between IA genes were studied through an analysis of co-expression network. We then searched for a link between IA genes and patient survival according to 3 statistical methods, before defining a mathematical risk model based on different data sets. Finally, we studied the infiltrative immune population of 73 GBM by cytometry. Results: A significantly different profile of IA genes expression between healthy brains and GBM was consistently defined, but not among GBM. The analysis of co-expression network revealed 6 modules, 5 of which were enriched by genes associated with patient survival. 108 IA genes have a significant association with patient survival and the 6-IA gene risk predictor allowed us to distinguish two groups of patients according to their survival, including patients whose tumor had a methylated MGMT gene promoter and in the subset of proneural GBM. Finally, in every analyzed GBM sample, we have shown that there was a leukocyte infiltration by macrophages/microglial cells and sometimes by lymphocytes or granulocytes. Only the lymphocytes infiltration was significantly associated with the survival in our group of patients. Conclusion: IA genes that are involved in various immune functions are expressed differentially between healthy brains and GBM and amongst GBM. A robust 6-IA gene risk predictor was defined: it divides patients into two low and high risk groups, including those who have a good prognosis. Finally, we revealed an infiltration of immune cells in a series of GBM, only the lymphocytic infiltration was positively associated with patient survival.

Telomeres are protective structures at the end of our chromosomes, composed of multiple repeats of the DNA sequence TTAGGG. They are essential for maintaining chromosomal stability by preventing damage and degradation of the chromosome ends. Telomeres are normally shortened with each cell division until a critical length is reached, at which stage cell cycle arrest is induced. Telomere shortening can be prevented in the presence of the telomere-­‐elongating enzyme telomerase. Telomerase is expressed during embryogenesis and in certain normal cell types, but most somatic cells exhibit undetectable levels of telomerase activity. In contrast, most cancer cells express telomerase enabling them to proliferate indefinitely. There is a search for reliable molecular markers that can be used to help predict cancer risk and outcome. The interest of investigating telomere length as a potential biomarker in malignancy has grown rapidly, and both tumors and normal tissues have been in focus for telomere length measurements. In this thesis, telomere length was investigated in breast cancer patients and in patients with renal cell carcinoma (RCC). The breast cancer patients were found to have significantly longer mean telomere length in peripheral blood cells (i.e. immune cells) compared to a tumor-­‐free control group. Moreover, patients with the longest blood telomere length had a significantly worse outcome compared to patients with shorter blood telomeres. In a patient group with clear cell RCC, telomere length was investigated in peripheral blood cells, in tumors and in corresponding kidney cortex. Again, patients with the longest blood telomere length had a significantly worse prognosis compared to those with shorter blood telomeres. In contrast, telomere length in tumor and kidney cortex tissues did not predict outcome per se. Immunological components may play a role in telomere length dynamics as well as in cancer development. We aimed to investigate a possible association between telomere length and certain immunological parameters, including various cytokines and peripheral levels of a blood cell type with suppressor function [regulatory T cells (Tregs)]. In our patients with clear cell RCC, three cytokines correlated significantly with tumor telomere length, but not with telomere length in peripheral blood cells. In a separate patient group with various RCC tumors, blood telomere length correlated positively with the amount of Tregs. It might be speculated that a subset of patients with long blood telomeres has a less efficient immune response due to high Treg levels, contributing to a worse prognosis. Another aim of this thesis was to explore telomere length changes over time. Evaluation of blood samples collected at a 6-­‐month interval from 50 individuals, showed that half of the participants experienced a decline in mean telomere length during the time period. This group had longer telomere length at baseline compared to those who demonstrated increased/stable telomere length. In a separate group of five blood donors, a remarkable drop in telomere length was detected in one donor over a 6-­‐month period, whereas the other donors exhibited only small fluctuations in telomere length. In conclusion, the results of this thesis indicate that blood telomere length has potential to act as an independent prognostic marker in malignancy. Adding to the complexity is the fact that changes in blood telomere length might occur within relatively short time spans, indicating that telomere length is a dynamic character.

The vagina is the port of entry for sexually transmitted diseases in women. Its epithelium constitutes the luminal border, thus comprising an important defence barrier. The objective of this work was to investigate the mechanisms of importance in the immune defence of the vaginal epithelium of healthy, fertile women, and possible menstrual cycle changes. Effects of hormonal contraceptive usage on oestrogen receptor (ER) and progesterone receptor (PR) expression were studied. The contribution of epithelial cell to the immune defence was estimated by assaying their expression of antimicrobial defensins and the epithelial thickness. Vaginal biopsies and serum samples were collected during the follicular and luteal phases in regularly menstruating women (controls) and in users of combined oral contraceptives (COCs), levonorgestrel implants (LNGs), or depot-medroxyprogesterone acetate injections (DMPAs). Fifteen healthy women (aged 20–34 years) were enrolled in each group. Morphometry was performed on vaginal tissue stained with haematoxylin/eosin and by immunohistochemistry using monoclonal antibodies against immune cell markers, PR, and ER. Expression of mRNA for human α-defensins HD-5 and HD-6, and human β-defensins (HBD) 1 to 4 were determined by real-time qRT-PCR and in situ hybridization. In controls, the epithelium was 261 ± 16 μm thick and harboured 241 ± 35 leukocytes (CD45+) per mm2. T lymphocytes (CD3+) dominated. Both αβ T cells and γδ T cells were present with an approximate 4-fold dominance of αβ T cells. Cytotoxic T cells (CD8+) were more frequent than T helper cells (CD4:CD8 ratio: 0.7 ± 0.1). Macrophages (CD68+) constituted the second-largest population, followed by Langerhans cells (CD1a+). B cells, natural killer cells, monocytes and granulocytes were generally absent. No differences were found between the follicular and luteal phase. All four β-defensins analysed for were detected in vaginal epithelium and most samples expressed at least two. HBD-2 and HBD-3 were most frequent. HBD-3 and HBD-4 expressing cells were localized in the parabasal and intermediate cell layers. α-defensins were not detected. The epithelium was significantly thicker (333 ± 9 μm) in COC, LNG, and DMPA users than in controls, and commonly showed hyperplasia. In DMPA and LNG users the frequency of intraepithelial leukocytes (CD45+) was increased, explained by increased frequencies of both αβ and γδ T cells. In DMPA users there was also a selective increase in CD8+ T cells. PR expression was significantly reduced in DMPA users compared with controls, COC and LNG users. COC and particularly DMPA users often had undetectable levels of serum E2. In conclusion, both adaptive immunity, i.e. intraepithelial T cells, and innate defence mechanisms, i.e. intraepithelial macrophages and β-defensins, are believed to contribute to the immune defence in the human female lower genital tract. These parameters did not change during the menstrual cycle but hormonal contraceptive usage, especially DMPA, affected the quality of the epithelium. The use of DMPA and LNG was correlated with the accumulation of T cells within the epithelium. The effects of these changes on the risk of contracting infections are yet to be determined.

Human immunodeficiency virus infection is a chronic condition causing significant changes in the immune system, which are reflected in the altered gene expression patterns of the immune cells. By studying these patterns through gene expression profiling it is possible to describe not only the current states the cells are in, but also to extrapolate the proximal signals that resulted in the observed patterns. In the studies described herein, we have applied this approach to better understand the alterations in the immune function that occur in HIV infection. First, we have obtained transcriptional profiles of CD4+ and CD8+ T cells from patients in early infection, in chronic infection, and in non-progressive infection, and we compared these profiles to each other and to the profiles from uninfected donors. The analyses of the profiles revealed no discernable changes in the T cells of the non-progressive patients when compared to the uninfected individuals. On the other hand, T cells from patients with progressive infection, both early and late, showed patterns characteristic of type I interferon (IFN) exposure. We next examined experimentally the possible proximal causes of the observed transcriptional profiles. We analyzed the gene expression patterns induced by TGFβ, 13 type I interferons, as well as recombinant HIV Tat protein, in T cells and peripheral blood mononuclear cells. The TGFβ responses were inconsistent with the transcriptional profiles seen in HIV-infected patients, whereas both type I IFNs and HIV Tat induced genes in patterns consistent with those seen in patients. In fact, the thirteen IFN-induced patterns were indistinguishable from each other. Tat treatments induced interferon-stimulated genes (ISGs) as well as other genes and the response was not dependent on the presence of plasmacytoid dendritic cells (pDCs), suggesting monocytes as the possible source of the interferon response. In the last study, we examined the responses of plasmacytoid dendritic cells (pDCs) to HIV and other stimuli in healthy and HIV-infected subjects. We observed induction of IFN genes in pDCs of all subjects in response to influenza virus and TLR7 agonist imiquimod, but not to HIV virus. In summary, HIV infection results in chronic induction of type I IFN response in cells of the immune system. The source of this response is likely to be type I IFNs produced by monocytes/macrophages rather than plasmacytoid cells. The monocytic production of type I IFN may be a Tat-dependent response.

Protein molecules and their interactions via protein-protein interactions (PPIs) are at the core of cellular functions. While such global PPI networks have been useful for analyzing gene function and effects of genetic variants, they do not resolve tissue and cell-typespecific interactions. Here we leverage recent advances in single-cell RNA sequencing (scRNA-seq) to reconstruct cell-type-specific PPI networks across different tissues to enable a context-sensitive analysis of immune cells’ gene-protein pathways. Targeting B cells, T cells, and macrophage cells as a proof-of-principle, we used scRNA-seq data across different tissues from the Tabula Muris mouse consortium. We mapped the protein-coding DEGs to a protein-protein interaction network database (STRING v.11). Topological and global similarity analysis of the networks revealed distinct properties between tissues highlighting tissue-specific behaviors for each cell type. For example, we found that degree and clustering coefficients distributions were tissue-specific. Different cell types and tissues displayed specific characteristics, and in particular, the splenic PPI networks were different compared to other analyzed tissues for all the immune cell types examined. For example, the pairwise comparison of the Jaccard index for node similarity and the mantel test correlation analysis showed that the spleen’ node and PPI networks are more different than any other tissues for each cell type examined. The physiological and anatomical properties that distinguish the spleen from other examined tissues might explain why the splenic PPI networks tend to be less similar compared to other tissues. The cell-type-specific network analyses using the different distance measures between the adjacency matrices on the hub nodes such as Euclidean, Manhattan, Jaccard, and Hamming distances showed a macrophage-specific behavior not observed in B cells and T cells, confirming their lineage differences. Finally, we explored the rewiring of selected hub nodes and transcription factors in the PPI networks along with their biological enrichments to validate our observations. The suggested biological validity of our results confirms the relevance of data-driven reconstruction of these context-sensitive networks using more advanced network inference algorithms. In conclusion, scRNA-seq enables the reconstruction of global unspecific PPI networks into cell and tissue-specific networks, thereby providing an increased resolution of the biological context.

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Advanced liver fibrosis in chronic hepatitis C infection (HCV) is associated with a generalized impaired immune system. Many immune cells are affected in chronic liver disease, including CD8+ T cells. The Crawley lab reported CD8+ T cell hyperfunction in cirrhotic HCV-infected individuals that persisted after effective antiviral therapy. To evaluate the link between CD8+ T cell dysfunction in advanced fibrosis, we adapted a hepatotoxic carbon tetrachloride (CCl4) murine model. We consistently observed severe fibrosis in CCl4-treated mice resembling fibrosis in chronic HCV infected individuals. After stimulation of PBMC, the proportion of granzyme B+, and IFN-γ+ CD8+ T cells in fibrotic mice was significantly higher than the controls, particularly naïve and central memory CD8+ T cells. This state of hyperfunction was sustained after liver insult removal and significant fibrosis regression to near normal tissue integrity. Sex differences were also studied in this model and were apparent after prolonged exposure to CCl4 and in the capacity to repair liver fibrosis. Following an ectopic challenge with cancer cells, tumor growth was significantly greater in fibrotic mice. Moreover, the response to immunotherapy was significantly delayed in CCl4-treated mice. In summary, we reported for the first time that circulating CD8+ T cells are hyperfunctional in a murine model of advanced liver fibrosis in response to a hepatotoxin. In this context, affected mice failed to control the growth of a tumor whose growth is known to be controlled by a robust CD8+ T cell response. In addition, the reduced responses to immunotherapeutic effects suggest deficiencies in antigen-specific CD8+ T cell responses. Therefore, this animal model might be useful to identify mechanistic targets with translational potential for immune restoring treatments in human chronic liver diseases with advanced liver fibrosis.

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No description available.