Contexte: La leucémie lymphoïde chronique (LLC) est une hémopathie maligne associée à des anomalies dans les réponses immunitaires. Ce qui contribue à la progression de la maladie. Certains LB peuvent induire une inhibition de la réponse anti-tumorale des LT et ainsi promouvoir la progression des tumeurs. Ces LB appelés LB régulateurs partagent des caractéristiques communes avec les LB de LLC.Objectif: L'objectif principal de ce projet est d'évaluer la fonction régulatrice des LB de LLC, afin d’estimer leur influence sur l'absence de réponses anti-tumorales par les cellules T.Méthode: Des modèles de co-culture in vitro ont été développés pour évaluer la capacité desLB à inhiber la prolifération des LT.Résultats: Nos résultats montrent qu’en absence de stimulation, les LB de LLC sont incapables d’inhiber la prolifération des LT contrairement aux LB de témoins. Cependant, deux groupes de patients ont été identifiés après stimulation du TLR-9 des LB. Dans le premier groupe, les LB présentent des fonctions régulatrices défectueuses par rapport aux LB de témoins. Dans le second groupe, aucune activité inhibitrice n’est détectée. L’expression différentielle des gènes associés à la voie TLR-9 entre les deux groupes de patients semble pouvoir expliquer cette dichotomie de réponse. Une corrélation a par ailleurs été retrouvée entre le doublement lymphocytaire et la faible activité régulatrice des LB de LLC.Conclusion: Pour aller plus loin, il convient d'identifier les mécanismes moléculaires endommageant la voie TLR-9. Ces nouvelles données aideront à la compréhension de l’absence de réponse anti-tumorale et des dysfonctionnements immunitaires retrouvés chez les patients. / Background: Chronic lymphocytic leukemia (CLL) is characterized by expansion of CD5+B cells associated with disruption of immune responses, contributing to the immunodeficiency and the disease progression. Regulatory B (Breg) cells may control the anti-tumor responses favoring tumor escape. Intriguingly, CLL B cells share phenotypical characteristics with these cells.Aims: The main focus of this project is to evaluate the regulatory function of CLL B cells, aiming to estimate their influence on the lack of anti-tumor responses mediated by T cells.Methods: In vitro models of co-cultures between T and B cells are used to appraise the regulatory capacity of CLL B cells on T cell proliferation.Results: We determined a defective spontaneous regulatory function for CLL B cells. Two groups of patients have been identified following CpG-ODN stimulation. The first group presents defective regulatory B cell functions compared with control B cells. In the second group, no inhibitory activity is detected. TLR-9 gene expression analysis highlighted differential gene expression between controls and the two groups of CLL patients. Moreover, ours observations indicate that patients with low Breg activity have more aggressive disease.Conclusion: These results suggest alteration of the TLR-9 pathway in CLL B cells. To go further, it will be of interest to identify the molecular mechanisms damaging the TLR-9 pathway. These results would contribute to clarify the lack of anti-tumor immune response found in the CLL patients.

Aim: The aim of this study is to determine the frequency of single nucleotide polymorphisms (SNPs) in various pattern recognition receptor (PRR) genes, including Toll like receptors (TLR) -2, -4, -9, and CD14 in chronic (CP), localized (LAP) and generalized aggressive (GAP) periodontitis and periodontally healthy (NP) patients in an African American population. Methods: A total of 205 subjects were involved in the study. The LAP group consists of 25 subjects, the GAP group 50 subjects, the CP group 73 subjects and the NP group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. Results: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between diseased-groups versus reference group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in healthy group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD 14 polymorphism. Conclusion: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to the periodontal disease susceptibility in African Americans. Additionally, presence of C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD 14 genes are not related to periodontitis in African Americans. However, low copy number of certain alleles warrants further investigations with increased sample size to explore the role of SNPs in periodontal disease. This study was supported by the Alexander Fellowship.

AIM: to determine if a relationship exists between TLR-2, TLR-4, TLR-9, or CD14 polymorphisms and risk for developing chronic periodontal disease in an African-American population. This is the first study conducted to determine role of SNPs in TLR genes and CD14 gene in a periodontally-diseased African-American population. Additionally, this is the first study to assess the role of TLR-9 polymorphism in periodontitis patients. METHODS: A total of 130 subjects were involved in the study. The chronic periodontitis (CP) group contained 73 subjects, and the healthy control (NP) group 57subjects. Genotyping was performed in TLR2 (G2408A), TLR4 (A896G),TLR9 (T1486C) and CD14 (C260T) genes by TaqMan® allelic discrimination using Assay-by-DesignSM SNP Genotyping Assays (Applied Biosystems). Accuracy of genotyping was confirmed by known DNA samples of each genotype and by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses on selected samples. Fisher’s exact test and chi-square analyses were performed to compare genotype and allele frequencies. Within disease groups, we investigated whether SNPs were related to disease severity by step-wise logistic regression adjusted for age, gender, and smoking status. RESULTS: There was a significant difference in the distribution of specific TLR9 (T1486C) genotypes between the periodontally diseased group versus the control group. Expression of TT genotype was more prevelant in periodontally-diseased individuals compared to periodontally-healthy subjects (p<0.0001) whereas individuals expressing C allele of the TLR9 SNP (CC&CT) were more frequently found in the control group after adjusting for age, gender, and smoking status (p<0.0001) There was no statistically significant difference in the distribution of genotypes between groups for any other TLRs or CD14 polymorphism. CONCLUSION: Based on findings of this study, homozygocity for the T allele of TLR 9 polymorphism was related to chronic periodontal disease susceptibility in African Americans. Additionally, presence of the C allele at TLR-9 appeared to confer resistance to periodontal destruction. Our results showed that specific SNPs in TLR-2, -4 and CD14 genes are not related to periodontitis in African Americans.

Increasing evidence implicates the presence of bacteria in intrauterine tissues as an important risk factor for spontaneous preterm labor. Epigenetic alterations of innate immunity genes may increase the mother’s sensitivity to subclinical levels of bacteria. This study examined the presence of TET2, TLR-2, and TLR-9 in intrauterine tissue, and evaluated whether epigenetic alterations of these genes, as well as IL-8, changed their expression in human decidual tissue and a macrophage cell culture. Immunohistochemicalstaining was used to detect the presence of these proteins in intrauterine tissue. Gene expression changes were evaluated in stimulated monocytes and macrophages. Fluorescence immunohistochemistry was used to track translocation of TET2 in stimulated monocytes and macrophages. Secreted IL-8 concentration was detected with ELISA. Decidual expression of TET2, TLR-2, and TLR-9 increased in the order TNL < TL < sPTL < iPTL. This study found that TET2, TLR-2, TLR-9, and IL-8 are regulated by epigenetic mechanisms. This study was the first to report activation of TET2 involves its translocation from the cytosol to the nucleus in macrophages.

This thesis concerns the investigation of novel immunotherapies for cancer eradication. CpG therapy was used in order to target antigen-presenting cells (APCs), facilitating antigen presentation and activation of T cells. Blockade of the two major immune checkpoint regulators (CTLA-4 and PD-1) was also studied to ensure proper and sustained T cell activation. The therapies were investigated alone and compared to BCG, the standard immunotherapy in the clinic today for bladder cancer. In addition, CpG as well as BCG was combined with CTLA-4 or PD-1 blockade to examine if the combination could improve therapy. Single and combination strategies were assessed in an experimental bladder cancer model. In addition, one of the therapies (local aCTLA-4 administration) was evaluated in an experimental pancreatic cancer model. To be able to study the effects of CpG in humans, a human whole blood loop system has been used. This allowed us to dissect the potential interplay between CpG and complement. CpG was found to be superior to the conventional therapy, BCG, in our experimental model and T cells were required in order for effective therapy to occur. Used as a monotherapy, CTLA-4 blockade but not PD-1 blockade, prolonged survival of mice. When CTLA-4 or PD-1 blockade was combined with CpG, survival was enhanced and elevated levels of activated T cells were found in treated mice. In addition, Treg levels were decreased in the tumor area compared to tumors in control treated mice. CTLA-4 blockade was also effective when administrated locally, in proximity to the tumor. Compared to systemic CTLA-4 blockade, local administration gave less adverse events and sustained therapeutic success. When CpG was investigated in a human whole blood loop system it was found to tightly interact with complement proteins. This is an interesting finding which warrants further investigation into the role of TLRs in complement biology. Tumor therapy could be affected either negatively or positively by this interaction. The results presented herein are a foundation for incorporating these combination therapies into the clinic, specifically for bladder cancer but in a broader perspective, also for other solid tumors such as pancreatic cancer.