by Li Pok Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 94-114). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abbreviations --- p.II / Abstract --- p.V / 摘要 --- p.VIII / Publications --- p.XI / Table of contents --- p.XII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Human Th lymphocytes and their immunopathogenic roles --- p.1 / Chapter 1.1.1 --- Characteristics of Th lymphocytes --- p.1 / Chapter 1.1.2 --- Migration and activation --- p.1 / Chapter 1.1.3 --- Th cell differentiation --- p.2 / Chapter 1.1.4 --- Pathological roles --- p.4 / Chapter 1.2 --- Cytokines as modulator in Th lymphocyte activation --- p.6 / Chapter 1.2.1 --- IL-18 --- p.6 / Chapter 1.2.2 --- IL-25 --- p.7 / Chapter 1.3 --- Surface marker expression in Th lymphocytes --- p.8 / Chapter 1.3.1 --- Adhesion molecules --- p.8 / Chapter 1.3.2 --- Cytokine and chemokine receptors --- p.9 / Chapter 1.3.3 --- Costimulatory molecules --- p.11 / Chapter 1.4 --- Cytokine and chemokine release from Th lymphocytes / Chapter 1.4.1 --- Thl cytokines --- p.13 / Chapter 1.4.2 --- Th2 cytokines --- p.14 / Chapter 1.4.3 --- Chemokines --- p.15 / Chapter 1.5 --- Intracellular signaling pathways in Th lymphocytes --- p.19 / Chapter 1.5.1 --- p38 MAPK pathway --- p.19 / Chapter 1.5.2 --- ERK pathway --- p.20 / Chapter 1.5.3 --- JNK pathway --- p.20 / Chapter 1.5.4 --- NF- k B pathway --- p.21 / Chapter 1.6 --- Pharmacological intervention of signaling pathways --- p.22 / Chapter 1.7 --- Aims and scope of the study --- p.24 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Blood samples --- p.26 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.26 / Chapter 2.1.3 --- Antibodies for costimulation of Th cells --- p.28 / Chapter 2.1.4 --- Recombinant human cytokines --- p.28 / Chapter 2.1.5 --- "Signaling pathway inhibitors: SB203580, PD98035, SP600125 and BAY117082" --- p.28 / Chapter 2.1.6 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.29 / Chapter 2.1.7 --- Reagents and buffers for the purification of human Th lymphocytes --- p.31 / Chapter 2.1.8 --- Reagents and buffers for protein array --- p.32 / Chapter 2.1.9 --- Reagents and buffers for Thl/2 cytokine and chemokine detection --- p.32 / Chapter 2.1.10 --- Reagents and buffers for protein extraction --- p.32 / Chapter 2.1.11 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis --- p.33 / Chapter 2.1.12 --- Reagents and buffers for Western blot analysis --- p.35 / Chapter 2.1.13 --- Reagents and buffers for non-radioactive electromobility shift assay (EMSA) --- p.37 / Chapter 2.1.14 --- Reagents and buffers for cell viability and proliferation assay --- p.39 / Chapter 2.1.15 --- Reagent kit for endotoxin level assay --- p.39 / Chapter 2.1.16 --- Other reagent kits --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Purification of human Th lymphocytes and cell culture --- p.41 / Chapter 2.2.2 --- Measurement of total and allergen-specific IgE concentrations --- p.41 / Chapter 2.2.3 --- Immunophenotyping of cells by flow cytometry --- p.42 / Chapter 2.2.4 --- Protein array --- p.42 / Chapter 2.2.5 --- Quantitative analysis of cytokines and chemokines by flow cytometry --- p.43 / Chapter 2.2.6 --- Quantitative analysis of IFN-γ by ELISA --- p.43 / Chapter 2.2.7 --- SDS-PAGE --- p.44 / Chapter 2.2.8 --- Western blot analysis --- p.44 / Chapter 2.2.9 --- EMSA / gel shift assay --- p.45 / Chapter 2.2.10 --- MTT assay --- p.46 / Chapter 2.2.11 --- Cell proliferation assay --- p.46 / Chapter 2.2.12 --- Endotoxin level assay --- p.47 / Chapter 2.2.13 --- Statistical analysis --- p.47 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of IL-18 and IL-25 on the induction of Thl/2 cytokine and chemokine release from costimulated Th lymphocytes --- p.48 / Chapter 3.1.1 --- IL-18 and IL-25 could up-regulate the protein expression of cytokines and chemokines --- p.48 / Chapter 3.1.2 --- IL-18 but not IL-25 induced the release of IFN-γ and TNF-α --- p.48 / Chapter 3.1.3 --- "IL-18 and IL-25 induced the release of IL-5, IL-6 and IL-10" --- p.49 / Chapter 3.1.4 --- "IL-18 induced the release of IP-10, MIG, RANTES, MlP-lα and IL-8" --- p.49 / Chapter 3.1.5 --- "IL-25 induced the release of IP-10, MIG and RANTES" --- p.49 / Chapter 3.1.6 --- IL-18 and IL-25 did not enhance the proliferation of costimulated Th cells --- p.49 / Chapter 3.2 --- "Effects of IL-18 and IL-25 on the activation of p38 MAPK, ERK, JNK and NF- k B" --- p.58 / Chapter 3.2.1 --- "Costimulation with or without IL-18 and IL-25 could activate p38 MAPK, ERK and JNK" --- p.58 / Chapter 3.2.2 --- Costimulation with or without IL-18 and IL-25 could induce NF- k B activity --- p.58 / Chapter 3.3 --- Effects of inhibitors on the IL-18 and IL-25-induced release of Thl/2 cytokines and chemokines --- p.63 / Chapter 3.3.1 --- "Optimal dosage of SB203580, PD98035, SP600125 and BAY117082" --- p.63 / Chapter 3.3.2 --- "SB203580, PD98035 and BAY 117082 but not SP600125 suppressed the IL-18 and IL-25-induced release of Thl/2 cytokines" --- p.63 / Chapter 3.3.3 --- SP600125 suppressed the IL-18 and IL-25-induced release of chemokines --- p.64 / Chapter 3.4 --- Effects of inhibitors on the cell surface expression of IL-18 and IL-25 receptors --- p.72 / Chapter 3.4.1 --- "SB203580, PD98035, BAY 117082 but not SP600125 could suppress IL-18 receptor on costimulated Th cells" --- p.72 / Chapter 3.4.2 --- "SB203580, SP600125, PD98035 and BAY 117082 could not suppress IL-25 receptor on costimulated Th cells" --- p.72 / Chapter 3.5 --- Effects of costimulation on the expression of cell surface markers on Th lymphocytes --- p.75 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effects of IL-18 and IL-25 on the release of Th1/2 cytokines and chemokines --- p.80 / Chapter 4.2 --- "Regulation of Thl/2 cytokines and chemokines through intracellular p38 MAPK, ERK, JNKand NF-kB" --- p.83 / Chapter 4.3 --- Effects of costimulation on different surface markers in Th cells --- p.87 / Chapter 4.4 --- Concluding remarks and future perspectives --- p.90 / References --- p.94

Dietary n-3 polyunsaturated fatty acids (PUFA) have been shown to potently attenuate T cell-mediated inflammation, in part, by suppressing T cell activation and proliferation. Apoptosis is an important mechanism for preventing chronic inflammation by maintaining T cell homeostasis through the contraction of populations of activated T cells. We hypothesized that dietary n-3 PUFA would promote T cell apoptosis, thus, providing an additional mechanism to explain the anti-inflammatory effects. We specifically examined activation-induced cell death (AICD) since it is the form of apoptosis associated with peripheral T cell deletion involved in immunological tolerance and T cell homeostasis. Female C57BL/6 mice were fed diets containing either n-6 PUFA (control) or n-3 PUFA for 14 d. Splenic T cells were stimulated with CD3/CD28, CD3/PMA, or PMA/Ionomycin for 48 h followed by reactivation with the same stimuli for 5 h. Apoptosis was measured using Annexin V/propidium iodide and flow cytometry. Cytokine analyses revealed that n-3 PUFA enhanced AICD only in T cells expressing a Th1-like cytokine profile (high IFN, low IL-4) compared to mice fed the n-6 PUFA control diet. Dietary n-3 PUFA significantly altered the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in T cell membranes. To examine the apparently selective effect of dietary n-3 PUFA on AICD in Th1 cells, CD4+ T cells were polarized in vitro to a Th1 phenotype by culture with IL-4, IL-2, and IL-12 for 2 d, followed by culture with IL-2 and IL-12 for 3 d in the presence of diet-matched homologous mouse serum (MS) to prevent loss of cell membrane fatty acids. Following polarization and reactivation, we observed that n-3 PUFA enhanced Th1 polarization and AICD only in cells cultured in the presence of MS, but not in fetal bovine serum. The n-3 PUFA enhancement of Th1 polarization and AICD was associated with the maintenance of diet-induced changes in EPA (20:5n-3) and DHA (22:6n-3) in plasma T cell membrane lipid rafts. Overall, these results suggest that dietary n-3 PUFA enhance both the polarization and deletion of pro-inflammatory Th1 cells, possibly as a result of alterations in lipid raft fatty acid composition.

ABSTRACT DEVELOPING A PEER HELPING PROGRAM AND TESTING ITS EFFECTIVENESS Aladag, Mine Ph. D., Educational Sciences Supervisor: Prof. Dr. Esin Tezer 2005, 290 pages The main purpose of this three-phase study was to develop a peer helping program. The purpose of the first phase was to plan a peer helping program with its goal and content. The purpose of the second phase was to develop a peer helping training program and to investigate its effectiveness on improving the helping skills and self-growth of the peer helpers. The purpose of the third phase was to understand the peer helpers&rsquo / and helpees&rsquo / evaluations of the peer helping program. In the first phase of the study, a need assessment study was carried out with a sample of 316 (168 females, 148 males) students from Faculty of Education of Ege University. Results indicated that students preferred peer helpers among other helping agents / they preferred both non-directive helping styles (empathic responses) and directive helping styles (instrumental responses) / they seemed to believe the helpfulness of the program / they tended to apply to the program / and they wanted the program to concentrate on their career, academic, and interpersonal relationships concerns. In the second phase of the study, in order to investigate the effect of peer helping training program on improving peer helpers&rsquo / helping skills and the self-growth, a pre-test post-test control group experimental design study was carried out with a sample of 31 participants (15 experimental group and 16 control group). The participants were also administered six-month follow-up test. The data were gathered by administering five instruments: Communication Skills Evaluation Scale (CSES), Empathic Skill Scale-B Form (ESS-B), Reflection Skills Evaluation Form (RSEF), Rosenberg Self-Esteem Scale (RSS) and Self-Acceptance Inventory (SAI). After testing the equivalence of the pre-test scores of CSES, ESS-B, RSS, and SAI of the experimental and the control group participants, a series of 2 (experimental-control groups) X 3 (pre-post-follow up tests) repeated measure of analysis of variance (ANOVA) was employed to the CSES, ESS-B, RSS, and SAI scores of the participants. An analysis of covariance (ANCOVA) using pre-test as covariate was employed to the post-test and follow-up test scores of the RSEF of the participants. The results indicated that a peer helping training program was more effective on improving both the empathic and the reflection skills of the experimental group participants as compared to the control group participants. The results also indicated that there was a significant increase in the experimental group participants&rsquo / self-esteem scores from the pre-test to the follow-up test and also a significant increase in the experimental group participants&rsquo / self-acceptance scores from the pretest to the posttest. In the third phase of the research, a study, mainly based on qualitative data, was carried out with 15 peer helpers and 33 volunteered students/peer helpees (17 females, 16 males) who received peer helping services. All peer helpees were administered Peer Helping Service Evaluation Form and 15 peer helpers were also administered Peer Helping Program Evaluation Form. The analysis of the qualitative data indicated that most of the above mentioned areas were positively evaluated by both peer helpers and peer helpees.

The purpose of this study is to investigate how professional career counselors look at the concepts self-reflection, self-awareness and the shadow side; “what happens underneath the surface of a conversation”. Furthermore, if they believed that this was something that was highlighted as important during their education and if they have the necessary tools for self-reflection. I also wanted to find out if they spent time self-reflecting in their daily work, and if so, in what way. The study has resulted in the following questions: How do career counselors argue about the concept of self-reflection, self-awareness and the shadow side of a conversation? How do career counselors view the importance of reflecting? What role does self-reflection play for the career counselors in their daily work?  In order to answer these questions, I have used a qualitative approach with in-depth interviews. These in-depth interviews have been performed with professional career counselors in the municipality of Gothenburg. The collected data has then been analyzed, with previous research as a theoretical starting point. The result of the study illustrates the views of these three professional career counselors on the concepts; self-reflection, self-awareness and the shadow side; “what happens underneath the surface of a conversation”. The study found that self-reflection is a central part of the work for all three career counselors. The three counselors believe self-reflection is a prerequisite for self-awareness. They also believe that in order to be a good career counselor, it is important to be aware of yourself and your behavior. Taking time to reflect and to get to know yourself also helped develop their professional roles. The professional development of self-reflection was beneficial for both themselves and their students. The result also shows that an encouraging and positive attitude is required for self-reflection to be part of the work. / Syftet med denna uppsats är att undersöka hur verksamma studie- och yrkesvägledare ser på begreppen självreflektion, självkännedom och skuggsidan, det som händer under ytan i ett samtal. Vidare om de upplever att detta var något som lyftes fram som viktigt under deras utbildning och om de upplever sig ha redskap för att själva kunna reflektera. Jag vill också ta reda på om de ägnar tid åt självreflektion och på vilket sätt i sitt arbete. Studiens syfte har utmynnat i följande frågeställningar: Hur resonerar studie- och yrkesvägledare kring begreppet självreflektion, självkännedom och skuggsidan av ett samtal? Hur ser studie- och yrkesvägledare på vikten av att reflektera? Vilken roll spelar självreflektion i studie- och yrkesvägledarnas yrkesutövande? För att besvara mitt syfte och frågeställningar har jag använt mig en kvalitativ metod med djupintervjuer. Dessa djupintervjuer har gjorts med verksamma studie- och yrkesvägledare i Göteborgs kommun. Empirin har sedan analyserats med tidigare forskning som teoretisk utgångspunkt. Resultatet av studien belyser dessa tre verksamma studie- och yrkesvägledares syn på begreppen; självreflektion, självkännedom och skuggsidan; ”det som händer under ytan i ett samtal”. I studien har det framkommit att självreflektion är en central del av arbetet för de tre studie- och yrkesvägledare som intervjuats. Dessa vägledare menar att självreflektion är en förutsättning för självkännedom. Att ha god självkännedom anser de är avgörande för att vara en bra vägledare. Att ägna tid åt att reflektera och lära känna sig själv leder också till att de utvecklas i sin yrkesroll. Den professionella utvecklingen som självreflektion leder till gynnar både dem själva och deras elever. Resultatet visar också att för att självreflektion ska vara en del av arbetet, så krävs en uppmuntrande och positivt inställd organisation.

Mental hygiene is the discipline that deals with developing and maintaining mental healt and preventing mental disorders and illnesses. It is a system of scientifically elaborated measures and instructions that help maintain, improve and restore mental health. Assistant branches and sciences are generally and undoubtedlly regarded as very demanding. The staff must meet high demands and be ready at all times to help others sort out their problems and difficult life situation. To keep their mental balance, these staff members should follow the principles of mental hygiene. The target of the diploma thesis at hand is to find out how an assistant employee subjectively assesses his or her workload and if they know how to tackle it. The thesis also aims at exploring particular measures of mental hygiene through which the staff cope with the workload arising from thein work. Before the survey was carried out, three hypotheses were formulated: H1: The employees in assistant positions in facilities providing social services in Pelhřimov region are aware of the degree of their workload, H2: The employees in assistant positions are informed about how to deal with workload, H3: The employees in assistant positions eliminate the workload through active and passive elements of mental hygiene. To complete the research part of the thesis, the quantitative survey employed questioning as the method of data collection using questionnaires as the technique. The survey was carried out in all 16 facilities providing social services in Pehřimov region. The research sample consisted of 224 employees in assistant positions in the said facilities. The research confirmed all formulated hypotheses. The research result can be made available to managements of facilities providing social services in order to serve as a stimulus for finding a more active approach to solving the issue at hand. Furthermore, the thesis might serve as a source of important data related to mental hygiene that may be distributed to all staff in assistant positions as well as to those who are interested in this topic.

Le syndrome de Sjögren primitif (SJp) est une pathologie auto-immune caractérisée par une sécheresse occulobuccale, un infiltrat lymphocytaire des glandes salivaires, ainsi qu'une production d'auto-anticorps. Les cellules épithéliales salivaires (SGEC) des patients atteints de SSp expriment les molécules impliquées dans les réponses immunitaires et jouent le rôle des cellules présentatrices d’antigènes. Les lymphocytes T folliculaires (LTf) jouent un rôle important en activant les lymphocytes B via la sécrétion d’interleukine (IL)-21. Une augmentation de la proportion de LTf est observée dans le sang des patients ayant un SJp. Nous avons fait l’hypothèse que les SGECs des patients pouvaient induire la différenciation des lymphocytes T naïfs (LTn) en LTf. Nous avons montré que les SGECs sont capables d’induire la différenciation des LTn en LTf via des facteurs solubles tel l’IL-6. La sécrétion d’IL-21 par les LTf nécessite un contact cellulaire impliquant en partie ICOSL.La voie de costimulation OX40/OX40L est impliquée dans plusieurs maladies autoimmunes. Les polymorphismes d’OX40L sont une prédisposent au SJp. Nous avons étudié le rôle pathogène de la voie OX40/OX40L chez les patients SJp. Notre résultats ont montrés une surexpression d’OX40L et d’OX40 dans les glandes salivaires des patients atteint de SJp. Les cocultures des LTn avec les SS SGECs ou contrôle SGECs augmentent l'expression d’OX40 par les LT. Les SS SGECs favorisent la survie et la prolifération des LT via la voie d’OX40/OX40L. Ces résultats démontrent l'implication d’OX40 et d’OX40L dans la pathogénie du SJp et confirment le rôle important des SGECs dans l’épithelite auto-immune du SJp. / The primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by dry mouth and dry eyes. Salivary gland epithelial cells (SGECs) of patients with pSS express the molecules involved in immune responses and act as antigen presenting cells. Follicular helper T cells (Tfh) secrete IL-21 whose augmented secretion is a hallmark of several autoimmunediseases. Here we investigated whether SGECs were capable to induce Tfh differentiation. We report that IL-6 and ICOSL expression by SGECs contributes to naïve CD4+ T differentiation into Tfh cells, as evidenced by their acquisition of a specific phenotype, characterized by Bcl-6, ICOS and CXCR5 expression and IL-21 secretion, but also but by their main functional feature: the capacity to enhance B lymphocytes survival. OX40/OX40L interaction is a pivotal costimulatory pathway. Polymorphisms of OX40L are involved in the genetic predisposition to pSS. We therefore investigated the pathogenic role of OX40/OX40L pathway in pSS. We demonstrated that the proportion of circulating CD4+ T cells expressing OX40 was elevated in patients with pSS and correlated with systemic disease activity. In salivary glands of patients with pSS, epithelial cells overexpressed OX40L and the expression of OX40L and OX40 was respectively evidenced on infiltrating B and T cells. Coculture of T cells with SGECs increased the expression of OX40 by CD4+ T cells promoted T cell survival and proliferation through OX40/OX40L interaction. These studies demonstrate emphasizes unknown pathogenic roles of SGECs and suggests that Tfh, IL-21 and OX40L might be therapeutic targets in pSS.

O Adenovírus (Ad) é um agente etiológico que causa infecções em diversas espécies e também pode ser utilizado na forma de vetor como ferramenta tecnológica para terapia gênica. O Controle sobre a replicação de Ad pode trazer beneficio para o combate de infecções e para as tecnologias de transferência genica. Porém, poucas ferramentas existem que podem inibir a replicação de Ad. Uma aplicação importante seria a inibição da replicação de Adenovírus helper utilizado na produção de Vírus Adenoassociado recombinante (rAAV), assim minimizando contaminação da produção de rAAV com o virus helper. Dessa maneira o objetivo desse trabalho foi investigar se há inibição da replicação do Ad mediada por RNA de interferência (RNAi) direcionada para alvos adenovirais chaves. Para isso foram construídos vetores lentivirais que codificam shRNAs para os genes hexon, IVa2 e pol. Em seguida foram criadas linhagens que expressam constitutivamente os shRNAs em 293T, células onde os vetores adenovirais conseguem se replicar. Os shRNAs específicos para hexon e IVa2 promoverem significantemente a redução dos níveis destes mRNAs conforme revelado utilizando RT-qPCR para quantificação dos transcritos adenovirais. Em seguida, knockdown do gene hexon se mostrou promissor em inibir a replicação do Ad, visto como redução de vírus produzido em células 293T anti-hexon. O knockdown do transcrito de hexon e a redução em replicação de Adenovírus foram mais acentuados após cell sorting e obtenção de clones celulares a partir da linhagem anti-hexon. O clone anti-hexon mostrou significante redução na quantidade de partículas adenovirais visualizadas por microscopia eletrônica e redução de 92% das partículas infecciosas em relação a 293T quando a produção foi realizada em larga escala. Esses resultados indicam que a tecnologia de shRNA para inibir a replicação do Ad é promissora e representa o primeiro passo de desenvolvimento de uma estratégia para a produção de rAAV livre de contaminação com Ad helper / Adenovirus (ad) is an etiologic agent that causes infections in diverse species and can also be used as a technologic resource, such as a vector applied in gene therapy. Control over Ad replication could be beneficial for the combat of infections and for the technology of gene transfer. However, few tools exist that may useful for the inhibition of Ad replication. One important application would be to impede replication of helper adenovirus utilized in the production of recombinant Adenoassociated Virus (rAAV), thus minimizing the contamination of the rAAV production with helper virus. The objective of the study was to investigate the use of RNA interference (RNAi) directed against key adenoviral targets as an inhibitor of Ad replication. For this, lentiviral vectors encoding shRNAs for hexon, IVa2 and pol were constructed. Next, constitutive expression of the shRNAs was established in 293T cells, the parental cell line that is permissive for adenovirus replication. The shRNAs specific for hexon or IVa2 significantly promoted reduction in the level of these mRNAs as revealed by RT-qPCR quantification of the adenoviral transcripts. Next, knockdown of hexon was shown to be promising as an inhibitor of Ad replication, seen as the reduction of Ad produced in the 293T anti-hexon cell line. Both the knockdown of the hexon transcript and reduction in adenovirus replication were accentuated after cell sorting and isolation of cellular clones from the anti-hexon cell line. The anti-hexon clone showed significant reduction in the quantity of adenovirus particles when visualized by electron microscopy and 92% fewer infectious particles as compared to the parental 293T cells when full scale production was made. These results indicate that the use of shRNA technology for the inhibition of Ad replication is promising and represents the first step for the development of a strategy for the production of rAAV free from helper virus contamination

<p>Over the past decade, studies using murine models have led to the demonstration that CD4+ T helper (Th) cells play a critical role in the control of cancer progression. Additional support for their importance comes from the growing body of recent clinical/translational research data demonstrating the importance of tumor-infiltrating T and B lymphocytes in long-term patient survival for various types of cancer, including breast cancer (BC). As the key population coordinating adaptive immune responses, the role(s) played by individual Th subsets in cancer immunity remains largely controversial. The Th1 subset has uniquely been shown to have a clear anti-tumor effect, guiding CD8+ cytotoxic T cells-mediated direct tumor cell lysis through IFN-γ secretion. Although the negative regulatory role played by Treg cells has been extensively studied in cancer, its prognostic value along with that of Th2 and Th17 cells have not been clearly demonstrated in patients. T follicular helper (Tfh) cells, a recently characterized Th subset that plays a primary role in the generation of B cell memory in secondary lymphoid organs, have not been previously described infiltrating solid tumors. The principal objective of this thesis was to perform an in-depth characterization of tumor-infiltrating CD4+ T cells (TIL) and Th subsets in human BC, where very little is currently known.<p>Using whole genome microarrays, we analyzed the gene expression profiles of TIL relative to their counterparts from the axillary lymph nodes and peripheral blood. Applying a novel approach, we compared TIL profiles with public microarray data for Th subsets, demonstrating: 1) the presence of all major Th subsets (Th1, Th2, Th17, Treg as well as Tfh) in the TIL, 2) the TIL are effector memory rather than central memory cells, 3) the TIL are concomitantly activated and suppressed and 4) TIL from tumors with extensive lymphoid infiltrates are more activated/less suppressed in the TCR/CD3 signaling pathway, producing higher levels and a wider panel of Th cytokines than TIL from minimally-infiltrated tumors.<p>We also performed in vitro experiments to study tumor microenvironment effects on TIL by treating normal CD4+ T cells from healthy donor blood with primary tumor supernatants (SN). Tumor SN largely reproduces the TIL profile in normal Th cells, totally suppressing their activation and inhibiting their cytokine production. Intriguingly, the highly restricted number of cytokines induced by tumor SN included several tumor-promoting factors, such as IL-8 and TNF. SN from an extensively-infiltrated tumor was found to be less immune-suppressive than SN from minimally-infiltrated tumors. In line with this, TIL from minimally-infiltrated tumors are closer to SN-treated (suppressed) activated donor cells whereas TIL from extensively-infiltrated tumors are more similar to activated cells without SN treatment.<p>These results led us to further investigate the observed differences between TIL from extensive and minimally-infiltrated tumors. Genes characterizing Th1 and Tfh cells were enriched in the extensively-infiltrated tumors. PD-1hiCD200hi Tfh cells were specifically detected in extensively-infiltrated tumors by flow cytometry and these cells were determined to be the major source of the chemokine CXCL13. Immunohistochemical analysis demonstrated highly-organized tertiary lymphoid structures (TLS) within the tumor, containing a CD4+/CD8+ T cell zone and a B cell zone with reactive germinal centers where Tfh cells and follicular dendritic cells (FDC) are resident. Their presence suggests the origin of an effective memory anti-tumor immune response.<p>Finally, we generated Tfh- and Th1-specific gene signatures reflecting differences between extensive and minimal TIL and tested their prognostic value in large-patient-scale public data sets. Our Tfh signature predicts better 10-year disease-free survival for all BC subtypes, outperforming the Th1 signature, suggesting that Tfh cells play a more central role than Th1 cells in anti-tumor immunity. CXCL13 is the determinant gene of our Tfh signature, showing particularly strong prognostic power for the HER2+ subtype. Additionally, these signatures also predict a better response to neoadjuvant chemotherapy.<p>This thesis research has demonstrated that a previously undetected Th subset, Tfh cells, infiltrates solid tumors and shown that their presence signals enhanced anti-tumor immunity.<p><p> <p>Durant cette dernière décennie, des travaux menés dans des modèles murins ont permis de mettre en évidence le rôle crucial joué par les lymphocytes T auxiliaires CD4+ (Th) dans le contrôle de la progression des cancers. De plus, de nombreuses études cliniques et/ou translationnelles récentes corroborent ces observations en montrant une corrélation entre l’importance de l’infiltration intra-tumorale par les lymphocytes T et B et la survie à long terme des patients atteints de différents types de cancer, dont le cancer du sein. En tant que chefs d’orchestre de la réponse immune adaptative, les rôles spécifiques des sous-populations des cellules Th restent controversés. Les Th1 sont la seule population exerçant une claire réponse anti-tumorale, qui est liée à la sécrétion d’IFN-γ, une cytokine primordiale à l’action des lymphocytes T cytotoxiques CD8+. Bien que le rôle néfaste des T régulateurs (Treg) a été largement étudié dans le cancer, leur implication pronostique ainsi que celle des Th2 et Th17 n’ont pas encore été clairement démontrées. La présence d’une sous-population de CD4, les T auxiliaires folliculaires (Tfh), cellules clés dans la différenciation des lymphocytes B mémoires au sein des organes lymphoïdes secondaires, n’a jamais été décrite dans les cancers solides. Le but principal de ce travail est de caractériser les sous-populations des lymphocytes T CD4+ infiltrant la tumeur (TIL) en prenant comme modèle le cancer du sein humain. A l’heure actuelle, il existe très peu de données sur les TIL CD4 dans ce type de cancer.<p>Nous avons d’abord établi le profil génique des TIL en les comparant avec ceux provenant des ganglions axillaires ou du sang périphérique. En appliquant une nouvelle approche, nous avons comparé les profils des TIL avec les données publiques de sous-populations de Th et démontré que :1) toutes les sous-populations de cellules Th (Th1, Th2, Th17, Treg et Tfh) infiltrent la tumeur, 2) les TIL ont un phénotype plus proche de celui des cellules mémoires effectrices que des cellules mémoires centrales, 3) les TIL sont simultanément activés et supprimés et 4) les TIL provenant des tumeurs massivement infiltrées («extensives») par des lymphocytes sont mieux activés et moins supprimés que les TIL des tumeurs peu infiltrées («minimales») dans la voie de signalisation TCR et produisent des cytokines d’une quantité plus élevée et d’une répertoire plus large.<p>Nous avons également effectué des expériences in vitro pour étudier l’effet de l’environnement tumoral sur les TIL en traitant des CD4 normaux (provenant des donneuses saines) par le surnageant (SN) extrait des tumeurs fraiches. Le SN tumoral induit un profil génique proche de celui des TIL en inhibant l’activation et la production de cytokines de ces cellules stimulées. Curieusement, parmi le peu de cytokines induites par le SN tumoral, des facteurs pro-tumoraux comme IL-8 et TNF sont détectés. Le surnageant provenant d’une tumeur «extensive» est moins immunosuppresseur que ceux des tumeurs «minimales». Conformément, les TIL provenant des tumeurs «minimales» ont un profil génique proche des cellules normales activées et traitées (supprimées) par le SN tumoral tandis que les TIL des tumeurs «extensives» ressemblent aux cellules activées non traitées.<p>Ces résultats nous avaient guidés à investiguer plus profondément les différences observées entre les TIL des tumeurs «extensives» et «minimales». Les gènes caractéristiques des Th1 et Tfh sont enrichis dans les tumeurs «extensives». Les cellules Tfh PD1hiCD200hi sont spécifiquement détectées par cytométrie de flux dans les tumeurs «extensives» et sont identifiées comme les producteurs principaux de la chimiokine CXCL13. L’examen par immunohistochimie a permis de détecter des structures lymphoïdes tertiaires (TLS) dans la tumeur, composées d’une zone T (CD4 et CD8) et d’une zone B au sein de laquelle se trouve parfois un centre germinatif actif contenant des Tfh et des cellules dendritiques folliculaires (FDC). La présence de ces structures suggère l’origine d’une réponse immune mémoire anti-tumorale.<p>Finalement, nous avons établi des signatures géniques spécifiques aux Tfh et Th1 et recherché leur impact pronostique dans deux bases de données publiques à grande échelle. Notre signature Tfh est positivement corrélée avec la survie à 10 ans des patientes de tous les sous-types de cancer du sein, et est plus performante que la signature Th1. Ceci suggère que les Tfh pourraient jouer un rôle plus crucial que les Th1 dans la réponse immune anti-tumorale. CXCL13 est le gène déterminant de notre signature Tfh et son expression est fortement associée à une meilleure survie chez les patientes du sous-type HER2+. De plus, ces signatures prévoient également une meilleure réponse à la chimiothérapie néoadjuvante (préopératoire).<p>Cette étude a démontré qu’une nouvelle sous-population de CD4, les Tfh, infiltre la tumeur solide et leur présence indique l’existence d’une immunité anti-tumorale renforcée.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Le système immunitaire comporte différents mécanismes de tolérance périphérique permettant de contrôler la réponse des cellules T CD8+. Dans certaines conditions encore peu connues, des cellules T potentiellement auto-réactives peuvent contourner les mécanismes de tolérance et se différencier en cellules effectrices, capables d’attaquer différentes organes de l’organisme, dans un processus d’auto-réactivité. En utilisant une souris transgénique exprimant un antigène modèle dans les cellules bêta du pancréas, j’ai étudié deux processus fondamentaux impliqués dans la différenciation des cellules T CD8+ en réponse aux antigènes du soi.1) Rôle de la translocation des lipopolysaccharides (LPS) dans la rupture de la tolérance. Nous avons préalablement démontré dans le laboratoire que des protocoles de lympho-déplétions, tels l’irradiation, étaient capables d’induire une rupture de la tolérance périphérique dans les cellules T CD8+. L’irradiation provoque la translocation des LPS des bactéries commensales vers la circulation sanguine, ce qui induit une activation du système immunitaire inné. Mes données ont montré que la translocation des LPS était corrélée avec l’activation systémique des cellules dendritiques (DC) CD11c+, en particulier les DC CD8+, responsables de la cross-présentation des auto-antigènes pancréatiques dans les tissus lymphoïdes. Alors que le traitement par des antibiotiques avant l’irradiation permet de prévenir la translocation des LPS, l’activation des DC n’est que partiellement affectée, et le développement de l’auto-immunité résultant d’une rupture de la tolérance périphérique des cellules T CD8+ ne peut pas être empêchée par le traitement.2) Visualisation de la coopération entre cellules T CD4+ et CD8+ effectrices dans la destruction des cellules bêta pancréatiques in vivo. En utilisant la microscopie intra-vitale à 2-photons, j’ai pu analyser, pour la première fois, la dynamiques des cellules T CD4+ et CD8+ auto-réactives exprimant un marqueur fluorescent, lors de l’infiltration du pancréas et du développement du diabète auto-immun. J’ai mis en évidence que l’infiltration des cellules T était accompagnée d’un remodelage de la matrice extracellulaire du pancréas, permettant la migration dirigée des lymphocytes. De plus, j’ai montré que l’arrêt MHC classe II-dépendant des cellules T CD4+, dû à des interactions avec des cellules présentatrices d’antigène recrutées au site d’inflammation et impliquant dans certains cas également les cellules T CD8+, contribuait au maintien des fonctions effectrices des cellules T CD8+. / The immune system has evolved multiple mechanisms of peripheral tolerance to control CD8+ T cell responses. Under particular conditions that are not yet well understood, potentially autoreactive T cells may override tolerance and differentiate into effector cells capable of targeting the own components of the organism resulting in self-reactivity. Utilizing transgenic mice expressing a model antigen in the beta cells of the pancreas, I have studied two important processes involved in CD8+ T cells differentiation in response to self-antigens. 1) Role of lipopolysaccharides (LPS) translocation in the breakdown of CD8+ T cell tolerance. It has been previously shown in our laboratory that lymphodepleting protocols, such as total body irradiation, promote breakdown of peripheral CD8+ T cell tolerance. Irradiation induces translocation of commensal bacteria LPS, a potent innate immune system activator, into the bloodstream. My data demonstrated that LPS translocation correlated with systemic activation of CD11c+ dendritic cells (DC), in particular CD8+ DC, responsible for pancreatic self-antigen cross-presentation, in lymphoid tissue. While antibiotic treatment of mice before irradiation prevented LPS translocation, DC activation was only partially affected, and onset of autoimmunity and breakdown of CD8+ T cell tolerance could not be prevented.2) Intra-vital visualization of effector CD8+ and CD4+ T cell cooperation in beta cell destruction in the pancreas. Using two-photon microscopy, I have been able, for the first time, to simultaneously analyze dynamics of fluorescently tagged autoreactive CD8+ and CD4+ T cells as they infiltrated the pancreas and induced autoimmune diabetes. I found that T cell infiltration promoted extracellular matrix remodeling in the pancreas, which in turn served as a scaffold for T cell migration. In addition, I showed that MHC class II dependent arrest of effector CD4+ T cells, due to interactions with antigen presenting cells, occasionally also implicating CD8+ T cells, provided help to effector CD8+ T cells in maintaining their effector functions.

L'objectif de cette thèse est de contribuer à faire progresser les connaissances sur les interactions bénéfiques entre les plantes et les microorganismes en évaluant la contribution des systèmes de sécrétion de type III (SST3). Une synthèse des connaissances disponibles relatives aux SST3 chez les Pseudomonas non pathogènes, saprotrophes ou mutualistes, présentée chapitre I, montre que les SST3 ne sont pas cantonnés aux interactions parasites ou pathogènes avec les plantes. Dans l’étude expérimentale présentée chapitre II, nous avons utilisé différents génotypes de Medicago truncatula Gaertn. cv. Jemalong capables (Myc+) ou non (Myc-) d’établir une symbiose mycorhizienne. Ce travail nous a permis de montrer que les Pseudomonas spp. fluorescents possédant un SST3 (SST3+) sont préférentiellement associés aux racines mycorhizées des génotypes Myc+ de M. truncatula (J5 et TRV48) plutôt qu’aux racines du mutant Myc- (TRV25) et au sol nu. Ainsi, la plante seule n’est pas à l’origine de la présence accrue des Pseudomonas SST3+. La colonisation de la racine par les champignons mycorhizogènes à arbuscules (CMA), le développement du mycélium intraradiculaire et/ou la formation associée d’arbuscules, sont également déterminants. Dans l’étude présentée chapitre III, nous avons comparé les effets de la souche modèle promotrice de mycorhization (MHB) P. fluorescens C7R12 (SST3+) et de son mutant C7SM7 (SST3-), sur la mycorhization et la croissance de M. truncatula dans un sol non stérile. Ce travail a permis de montrer que le SST3 de C7R12 contribue à l’effet MHB de la bactérie. La promotion de la colonisation de la racine de M. truncatula par les CMA indigènes induite par le SST3 de C7R12 s’est traduite par une amélioration de la croissance de la plante. En revanche, l’inactivation du SST3 chez C7SM7 a eu un impact délétère sur la colonisation de la racine de M. truncatula par les CMA du sol étudié et sur la croissance de la plante. L’observation d’effets quantitatifs opposés entre C7R12 et C7SM7, nous a conduits à nous interroger sur l’existence d’un effet différentiel de l’inoculation de ces bactéries sur la structure et la diversité des communautés des microorganismes associés. Dans une étude présentée chapitre IV, le suivi dynamique en parallèle de la structure des communautés totales bactériennes (B-RISA) et fongiques (F-RISA) et de la colonisation de la racine par les CMA a été réalisée. Aucun effet de l’inoculation n’a été observé sur la structure des communautés fongiques de la rhizosphère ou des racines. En revanche, la structure des communautés bactériennes a varié selon que les plantes aient été inoculées ou non et selon la souche inoculée. Néanmoins, ces différences ont été observées plusieurs semaines après les effets de l’inoculation de C7R12 ou de C7SM7 sur la colonisation de la racine par les CMA. Ce décalage dans le temps, suggère que les différences observées dans la structure des communautés bactériennes pourraient être une conséquence plutôt qu’une cause des variations observées sur la mycorhization de M. truncatula. Nos résultats n’ont pas permis de mettre en évidence d’effets de l’inoculation sur la diversité des populations des bactéries fixatrices d’azote présentes dans les nodosités de M. truncatula. L’analyse des séquences de la grande sous-unité de l’ADN ribosomique (LSU rDNA) amplifiées à partir d’ADN extrait des racines, a montré pour les plantes inoculées et non inoculées, que les populations de CMA étaient majoritairement apparentées à Glomus intraradices. Un groupe d’isolats spécifiquement associé aux racines inoculées avec C7R12 et apparenté à G. claroideum a été décrit. Le groupe spécifique pourrait être associé à l’amélioration de la mycorhization observée dans les racines inoculées avec C7R12. Néanmoins, compte tenu de sa faible représentation numérique (8%), il semble probable que l’inoculation de C7R12 ait aussi un effet quantitatif sur la colonisation de la racine de M. truncatula par les CMA. etc / No abstract