T cell activation and thymic selection are thought to be determined by the binding propensity (avidity or affinity) of the T cell receptor (TCR) to its ligands. However, binding propensity quantified by previous 3D TCR–pMHC kinetics such as using tetramer staining or surface plasmon resonance (SPR) under estimate TCR–pMHC interaction due to neglecting physiological conditions. Recent studies considering membrane contribution in TCR–pMHC interaction reported 2D kinetics and force regulated bond dissociation kinetics have better prediction to biological responses in CD8+ T cells. In this study, we further tested the findings in CD4+ T cells and CD4+ CD8+ (double-positive, DP) thymocytes. We analyzed TCR–pMHC interaction for a well-characterized panel of altered peptide ligands (APLs) on multiple transgenic mouse TCR systems. Using ultrasensitive 2D mechanical assays, in situ 2D kinetic measurements show better sensitivity than the SPR 3D kinetic measurements in gauging the ligand potency and thymic selection. Furthermore, force-regulated bond lifetime of TCR–pMHC interaction amplifies the discrimination in recognition of APLs and thymic selection. When force was applied to TCR–pMHC–CD4/8 bonds, two distinct patterns emerged: agonist/negative selecting ligands formed CD4/8-dependent catch-slip bonds where lifetime first increased, reached a maximum, then decreased with increasing force, whereas antagonist/positive selecting ligands formed slip-only bonds where lifetime monotonically decreases with increasing force. Our results highlight an important role of mechanical force in ligand discrimination and suggest a new mechanism for T cell activation and thymic selection that is distinct from previous models based on 3D measurements.

Thy-1 is a glycosylphosphatidylinositol-anchored protein that is expressed on murine T lymphocytes and is involved in T cell-mediated immune responses. In the presence of costimulatory signals, monoclonal antibody (mAb)-induced signaling through Thy-1 is associated with hallmarks of T cell activation, including IL-2 production and T cell proliferation. Thy-1-induced signaling promotes cytotoxic effector molecule expression, but is unable to trigger delivery of the lethal hit to target cells, suggesting that Thy-1 provides an incomplete T cell receptor (TcR)-like signal. However, the effect of Thy-1 signaling on cytokine production and the development of T helper (Th) cell phenotypes (Th1, Th2, Th17) remains unclear. The purpose of this work was to further our understanding of Thy-1-mediated signal transduction and the role that Thy-1 plays in the development of effector T cell responses. I found that, in the context of costimulatory signals, anti-Thy-1 mAb induced significantly less IL-2 production, CD25 expression and T cell proliferation than anti-TcR? mAb. Several key signaling molecules, including protein tyrosine kinases, zeta chain-associated protein-70 and extracellular signal-regulated kinase were activated with delayed kinetics during Thy-1-mediated T cell activation. The delayed signaling kinetics resulted in the delayed acquisition of cytotoxic effector function and also delayed delivery of the lethal hit to target cells. Interestingly, Thy-1-mediated signaling induced significantly more IL-17 and IL-4 synthesis and less IFN-? synthesis in comparison to TcR-mediated signaling. Moreover, Thy-1-activated CD4+ T cells produced high levels of IL-17 and IL-4 but minimal IFN? when restimulated with anti-Thy-1 mAb or anti-TcR? mAb with or without costimulatory signals. The unique ability of Thy-1 signaling to induce IL-17 production correlated with the expression of the Th17 lineage-specific transcription factor, retinoic orphan receptor gamma t. These observations show that Thy-1 signaling differs from TcR signaling in its ability to induce Th cell cytokines. Taken together, my findings show that Thy-1 signaling can provide the full TcR-like signal required for both the differentiation and triggering of Th cells and cytotoxic T lymphocytes, albeit with delayed kinetics in comparison to TcR signaling. They also suggest that Thy-1 signaling may be important in the development of Th2 and Th17 responses.

The surface of biomaterials can induce contacting blood to coagulate, similar to the response initiated by injured blood vessels to control blood loss. This poses a challenge to the use of biomaterials as the resulting coagulation can impair the performance of hemocompatible devices such as catheters, vascular stents and various extracorporeal tubings [1], what can moreover cause severe host reactions like embolism and infarction. Biomaterial induced coagulation processes limit the therapeutic use of medical products, what motivates the need for a better understanding of the basic mechanisms leading to this bio-incompatibility [2] in order to define modification strategies towards improved biomaterials [3]. Several approaches for the enhancement of hemocompatible surfaces include passive and active strategies for surface modifications. The materials’ chemical-physical properties like surface chemistry, wettability and polarity are parameters of passive modification approaches for improved hemocompatibility and are the focus of the present work. In the present study self assembled monolayers with different surface functionalities (-COOH, -OH, -CH3) were applied as well as two-component-layers with varying fractions of these, as they allow a defined graduation of surface wettability and charge. The ease of control over these parameters given by these model surfaces enables the evaluation of the influence of specific surface-properties on biological responses. To evaluate the effects of different surface chemistry on initial mechanisms of biomaterial induced coagulation, the surfaces were incubated with protein solution, human plasma, blood cell fractions or fresh heparinised human whole blood. Indicative hemocompatibility parameters were subsequently analysed focusing on protein adsorption, coagulation activation, contact activation (intrinsic/ enhancer pathway), impact of tissue factor (extrinsic/ activator pathway) and cellular systems (blood platelets and leukocytes).

Orientador: Beatriz Maria Machado de Medeiros / Banca: Beatriz Maria Machado de Medeiros / Banca: Maria Terezinha Serrão Peraçoli / Banca: Iracilda Zeppone Carlos / Banca: Cleni Mara Marzocchi Machado / Banca: Fernanda de Freitas Anibal / Resumo: Yersinia pseudotuberculosis e Y. enterocolitica são patógenos que causam desordens gastrintestinais. Estudos utilizando infecção in vitro demonstraram que Y. enterocolitica pode ter como alvo as células dendríticas (DCs), afetando várias de suas funções, incluindo sua maturação e produção de citocinas, e, conseqüentemente, contribuindo para a diminuição da ativação de células T CD4+. O objetivo deste estudo foi investigar o papel das células dendríticas das placas de Peyer (PP) na determinação do padrão de resposta imune, Th1 e Th2, durante a infecção por via intragástrica de camundongos suscetíveis (BALB/c) e resistentes (C57BL/6) com a amostra virulenta de Y. pseudotuberculosis (YpIII pIB1 - Yp+) ou seu par isogênico, curado do plasmídeo de virulência (YpIII - Yp-). As DCs das PP foram obtidas no 1°, 3° e 5° dia pós-infecção, quantificadas e analisadas quanto às suas subpopulações, expressões de moléculas de superfície e capacidade imunoestimulatória por citometria de fluxo, e quanto à secreção de citocinas (IL-4, IL-10, IL-12 e TNF-α) por ELISA. Os linfócitos das PP também foram obtidos no mesmo período e tiveram suas sub-populações e o padrão de citocinas intracelulares Th1/Th2 (IL-2, IL-4, IL-10 e IFN-γ) analisado por citometria de fluxo. A infecção por Yp+ reduziu o número de DCs no 1° dia pós-infecção e aumentou, no período inicial, a expressão de B7.1 e B7.2 nos camundongos BALB/c. Nos camundongos C57BL/6 reduziu o número de DCs durante todo o período analisado, aumentou a expressão de B7.1 e B7.2 no período inicial e a expressão de ICAM-1. A infecção por ambas as amostras provocou redução da sub-população CD8α+ e da expressão de MHC II nas duas linhagens de animais, aumentou a sub-população CD11b+ nos animais suscetíveis e diminuiu nos animais resistentes. Os animais estudados não apresentaram... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Yersinia pseudotuberculosis and Y. enterocolitica are pathogens that cause gastrointestinal disorders. Studies using in vitro infection demonstrated that Y. enterocolitica can have as a target dendritic cells (DCs), affecting several of its functions, including their maturation and production of cytokines, and, consequently, contributing to the diminished activation of the T CD4+ cells. The aim of this study was to investigate the role of dendritic cell from Peyer's patches (PP) in determining of immune response pattern, Th1 and Th2, during infection by the intragastric route in susceptible (BALB/c) and resistant (C57BL/6) mice with a virulent sample of Yersinia pseudotuberculosis (YpIII pIB1 - Yp+) or its isogenic pair, cured of the virulence plasmid (YpIII - Yp-). The PP DCs were obtained on the 1st, 3rd and 5th days postinfection, quantified and analyzed as far as their subpopulations, expressions of surface molecules and immunostimulatory capacity by flow cytometry, and the cytokines secretion (IL-4, IL-10, IL-12 and TNF-α) by ELISA. The PP lymphocytes were also obtained in the same period, and had their subpopulations and the pattern of intracellular Th1/Th2 cytokines (IL-2, IL-4, IL-10 and IFN-γ) analysed by flow cytometry. The infection by Yp+ reduced the number of DCs on the 1st day post-infection and increased, in the initial period, the expression of B7.1 and B7.2 in BALB/c. In C57BL/6 mice reduced the number of DCs throughout the study period, increased the expression of B7.1 and B7.2 in the initial period and the expression of ICAM-1. The infection by both samples reduced CD8α+ subpopulation and expression of MHC II in both animals, increased CD11b+ sub-population in susceptible animals and reduced the same sub-population in resistant animals. The studied animals did not present important differences as far as secretion of cytokines by the DCs of PP and both... (Complete abstract click electronic access below) / Doutor

As células T Natural Killer (NKT) são linfócitos que expressam o rearranjo V24V11 com TCR invariante e reconhecem glicolipídeos como o alfagalactosilceramida (-GalCer) apresentado no contexto da molécula MHC-não clássica chamada CD1d. As NKT são divididas em subgrupos distintos: os fenótipos CD4+ e CD4- em camundongos e humanos, e CD8+ e CD4-CD8- em humanos. Após estímulo, as NKT secretam citocinas Th1 e Th2. A frequência das NKT representa menos de 1% da população de linfócitos T no sangue periférico humano. O objetivo deste trabalho é primeiro, identificar a frequência, o fenótipo e a função da população de células NKT nos grupos de indivíduos saudáveis de procedências distintas. Segundo, avaliar a influência da idade, gênero e país de moradia na frequência das NKT, incluindo suas subpopulações, no sangue periférico; Terceiro, verificar se há variabilidade ou não na apresentação da molécula CD1d. Quarto, verificar se há alteração na função das NKT utilizando o estímulo -GalCer e por fim, verificar o perfil das NKT quanto ao seu estado de ativação e migração celular quando submetidas a expansão. As amostras são provenientes de São Paulo, Brasil; São Francisco, Estado Unidos da América e Estocolmo, Suécia. Os parâmetros imunológicos foram analisados por citometria de fluxo. Demonstramos que o grupo de São Paulo, não apresentou maiores valores de linfócitos T CD3+, no entanto, apresentou valores significantes de células NKT V24+/V11+. Não estabelecemos a expressão dos marcadores de ativação e migração nas NKT. Estabelecemos, no grupo de São Paulo, a capacidade funcional das NKT que expressaram IFN- e TNF- assim como de expansão, sob estímulo de -GalCer. Os resultados sugerem que vários fatores podem alterar a quantidade das células NKT na periferia. Mais estudos devem ser endereçados para esclarecer melhor estes fatos / Natural Killer T cells (NKT cells) are lymphocytes that express the invariant TCR rearrangement V24V11 and recognize glycolipid as alpha-galactosylceramide (-GalCer) presented in conjunction with a non-MHC pathway molecule named CD1d. The NKT cells are divided into distinct subsets: the CD4+ and CD4- phenotype in mice and humans and the CD8+ and CD4-CD8- phenotype in humans. After stimulation, the NKT secrete Th1 and Th2 cytokines. The frequencies of NKT cells represent <1% of the population of T lymphocytes in human peripheral blood. The objective of this study is to 1) identify the frequency, phenotype and function of NKT cell population in groups of healthy individuals from different racial/ethnic origins, 2) evaluate the influence of age, gender and country of residence on the frequency of NKT cells/subpopulations, 3) investigate the variability of presentation of the CD1d molecule, 4) investigate the functional variability of NKT cells using -GalCer stimulation, and 5) study the activation and migration profile of NKT cells after expansion. Immunologic parameters of clinical samples from Sao Paulo, Brazil; San Francisco, California and Stockholm, Sweden were analyzed by flow cytometry. We demonstrate that the São Paulo group does not have an elevated number of CD3+ T lymphocytes, but does have significantly more NKT V24+/V11+ cells. We have not yet determined expression of NKT markers for activation and migration. Thus, we have shown that among sample obtained in São Paulo, there is increased functional capacity of IFN- and TNF--expressing NKT cells along with NKT cells expansion under stimulation with the -GalCer. These results suggest that there are several factors that can modulate NKT cell number and function in the peripheral blood and additional studies are needed to further clarify these findings

The spleen is a key participant in the pathophysiology of sepsis and inflammatory disease. Many splenocytes exhibit a cholinergic phenotype, but our knowledge regarding their cholinergic biology and how they are affected by sepsis is incomplete. We evaluated effects of acute sepsis on the spleen using the cecal ligation and puncture (CLP) model in C57BL/6 and ChATBAC-eGFP mice. Quantification of cholinergic gene expression showed that choline acetyltransferase and vesicular acetylcholine transporter (VAChT) are present and that VAChT is upregulated in sepsis, suggesting increased capacity for release of acetylcholine (ACh). High affinity choline transporter is not expressed but organic acid transporters are, providing additional mechanisms for release. Flow cytometry studies identified subpopulations of cholinergic T and B cells as well as monocytes/macrophages. Neither abundance nor GFP intensity of cholinergic T cells changed in sepsis, suggesting that ACh synthetic capacity was not altered. Spleens have low acetylcholinesterase activity, and the enzyme is localized primarily in red pulp, characteristics expected to favor cholinergic signaling. For cellular studies, ACh was quantified by mass spectroscopy using d4-ACh internal standard. Isolated splenocytes from male mice contain more ACh than females, suggesting the potential for gender-dependent differences in cholinergic immune function. Isolated splenocytes exhibit basal ACh release, which can be increased by isoproterenol (4 and 24 h) or by T cell activation with antibodies to CD3 and CD28 (24 h). Collectively, these data support the concept that sepsis enhances cholinergic function in the spleen and that release of ACh can be triggered by stimuli via different mechanisms.

Chronic hepatitis C virus (HCV) infection exhibits persistent high viral load, inducing T cells differentiation and dysfunction in chronically infected individuals. Recent longitude studies in both HCV specific- and bulk T cells reveal that chronic immune stimulation is the driving force for the impaired T cell functions, however, the underlying mechanisms remain elusive. Here, we show that peripheral CD4+ T cells from chronically HCV-infected patients exhibit lymphopenia with the reduction of naïve population and expansion of effector memory T cells. CD4+ T cells from HCV patient also display elevated activation markers. including HLA-DR, GLUT1, Granzyme B, and short-lived effector marker CD127- KLRG1+, whereas stem cell-liked transcription factor TCF1 and telomere sheterin subunit TRF2 are significant reduced, comparing to age- and gender-matched healthy controls. Mechanistically, ex vivo T cell differentiation revealed that CD4+ T cells from HCV patients exhibit PI3K/Akt/mTOR signaling hyperactivation upon TCR stimulation, favoring pro-inflammatory effector differentiation with TRF2 downregulation, rendering telomere dysfunction induced foci (TIFs) accumulation, resulting in telomeric DNA damage and cellular apoptosis. Importantly, exacerbation of telomere deprotection by knockdown of TRF2 expression in healthy T cells resulted in an increase in telomeric DNA damage and T cell apoptosis; whereas overexpression of TRF2 in HCV-T cells led to an alleviation of telomeric DNA damage and T cell death. Additionally, inhibition of Akt signaling during T cell activation can preserve precursor memory population, while limiting inflammatory effector expansion, DNA damage, and cell death. Taken together, these results suggest that modulation of immune activation by inhibiting Akt signaling and protection of telomeres by enforcing TRF2 expression could open new therapeutic strategies to balance adaptive immune responses in the setting of chronic immune activation and inflammatory in vulnerable populations such as chronically viral infected individuals.

Thesis (MScMed)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Introduction: Flow cytometry has progressively replaced many traditional laboratory tests due to its greater accuracy, sensitivity and rapidity in the routine clinical settings especially clinical trails. It is a powerful tool for the measuring of chemical (the fluorochrome we add) and physical (size and complexity) characteristics of individual cells. As these instruments became major diagnostic and prognostic tools, the need for more advanced quality control, standardized procedures and proficiency testing programs increased as these instrumentations and their methodology evolve. Minor instrument settings can affect the reliability, reproducibility and sensitivity of the cytometer and should be monitored and documented in order to ensure identical conditions of measurement on a daily basis. This can be accomplished by following an Internal Quality Assurance (IQA) and/ or External Quality Assurance (EQA) program. Currently there are no such programs available in South Africa and poorer Africa countries. HIV is a global concern and the laboratories and clinics in these places are in need of such IQA programs to ensure quality of their instrumentation and accurate patient results. Quality assurance programs such as CD Chex® and UK Nequas are available but due to bad sample transport, leave the receiving laboratories with nightmares. It would be best if there was a laboratory in South Africa that could provide the surrounding laboratories with stabilized whole blood samples that can be utilized as IQA. The transport of these samples can be more efficient due to shorter distance and thus the temperature variations limited. Aims and Objectives: The aim of Chapter one is to familiarize the reader with general terminology and concepts of immunology. Chapter two describes in detail the impact stabilized whole blood had on clinical immunology concerning Quality Control and Quality Assurance. The objective of this study is to stabilize whole blood with a shelf life of greater than 30 days to serve as reference control material for South African Immunophenotyping. It is further an objective to use these in-house stabilized control samples for poorer African countries as Internal Quality Assurance reference material. It is a still further objective to stimulate various lymphocyte subsets to express activation antigens and then stabilize these cells for more specialized immunological test and can serve as a QC for those required samples. Study design: In Chapter three, the method currently used to stabilize whole blood was modified. The stability of different concentrations of a first stabilizing agent (Chromium Chloride hexahydrate) was investigated. Incubation periods and concentrations of paraformaldehyde as second stabilizing agent were investigated. Blood samples from healthy individuals (n=10) were stabilized and monitored for the routine HIV phenotypic surface antigens over a period of 40 days. These samples (n=10) were compared on the Becton Dickinson Biosciences (BD) FACSCalibur™ versus BD FACSCount™ instrumentation. Blood samples (n=3) were stabilized and monitored to identify phenotypic cell surface molecules for as long as possible. They were quantified on both flow cytrometric instruments. In addition, these stabilized samples (n=3) were investigated as control blood for calibration purposes on the BD FACSCount™ instrument. In Chapter four, lymphocytes were isolated and activated with various stimuli to express sufficient activation antigens such as CD25, CD69, HLA-DR and CD40 Ligand on the T helper cell surfaces. These activated antigens were analyzed on the BD FACSCalibur™ and further stabilized to serve as possible IQA samples in future. Results: In Chapter three, the ten individual stabilized samples had non-significant P values (P > 0.05) for CD3, CD4 and CD8 percentages and absolute values comparing day 3 until day 40. Comparing the BD FACSCalibur™ versus BD FACSCount™, resulted in a R2 = 0.9848 for CD4 absolute values and a R2 = 0.9636 for CD8 absolute values. Stabilized blood samples (n=3) were monitored for routine HIV phenotypic markers until day 84. The cells populations were easily identifiable and could be quantified on both BD FACSCalibur™ and BD FACSCount™ instruments. In Chapter four; for the activation study purposes, activated T helper lymphocytes expressed approximately 25 to 35% CD40 Ligand cell surface molecules. The stimulant of choice was Ionomycin at a 4μM concentration. Cells were incubated for four hours at 37 degree Celsius in a 5% CO2 environment. For CD69 surface expression, 6 hour incubation was optimum. The stimulus of choice in this case was 4μM Ionomycin which induced 84.21% CD69 expression in the test samples. For CD25 expression; 6 hour incubation with PHA resulted in approximately 43% of CD25 expression. For HLA-DR surface expression; 6 hour incubation with PHA resulted in approximately 43.32% of HLA-DR expression. Activated lymphocytes expressing CD40 Ligand showed stability until day 23. Activated Lymphocytes expressing CD69, CD25 and HLA-DR were stabilized in the same manner and stability could be achieved until day 16. Conclusion: This thesis was related to the preparation of control samples (IQA) designed to simulate whole blood having defined properties in clinical laboratory situations. In future kits can be developed with a low, medium and high control sample for the various immunological phenotypic determinants. Another kit can be compiled where various activation markers can be identified, quantified with a “zero”, low and high control. These whole blood IQA kits and “activation IQA kits” can be implemented for training of newly qualified staff, competency testing of staff, method development, software testing, panel settings and instrument setting testing. Control samples ideally must have a number of properties in order to be effective. For instance stability during storage times, preferably lasting more than a few weeks, reproducibility and ease of handling. These will provide the information on day-to-day variation of the technique or equipment which will enhance accuracy and improve patient care. / AFRIKAANSE OPSOMMING: Inleiding: Vloeisitometrie tegnologie het verskeie tradisionele laboratorium toetse vervang as gevolg van beter akuraadheid, sensitiwiteit en vinniger beskikbaarheid van resultate in ‘n kliniese omgewing, veral kliniese proewe. Vloeisitometrie is ‘n kragtige tegniek om chemiese (fluorokroom byvoeging) en fisiese (sel grote en kompleksiteit) karakter eienskappe van individuele selle te meet. Met die toename in gebruik en gewildheid van hiedie instrumente, neem die behoefde toe vir gevorderde kwaliteit kontroles, gestandardiseerde prosedures, met profesionele toets programme tesame met metode ontwikkeling. Klein verstellings aan instrument parameters beinvloed die betroubaarheid, herhaalbaarheid en sensitiwiteit van ‘n sitometer en moet gemonitor (en dokumenteer) word om identiese kondisies van leesings op ‘n daaglikse basis te verseker. Dit kan bereik word deur in te skakel met ‘n interne kwaliteits versekerings program [IQA: “Internal Quality Control”] en/of ‘n eksterne kwaliteits versekerings program [EQA: “External Quality Control”] te volg. Op die oomblik is daar geen sulke kwaliteits versekerings programme in Suid Afrika en/of in die verarmende Afrika lande beskikbaar nie. MIV is ‘n wêreldwye bekommernis en laboratoriums en klinieke in hierdie gedeeltes van die land verlang ‘n dringende behoefdte vir sulke “IQA” programme om kwaliteit van instrumentasie en akkurate pasiënt resultate te verseker wat tot beter behandeling van pasiënte lei. Kwaliteit versekerings programme soos “CD Chex®” en “UK Nequas” is beskikbaar, maar baie probleme met verwysing na monster integriteit as gevolg van tydsame vervoer en aflewering kondisies word hiermee geassosieër. Die behoefte het ontstaan vir ‘n laboratorium in Suid Afrika wat direk die omliggende laboratoriums, hospitale en klinieke kan voorsien met gestabiliseerde blood monsters wat gebruik kan word as “IQA”. Die vervoer en aflewerings kondisies van hierdie monsters sal aansienlik verbeter as gevolg van die korter aflewerings afstand wat direk die beperkte temperatuur wisseling beinvloed. Doel van studie: Die doelwit van hoofstuk een is om vir die leser ‘n inleiding te gee tot terminologie en konsepte van immunologie en die immune sisteem. Hoofstuk twee beskyf die impak wat gestabiliseerde heelbloed het op die kliniese immunologie met betrekking tot kwaliteit beheer en kwaliteit versekering. Die doelwit van hierdie studie is om heelbloed te stabiliseer sodat die rakleeftyd meer as 30 dae is en sodoende as verwysings-materiaal kontroles vir Suid Afrikaanse immunofenotipering kan dien. Dit is ‘n verdere doelwit om hierdie tuis-gestabiliseerde kontrole monsters te gebruik as “IQA” verwysings materiaal in verarmende Afrika lande. Die doelwit van hoofstuk vier is om limfosiete te stimuleer om verskeie aktiverings merkers uit te druk op hul selmembrane en dan te stabiliseer en dié te gebruik as Kwaliteits Kontroles vir die meer gespesialiseerde immunologiese toetse. Studie ontwerp: Hoofstuk drie beskryf ‘n aangepaste en verbeterde metode van heel bloed stabiliseering. Stabiliteit word ondersoek in ‘n verskyndenheid konsentrasies van ‘n primêre stabiliseerings agent (chromium chloried heksahidraat) en inkubasie periodes met paraformaldehied as tweede stabiliseerings agent word deeglik gedokumenteer. Bloedmonsters van gesonde indiwidië (n=10) was gestabiliseer en gemonitor vir roetine MIV membraanoppervlak antigene oor ‘n periode van 40 dae. Hierdie monsters (n=10) was gelees en geanaliseer op ‘n BD FACSCalibur™ en vergelyk met ‘n BD FACSCount™ vloeisitometer instrument. Drie gestabiliseerde heelbloed monsters (n=3) was gemonitor vir ‘n periode vir so lank moontlik die fenotipiese selmembraan molekules identifiseerbaar was en die kwantiteit bepaalbaar was. Hierdie drie monsters was gemeet op beide instrumente. As ‘n addisionele doelwit, was hierdie drie gestabiliseerde monsters ondersoek om as moontlike kalibrasie materiaal (verteenwoordig ‘n normale bloedmonster) te dien vir die BD FACSCount™ instrument in die oggende voor pasiënt monsters gelees kan word. In hoofstuk vier was limfosiete geϊsoleer en geaktiveer met ‘n verskyndenheid stimulante om optimale aktiveerings-antigene uit te druk op T helper selmembrane (byvoorbeeld CD25, CD69, HLA-DR en CD40 Ligand). Hierdie geaktiveerde monsters was geanaliseer op die BD FACSCalibur™ en daarna gestabiliseer. Na stabilisasie van die geaktiveerde limfosiet monsters was dit gemonitor oor ‘n tydperk so lank moontlik data plotte leesbaar en selpopulasies identifiseerbaar was. Hierdie monsters kan dien as ‘n moontlike “IQA” toets stel vir ‘n meer gespesialiseerde immunologiese aktiveerings kontrole doeleindes. Resultate: In hoofstuk drie; tien individiële gestabiliseerde heelbloed monsters het gedui op geen-beduidende P waardes (P > 0.05) vir CD3, CD4 en CD8 persentasies en absolute waardes; gemeet vanaf DAG 3 vergelykbaar tot-en-met DAG 40. Met korrelasie statistiek en vergelyking van die BD FACSCalibur™ met die FACSCount™ instrumente, is die volgende opgemerk; R2 = 0.9848 vir die CD4 absolute waardes en ‘n R2 = 0.9636 vir die CD8 absolute waardes. Drie gestabiliseerde monsters (n=3) was gemonitor vir MIV roetine fenotipeering tot en met DAG 84. Die selpopulasies was duidelik identifiseerbaar en die kwantitatief meetbaar op albei instrumente (BD FACSCalibur™ en BD FACSCount™). Hoofstuk vier: geaktiveerde T helper lymphosiete het 25 – 35% membraan CD40 Ligand uitgedruk op hul selmembrane. Die stimulant van keuse was ionomysien teen ‘n optimale konsentrasie van 4μM. Die optimale inkubasie tydperk was vier ure by 37°C in 5% CO2 kondisie. Ses uur inkubasie in 4μM ionomysien by 37°C in ‘n 5% CO2 omgewing was optimal vir die CD69 selmembraan uitdrukking en het 84.21% opgelewer. Vir CD25 selmembraan uitdrukking was die selle vir ses ure met phietoheamagglutinin (PHA) gestimuleer by 37°C in 5% CO2 kondisie en het 43% CD25 selmembraan uitdrukking opgelewer. HLA-DR selmembraan uitdrukking: selle was vir ses ure saam met PHA by 37°C in 5% CO2 kondisie inkubeer en het 43.32% opgelewer. CD40 Ligand aktivering/gestabiliseerde limfosiete het tot en met dag 23 stabiliteit getoon. Die ligand was duidelik identifiseerbaar en kwantifiseerbaar. Geaktiveerde lymphosiete wat CD69, CD25 en HLA-DR selmembraan merkers uitdruk het na die stabiliseerings proses stabiliteit getoon tot-en-met dag 16. Gevolgtrekking: Die doel van hierdie studie was om verwysingskontroles voor te berei sodat dit vars heelbloed naboots met uitkenbare eienskappe vir kliniese situasies. ‘n Toets kontrolestel met verwysings materiaal vir drie vlakke (byvoorbeeld ‘n lae, medium en hoë kontrole) absolute selwaardes en persentasies kan voorberei word vir roetine immunologiese fenotiperings merkers (CD3/CD4/CD8/CD45). Meer gespesialiseerde kontrolestelle vir meer spesifieke doeleindes kan opgemaak word wat ‘n verskydenheid van limfosiet aktiveringsmerkers bevat met byvoorbeeld ‘n “nul”, lae en hoë verwysings kontrole daarin. Hierdie heelbloed kan dien as “aktiveerde interne kwaliteits verwysings materiaal” en kan gebruik word om nuut aangestelde laboratorium werkers en nuut gekwalifiseerde studente op te lei. Hierdie verwysings materiaal / kontroles kan aangewend word vir bevoegdheids doeleindes (byvoorbeeld vir SANAS akkreditasie doeleindes), vir metode ontwikkeling, vir sagteware toetsing, vir paneel opstelling en instrument verstellings doeleindes. Die kontroles moet ‘n verskydenheid eienskappe bevat om effektief te wees. Byvoorbeeld, stabiliteit tydens storing, gewenslik meer as ‘n paar weke, herhaalbaar en maklik handteerbaar. Hierdie kontroles sal inligting voorsien op ‘n daaglikse basis tydens wisseling van tegnieke of instrumentasie wat akuraatheid beinvloed en op die ou-end direk pasiënt versorging bevoordeel.

Les voies de signalisation des MAP kinases (MAPK) conventionnelles jouent des rôles essentiels pendant le développement des lymphocytes T (LT) ainsi que lors de leur activation suite à la reconnaissance antigénique. En raison de ses différences structurelles ainsi que de son mode de régulation, ERK3 fait partie des MAPK dites non-conventionnelles. Encore aujourd’hui, les événements menant à l’activation de ERK3, ses substrats ou partenaires ainsi que sa fonction physiologique demeurent peu caractérisés. Nous avons entrepris dans cette thèse d’étudier le rôle de ERK3 lors du développement et de l’activation des LT en utilisant un modèle de souris déficient pour l’expression de ERK3. Nous avons premièrement établi que ERK3 est exprimée chez les thymocytes. Ensuite, nous avons évalué le développement thymique chez la souris ERK3-déficiente et nous avons observé une diminution significative de la cellularité aux étapes DN1, DP et SP CD4+ du développement des LT. La création de chimères hématopoïétiques ERK3-déficientes nous a permis de démontrer que la diminution du nombre de cellules observée aux étapes DN1 et DP est autonome aux thymocytes alors que le phénotype observé à l’étape SP CD4+ est dépendant de l’abolition simultanée de ERK3 dans l’épithélium thymique et dans les thymocytes. Une étude plus approfondie de l’étape DP nous a permis de démontrer qu’en absence de ERK3, les cellules DP meurent plus abondamment et accumulent des cassures doubles brins (DSB) dans leur ADN. De plus, nous avons démontré que ces cassures dans l’ADN sont réalisées par les enzymes RAG et qu’en absence de ces dernières, la cellularité thymique est presque rétablie chez la souris ERK3-déficiente. Ces résultats suggèrent que ERK3 est impliquée dans un mécanisme essentiel à la régulation des DSB pendant le réarrangement V(D)J de la chaîne  du récepteur des cellules T (RCT). Dans le deuxième article présenté dans cette thèse, nous avons montré que ERK3 est exprimé chez les LT périphériques, mais seulement suite à leur activation via le RCT. Une fois activés in vitro les LT ERK3-déficients présentent une diminution marquée de leur prolifération et dans la production de cytokines. De plus, les LT ERK3-déficients survivent de façon équivalente aux LT normaux, mais étonnamment, ils expriment des niveaux plus faibles de la molécule anti-apoptotique Bcl-2. Ces résultats suggèrent que la prolifération réduite des LT ERK3-déficients est la conséquence d’une altération majeure de leur activation. Ainsi, nos résultats établissent que ERK3 est une MAPK qui joue des rôles essentiels et uniques dans le développement thymique et dans l’activation des lymphocytes T périphériques. Grâce à ces travaux, nous attribuons pour la toute première fois une fonction in vivo pour ERK3 au cours de deux différentes étapes de la vie d’un LT. / Classical MAP kinases (MAPK) play essential roles during T cell development and activation. ERK3 is a member of the MAPK family for which no physiological function has been described yet. Also, ERK3 is an atypical MAPK since its structure and mode of regulation are different from the conventional MAPK. Even today, the events leading to ERK3 activation and its substrates or partners are still largely unknown. We have studied in this thesis the role of ERK3 during T cell development and activation by using a mouse model in which ERK3 is not expressed. First, we have established that ERK3 is expressed in thymocytes. Next, we have evaluated thymic development in ERK3-deficient mice and we have observed a significant decrease in cell number at DN1, DP and CD4SP stages of T cell development. ERK3-deficient hematopoietic chimeras revealed that the DN1 and DP phenotype are T-cell autonomous, while abrogation of CD4SP development requires ERK3-deficiency in both thymocytes and thymic epithelium. By investigating further the DP stage, we have shown that ERK3-deficient DP thymocytes are more prone to apoptosis and also accumulate DNA double-strand breaks (DSBs). Moreover, we have shown that the increase DSBs are the direct consequence of RAG activity and that abolition of RAG almost restored thymic cellularity in ERK3-deficient mice. These results suggest that ERK3 is involved in an essential mechanism of DBSs regulation during TCR recombination. In the second article presented in this thesis, we have shown that ERK3 is expressed in peripheral T cell, but only when their TCR is activated. Also, ERK3-deficient T cells presented a strong reduction in proliferation and cytokine secretion following in vitro stimulation. Moreover, activated T cells lacking ERK3 are not more prone to death and surprisingly, they are unable to up-regulate the expression of the anti-apoptotic molecule Bcl-2 following TCR stimulation. These results suggest that the reduced proliferation of ERK3-deficient T cells is a consequence of their defective activation. Collectively, our results unveil essential and unsuspected roles for ERK3 in T cell development and activation. With this study, we establish for the first time an in vivo function for the atypical MAPK ERK3 in two different stages during T cell life.

La reconnaissance d'antigènes par le TCR active des protéines tyrosine kinases qui phosphorylent d'autres substrats intracellulaires dont LAT. Ceci engendre l'activation de molécules telles que PKCθ et CARMA-1. La mutation LatY136F associe des TCR "estropiés" dans le développement de cellules T effectrices générant des désordres lymphoprolifératifs. Nous avons essayé de comprendre les gènes aggravant ou empêchant cette lymphoprolifération en utilisant la mutagénèse ENU. Nous avons identifié une mutation appelée Basilic empêchant le déroulement de la pathologie LatY136F. Basilic est une mutation du gène Rltpr qui constitue une phénocopie de Cd28-/- sur fond sauvage et sur fond LatY136F. Rltpr est un une nouvelle protéine ayant de multiples domaines, qui appartient à la famille CARMIL et qui est exprimée dans les cellules T et B. L'objectif de ce travail était d'élucider les mécanismes au cours desquels CD28 et Rltpr coopèrent avec le TCR pour différencier des cellules T naïves en cellules T effectrices. Ce travail visait aussi à caractériser Rltpr, dont la structure/fonction et l'interactome sont encore inconnus. En utilisant des techniques de microscopie confocale, nous avons montré que la localisation et le recrutement de Rltpr et de RltprBas à la synapse immunologique sont tous deux CD28-dépendants. Les deux molécules colocalisent avec CD28 tout au long du processus d'activation. En outre, Rltpr est essentiel pour la translocation à la synapse de PKCθ et CARMA-1, qui sont induits lors de la co-stimulation par CD28. Ces résultats permettent une meilleure compréhension du fin réglage du système immunitaire adaptatif qui est mis en place lors de l'activation. / TCR recognition of antigens triggers the activation of protein tyrosine kinases that phosphorylate other intracellular substrates including LAT. LAT phosphorylation leads to the activation of PKCθ and CARMA-1. The point mutation LatY136F associates TCRs with crippled signaling abilities to the development of effector T cells generating lymphoproliferative disorders (LPDs). We tried to shed light on genes exacerbating or preventing the LatY136F LPD by using an ENU mutagenesis screening. We identified one point mutation called Basilic that prevents the unfolding of the LatY136F pathology. Basilic is a point mutation of the Rltpr gene and is a phenocopy of a Cd28-/- mutation both on a wild-type and on a LatY136F background. Rltpr is a newly-discovered, multidomain protein belonging to the CARMIL family that is expressed in T and B cells. The objective of the present work was to elucidate the mechanisms during which CD28 and Rltpr cooperate withthe TCR to differentiate naïve into effector T cells. I also aimed at characterizing the Rltpr molecule, whosestructure/function and interactome are still largely unknown. Using confocal microscopy in collaborationwith Takashi Saito's group and Christoph Wülfing we showed that the localization and the recruitment ofboth Rltpr and RltprBas at the immune synapse are CD28-dependent. The two molecules colocalize with CD28all along the activation process. Moreover, Rltpr is essential for the synapse translocation of PKCθ andCARMA-1, which are induced upon CD28 costimulation. Those results allow a better understanding of theadaptive immune system fine tuning upon activation.