L'importance de l'approche globale à deux paramètres dans l'analyse de mécanique linéaire élastique de la rupture est de plus en plus reconnue pour des évaluations de fracture dans des applications d'ingénierie. La considération du deuxième paramètre, à savoir la contrainte élastique T, ou T-stress en anglais, permet d'évaluer le niveau de confinement à la pointe de la fissure où d'entaille. Il est important de fournir des solutions de la contrainte T pour une pratique géométrie pour employer la mécanique de la rupture à base de contrainte de confinement. Dans la présente recherche, nous fournissant des solutions de la contrainte T pour une entaille en forme de U dans le cas de quatre éprouvettes : CT, DCB, SENT et Tuile Romaine. L'entaille en forme de U est analysée utilisant la méthode d'élément finie par le code de calcul Castem 2000 pour déterminer la distribution de contraintes à la pointe de l'entaille et le long du ligement. Le rapport du profondeur du défaut sur l'épaisseur a été varié : a/w = 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 et 0.7. Le rayon de l'acuité de l'entaille a été fixé pour toute la géométrie à 0.25 mm. Contrairement aux fissures, il a été trouvé que la contrainte T n'est pas constante et dépend de la distance de la pointe de l'entaille. Pour évaluer cette contrainte dans le cas d'une entaille, une nouvelle méthode, à savoir, la méthode de ligne, inspirée de l'approche de méthode volumétrique proposée par pluvinage a été développée. La méthode est basée sur la détermination d'une contrainte moyenne T sur une distance effective en avant de la pointe de l'entaille. Ainsi, l'approche à deux paramètres a été adoptée pour la mécanique de la rupture à deux paramètres pour les entailles en termes du Facteur d'Intensité de Contraintes d'entaille Kpc et la contrainte moyenne (effective) Teff. La courbe de transférabilité de ténacité à la rupture (Kpc -Teff) dans l'acier de pipeline X52 a été établi. Cette approche a été utilisée avec succès pour évaluer quantitativement le champ des confinements à la pointe de l'entaille pour des différentes géométries et conditions de chargements / The importance of the two-parameter approach in linear eleastic fracture mechanics analysis is increasingly being recognized for fracture assessments in engineering applications. The consideration of the second parameter, namely, the elastic T-stress, allows estimating the level of constraint at a crack or notch tip. It is important to provide T-stress solutions for practical geometries in order to employ the constraint-based fracture mechanics methodology. In the present research, T-stress solutions are provided for a U-shaped notch in the case of four specimens : CT, DCB, SENT and Romain Tile. The U-shaped notch is analyzed using the finite element method by the commercial Castem 2000 software to determine the stress distribution ahead of the notch tip. The notch aspect ratio was varied : a/w = 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7. The notch-tip radius was fixed for all geometries and equal 0.25 mm. In contrast to a crack, it was found that the T-stress is not constant and depends on distance from the notch-tip. To estimate the T-stress in the case of a notch, a novel method, namely, method of line, inspired from the volumetric effective distance ahead of the notch tip. The effective distance corresponds to the point with a minimum of the stress gradient in the fracture process zone. Thus, the two-parameter approach was adopted for the notch two-parameter fracture mechanics in terms of the notch stress intensity factor Kpc and the effective (average) T-stress, Teff. Fracture toughness transferability curve (Kpc-Teff) of X52 pipe steels has been established. This approach was successfully used to quantify the constraints of notch-tip fields for various geometries and loading conditions. Moreoveer, the proposed T-stress estimation creates a basis to analyse the crack path under mixed mode loading from viewpoint of the two-parameter fracture mechanics

The work reported here involves studies of suppressor T cells (T[sub=s]C) and their suppressor factor (SF) which specifically suppress the in vitro generation of cells cytotoxic for a syngeneic tumor, P815, in DBA/2 mice. This work can be divided into three sections: a) the immunogenetic properties and requirements of this T[sub=s]C and SF, b) the Lyt phenotype of the T[sub=s]C as well as that of the cells involved in the cytotoxic response to the syngeneic tumor, c) the properties of syngeneic and allogeneic antisera raised to the P815 specific SF. a) P815-antigen specific T[sub=s]C and suppressive extracts obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 tumors, were compared for their immunogenetic properties and requirements. It was shown that pretreatment of T[sub=s]C populations with anti-la[sub=d] antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the SF with anti-la[sup=d] antiserum removed the suppressive properties of the material. It was found that the T[sub=s]C and SF were capable of specifically suppressing the anti-P815 response of B6D2F₁ radiation chimeras possessing lymphoid cells of the H-2[sup=b] or H-2[sup=t2] haplotype equally as well as they could suppress the response of H-2[sup=d] bearing cells. This indicates that the T[sub=s]C and SF are not H-2 restricted with respect to K or D markers on responder cells in this system. b) T[sub=s]C were also identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells was determined. DBA/2 allogeneic killer cells were identified as Lyt-1⁺2⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-1⁺2⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-1⁺2⁻ cell is essential in this suppression. c) P815 tumor-specific SF was partially purified by passage of suppressive spleen extracts through an immunoadsorbent containing P815 membrane components. Antisera raised in syngeneic DBA/2 and allogeneic, C57BL/6, mice were tested. It was found that these antisera, but not their controls were capable of absorbing out the SF. The antisera were also capable, in the presence of complement, of eliminating T[sub=s]C from suppressive spleen cell populations. However, the antisera were not capable of eliminating syngeneic tumor specific in vitro generated killer cells, indicating that the receptor molecules on suppressor and effector cells in this system are distinct from each other. Only the antisera "raised in syngeneic DBA/2 mice had any observable effect on P815 tumor growth in vivo. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Due to their excellent tribological properties and potential to replace problematic lubricant additives currently in use, WS2 nanoparticles (NPs) have spurred considerable interest from academia and industry over the last two decades to decipher their mechanism of action. To elucidate the mechanism, this study carried out tribological tests at high pressures and low/high temperatures (40 and 100ºC) and investigated the tribofilm generated on the wear track and its wear and friction properties. It was found that WS2 NPs react with the metal substrate at high temperatures to form a chemical tribofilm covered with squashed NPs. The generation of this tribofilm accounts for their excellent tribological properties. By investigating the chemical and mechanical properties of the tribofilm, it was possible to explain the tribological properties of WS2 NPs. Based on chemical analysis results, a layered structure was proposed for the chemically formed tribofilms. The large amount of tungsten compounds in the composition may explain the excellent mechanical properties of the tribofilm, as revealed by nanoindentation tests. The importance of base oil polarity was investigated. It was found that the efficiency of the NPs is reduced in polar oils, because the oil molecules can compete with the nanoadditive by adsorbing on the metal surface in the tribological contact and impeding the formation of the tribofilm. To investigate which type of WS2 NPs (2H or IF) performs better in tribological applications and if other tungsten dichalcogenides (IF-WSe2) are also potential candidates as nanoadditives, tribological tests and analysis of the wear tracks were performed. 2H-WS2 showed superior friction and wear reducing properties in high-pressure high-temperature contacts. The tribological performance of 2H-WS2 NPs was compared to that of popular conventional additives, e.g. antiwear zinc dialkyldithiophosphates (ZDDPs) and organic friction modifiers (OFMs). At the end of three hour tests, 2H-WS2 NPs and ZDDP+OFM mixtures showed similar antiwear properties, but 2H-WS2 NPs induced a significant reduction of the friction coefficient in the mixed and boundary lubrication regimes. The ability of 2H-WS2 NPs to inhibit hydrogen permeation in high strength bearing steel used in fuel cells and wind turbines was also investigated. Thermal desorption spectroscopy revealed that the chemical tribofilm generated on the wear tracks can significantly reduce the concentration of hydrogen and water in the steel substrate after rolling contact fatigue tests performed in high-temperature high-pressure conditions.

La destruction du compartiment T CD4 induite par le VIH‐1 est essentiellement périphérique. Cependant, l’atteinte des précurseurs lymphocytaires T, pour laquelle existent des arguments, est encore mal connue. L’objectif de ce travail a été d’explorer le rôle de la protéine Nef, hors contexte d’infection virale, dans la différenciation lymphoïde. Nef, produite très tôt après l’infection par le VIH‐1, possède un rôle‐clé dans la pathogénie, comme en témoigne la reproduction, chez la souris transgénique pour Nef, d’une maladie proche de la maladie humaine. Dans les lymphocytes matures infectés, Nef module l’expression du récepteur CD4, des molécules du CMH de classe I et induit un état d’activation T. Nef interagit également avec de nombreux partenaires cellulaires impliqués dans des voies de signalisation cytokinique, de contrôle de l’apoptose et de l’ontogénie T. Au cours de ce travail, nous avons montré que l'expression de Nef en absence d'autres gènes viraux bloquait le potentiel T et NK des précurseurs hématopoïétiques humains par un mécanisme apoptotique indépendant des caspases et lié à une perturbation du potentiel mitochondrial. Grâce à l’utilisation du système de culture OP9‐DL1 permettant l’identification des premiers stades de différenciation des cellules T, nous avons montré que la génération de cellules T à partir de CD34+ est affectée à un stade CD3+CD5+CD1a+ qui a initié le réarrangement D‐J du TCRβ. Les stades suivant du développement T sont aussi affectés, en effet, une réduction quantitative du nombre de CD4 ISP et de DP CD4+CD8+ TCRαβ a été observée. Nef diminue aussi la génération de cellules NK CD56+ alors que la production de cellules B n’est pas affectée. Des analyses par dilution limite montrent une réduction significative de la fréquence des précurseurs T/NK parmi les cellules CD34+ exprimant Nef. Nous avons par ailleurs montré une augmentation de 15 à 25% de cellules apoptotiques dans les cellules exprimant Nef‐WT par rapport aux autres conditions. Cette apoptose n’est pas caspase dépendante et La voie Fas/Fas‐L ne semble pas entrer non plus en jeu. Nous avons par ailleurs pu déterminer que l’atteinte apoptotique des précurseurs passait par une déstabilisation mitochondriale, ceci grâce à un marquage DIOC6. L’ensemble des résultats regroupés dans de ce travail permet d’approfondir les connaissances des mécanismes d’interférence du VIH avec l’hématopoïèse et plus précisément des actions de Nef sur l’hématopoïèse humaine. / HIV-1 infection is characterized by a progressive loss of CD4 T cell leading to a severe immune deficiency. More than 20 years after the identification of HIV, the exact mechanisms by which HIV leads to the depletion of CD4 T cells are not completely understood and remain a matter of cntroversy. The purpose of this work has been to explore out of the viral context, the effect of the HIV-1 Nef protein on the T cell differentiation. The HIV-1Nef gene is essential for AIDS pathogenesis. HIV-1Nef protein is a 27‐kDa protein that is expressed early during cell infection. Nef increases viral replication in vivo and enhances viral infectivity. Nef interferes with the expression of crucial molecules at the surface of lymphocytes such as CD4, MHC class I and II, CD28, DC‐SIGN, and chemokine receptors. Nef also alters T-cell signaling pathways and immunological synapse formation. During this work, we found that Nef expression in hematopoietic precursors interfere by an apoptotic pathway in the differentiation of a T/NK progenitor. In order to study the differenciation, we used a recent model of murine stromal cells engineered to express the Notch ligand delta 1 (OP9‐DL1 system). This system allows the differentiation of hematopoietic progenitors to a DP CD4+CD8+ stage and led us to examine the effects of Nef expression on T‐cell development. For this, Nef was expressed in hematopoietic CD34+ precursors using lentiviral vectors. In this study, we demonstrate that HIV-1Nef expression in the absence of other viral genes hampers the T and NK cell potential of human hematopoietic progenitors. We show that the generation of T cells from CD34+ progenitors is affected at a CD3ε+CD5+CD1a+ precursor stage that has initiated a DJB rearrangement of the TCRB locus. Subsequent stages of T cell development were also affected with a quantitative reduction of CD4 ISP and DP CD4+CD8+ TCRαβ cells. Nef also decreased the generation of CD56+NK cells whereas B cell production was not affected. Limiting dilution analyses demonstrated a significant reduction in the frequency of T/NK progenitors among Nef‐expressing CD34+ cells. Collectively, these data indicate that Nef interferes with the differentiation of a primitive lymphoid precursor with a T/NK potential. This interference was caused by the triggering of an apoptotic pathway which triggered the mitochondrial depolarization. This study allows a better knowledge of the interference mechanisms used by HIV on the T cell hematopoïesis, more accurately on the effect of Nef on human T cell differentiation

5 Regulatory T cells (Tregs) are essential for the maintenance of peripheral self-tolerance and prevention of autoimmunity by suppressing the response of self-reactive CD8+ and CD4+ T cells. However, while interactions of Tregs with CD4+ T cells have been extensively studied, their effect on the self-tolerance of CD8+ T cells has not been explored in detail. The main aim of this diploma project was to provide evidence whether and how Tregs prevent autoimmunity induced by CD8+ T cells. We used an experimental mouse model of autoimmune diabetes allowing us to acutely deplete Tregs and titrate the number of self-reactive T cells, self- antigen affinity, and self-antigen doses. We found out that Tregs play an important role in the prevention of CD8+ T-cell mediated autoimmunity. Moreover, we revealed that Tregs suppress both high-affinity T cells that escape negative selection and relatively weakly self-reactive, but numerous, positively selected T cells. Tregs do so by increasing requirement for the number of self-reactive CD8+ T cells required for the autoimmunity induction. Intriguingly, presence of Tregs does not impact threshold for self-antigen. Moreover, for the first time, we showed that Tregs can suppress CD8+ T-cell-mediated autoimmunity in the absence of conventional CD4+ T cells. This means that...

Atherogenesis involves the activation of endothelial cells and the egress of atherogenic T lymphocytes and monocytes into the intima. Exercise training contributes to the arrest and even reversal of atherosclerosis by modifying risk factors and by inducing an atheroprotective phenotype in endothelial cells and T cells.

Interleukin-7 is an essential cytokine that plays a major role in the development and homeostatic maintenance of T-cells. The presence of soluble forms of various cytokine receptors have been proposed to be involved in the endogenous regulation of cytokine activity. Due to the natural ability of soluble CD127 (sCD127) to bind to IL-7, there is an interest in its potential application as an immunotherapeutic agent in diseases, where IL-7 has been found to be relevant, including HIV infection. In this study, I hypothesize that by administering sCD127 to healthy mice, IL-7 activity should be enhanced, thus enhancing T cell proliferation in vivo. The work presented here focuses on three main objectives: 1) evaluating the effect of IL-7 with or without sCD127 on T cell proliferation in healthy mice; 2) validating a mouse model of T cell depletion using anti-CD4 and CD8 antibodies; and 3) determining the effect of sCD127 treatment with or without IL-7 on T cell reconstitution and proliferation in the T cell depletion model. To assess the effect of administering exogenous sCD127, IL-7 or the combination on T cell proliferation, peripheral blood mononuclear cells and spleen were isolated, and stained to characterize T cell number, proliferation, and surface CD127 expression by flow cytometry. For the T cell depletion model, wild type C57BL/6 mice were injected intra-peritoneally with 150 μg single dose of anti-CD4 and anti-CD8 depleting antibodies. Consequently, mice were bled weekly to demonstrate the kinetics of T cell reconstitution following depletion (from d7 to d63). Our results demonstrated that in healthy mice daily treatment with murine IL-7 significantly stimulated T cell proliferation and consequently increased cell number. This observation was further boosted by pre-complexing IL-7 with sCD127. For T cell depletion experiments, the kinetics of T-cell reconstitution was different between the CD4+ and CD8+ T cells. CD4+ T cell reconstitution was almost complete 6 weeks following T cell depletion, while CD8+ T cells were only partially reconstituted at this time point. Treatment with IL-7 or combined therapy had a transient and significant effect on T cell proliferation and reconstitution, and this influence was abrogated after treatment discontinuation. Interestingly, CD8+ T cells exert greater responses to our treatments in that a more pronounced proliferation and significant increase in cell number was observed relative to the effect seen on CD4+ T cells in both healthy and depleted mice. In conclusion, antibody-mediated T cell depletion is a potentially valuable tool to investigate lymphopenia-induced proliferation and potential therapies thereof. This study suggests that combining sCD127 and IL-7 therapies enhances IL-7-mediated T cell proliferation, and provides important information for the potential therapeutic use of sCD127 and its impact on IL-7 function.

ABSTRACT Cases of HIV-1 infection are growing in large numbers, and deaths as a result of AIDS are escalating in South Africa. Understanding cellular immune responses to HIV-1 by exploring general effects and changes occurring at a cellular level, including direct engagement of T cells with the virus during exposure or infection will provide information on possible correlates of viral control. This dissertation focuses on three characteristics of T cells during HIV infection, dual HIV/TB co-infection and exposure to HIV. The characteristics examined are 1) memory and activation status of CD4+ and CD8+ T cell subsets; 2) T cell receptor repertoire and 3) HIV-1– specific T cell responses. There are two hypotheses in this dissertation. Firstly, that co-infection with TB leads to elevated T cell activation, disruption of the T cell receptor repertoire and altered patterns of immunodominance in HIV-1 subtype C-specific T cell responses in infected adults. Secondly, that T cell priming occurs in utero in HIV uninfected babies born to HIV infected mothers. Four cohorts were examined in this dissertation. Three were recruited from a clinic around the Welkom area and analysed in a cross-sectional and longitudinal manner. The cohorts consisted of HIV-1 infected adults, individuals dually infected with HIV and TB and healthy controls. Whole blood samples from the HIV and HIV/TB infected groups were analysed at baseline (before TB chemotherapy), at 2, 8 and 24 weeks. A further cohort consisted of babies born to HIV-1 infected mothers, with some being followed up at three months after birth. This dissertation consisted of five different methods: 1) the use of four colour flow cytometry to measure the frequency of naïve T cells (CD45RA+/CD62L+), memory (CD45RA-CD62L-) and activated (CD38) T cell populations in individuals singly infected with TB and dually with HIV and TB. This investigation was aimed at obtaining the overall representation of T cells involved in HIV-1 and TB co-infection. 2) the use of flow cytometry staining with monoclonal antibodies recognizing different T cell receptor (TCR) Vβ specificities for quantitation of the percentage of particular TCR families in pools of T cells. The aim was to provide an indication of TCR usage in different disease states. 3) the immunoscope assay was used to measure the different CDR3 lengths of the TCR and assess Vβ family repertoires in newborn babies. The aim was to show evidence of T cell maturity at birth and whether there was TCR engagement in utero by analysing cord blood cells. 4) the use of the IFN-γ ELISPOT to measure HIV-specific T cell responses in a cohort of HIV and HIV/TB co-infected individuals. The aim was to identify targeted immunodominant regions and to determine whether TB infection resulted in differing patterns of HIV-1 specific T cell immunity. 5) intracellular cytokine staining (ICS) was used to confirm the responses obtained after initial screening with the IFN-γ ELISPOT and was used to delineate CD4+ and CD8+ T cell responsiveness.Whole blood was stained with an array of monoclonal antibodies to measure various T cell subsets in HIV-1 and HIV/TB co-infected adults. The CD4+ T cells in HIV-1 infected individuals ranged between 245 - 436, those of HIV/TB co-infected patients were 157-840 and the TB group had CD4+ absolute counts ranging between 583-1757 cells/mm3. CD4+ T cells were reduced as a result of HIV-1 infection and HIV/TB coinfection, and no loss of these cells was seen as a result of single infection with TB. There was a loss of naïve T cells, with increased memory phenotypes in the presence of TB and HIV single infection, which was more pronounced in the presence of HIV and TB co-infection. The loss of naïve CD4+ and CD8+ T cells was associated with a high HIV-1 plasma RNA load in patients co-infected with HIV and TB. CD8+ T cells in HIV singly infected and HIV/TB co-infected individuals were highly activated when compared to those infected with TB only, which was likely due to the high HIV plasma RNA load. The standard course of six months of TB therapy in HIV/TB co-infected adults did not lead to recovery of absolute CD4 cells, nor did it stem the loss of naïve CD4+ and CD8+ T cells, which remained in a highly activated state: possibly due to unchanged HIV-1 RNA loads. The fine specific nature of T cell activation was investigated by examining TCR Vβ expansions in HIV and TB single and co-infected individuals. Whole blood was stained with CD3+, CD4+, CD8+, CD38+ and an array of Vβ-specific antibodies. Polyclonal skewing of the TCR Vβ repertoire, showing expansion of various Vβ−CD4+ and -CD8+ families was observed in TB and HIV-1 single and dual infection. A more restricted usage of the T cell repertoire was observed in both HIV and HIV/TB co-infected patients, where major and oligoclonal expansions of Vβ11, Vβ16, Vβ20 and Vβ22 were observed. No major expansions were observed in TB single infection. Overall, significantly greater use of TCR Vβ families were found in the CD8+ T cell compartment rather than by CD4+ T cells in both HIV-1 and HIV/TB co-infected adults. A cohort of neonates born to HIV-infected mothers was used to assess TCR usage using immunoscope analysis to support the hypothesis that the TCR repertoire skewing in cord blood cells can be a marker of T cell priming in-utero. The repertoire measured in HIV uninfected neonates born to HIV-1 infected mothers displayed a polyclonal skewing of various TCR families and oligoclonal distribution of Vβ5, Vβ6a, Vβ7, Vβ18, and Vβ23 families as compared to the Gaussian distributions seen in healthy controls. This study readily detected perturbations in the TCR repertoire in presumed HIV exposed babies and that newborns possess an intact TCR repertoire. Measurement of HIV-1-specific CD8+ T cells was made to identify which regions of the expressed HIV genome were immunodominant and what impact of co-infection with TB may have. PBMC samples were thawed and cultured in vitro using CD3+/CD4+ bispecific antibody to preferentially expand CD8+ T cells and measure IFN-γ producing cells in the ELISPOT and confirmed with the ICS. The ELISPOT results were interpreted in SFU/million PBMC and as percentages of T cell subsets in ICS assays. HIV-1 subtype C-specific CD8+ responses were readily detected in both HIV-1 and HIV/TB co-infected patients, however, patterns of peptide targeting were different between the two groups. Gag was targeted by 85% of HIV-1 infected patients, whereas only 27% of HIV/TB coinfected patients targeted Gag. Pol was targeted by 73% in the HIV/TB group. Gag and Nef responses observed in some (n =7) of the patients were confirmed using ICS. These data infer that TB co-infection may change patterns of targeting and in how CD8+ T cells recognize HIV antigens. Collectively, this dissertation demonstrated the existence of highly activated CD8+ T cells, most probably driven by high HIV-1 plasma RNA loads; restricted TCR usage by CD8+ T cells, predominantly in individuals dually infected with HIV and TB; possible shifting of immunodominant HIV-specific CD8+ T cell responses as a result of coinfection with TB. Despite successful treatment of TB with chemotherapy, these immunological observations remained unchanged.

T cells play an essential role in the immune system. Engaging the T cell receptor (TCR) initiates a cascade of signaling events that activates the T cells. Neutral sphingomyelinase (NSM) is a member of a superfamily of enzymes responsible for the hydrolysis of sphingomyelin into phosphocholine and ceramide. Sphingolipids are essential mediators in signaling cascades involved in apoptosis, proliferation, stress responses, necrosis, inflammation, autophagy, senescence, and differentiation. Upon specific ablation of NSM2, T cells proved to be hyper-responsive to CD3/CD28 co-stimulation, indicating that the enzyme acts to dampen early overshooting activation of these cells. It remained unclear whether a deregulated metabolic activity supports the hyper-reactivity of NSM2 deficient T cells. This work demonstrates that the ablation of NSM2 activity affects the metabolism of the quiescent CD4+ T cells. These accumulate ATP in mitochondria and increase basal glycolytic activity by increasing the basal glucose uptake and GLUT1 receptor expression, which, altogether, raises intracellular ATP levels and boosts cellular respiration. The increased basal metabolic activity is associated with rapid phosphorylation of S6, a mTORC1 target, as well as enhanced elevation total ATP levels within the first hour after CD3/CD28 costimulation. Increased metabolic activity in resting NSM2 deficient T cells does, however, not support sustained stimulated responses. While elevated under steady-state conditions and elevated early after co-stimulation in NSM2 deficient CD4+ T cells, the mTORC1 pathway regulating mitochondria size, oxidative phosphorylation, and ATP production is impaired after 24 hours of stimulation. Taken together, the absence of NSM2 promotes a hyperactive metabolic state in unstimulated CD4+ T cells yet fails to support sustained T cell responses upon antigenic stimulation without affecting T cell survival. / T-Zellen spielen eine wesentliche Rolle im Immunsystem. Die Aktivierung des T-Zell-Rezeptors (TCR) löst eine Kaskade von Signalereignissen aus, die die T-Zellen aktivieren. Neutrale Sphingomyelinase (NSM) gehört zu einer Superfamilie von Enzymen, die für die Aufspaltung von Sphingomyelin in Phosphocholin und Ceramid verantwortlich sind. Sphingolipide sind wesentliche Mediatoren in Signalkaskaden, die an Apoptose, Proliferation, Stressreaktionen, Nekrose, Entzündung, Autophagie, Seneszenz und Differenzierung beteiligt sind. NSM2-depletierte T-Zellen erwiesen sich als hyper-reaktiv gegenüber CD3/CD28-Kostimulation, was darauf hinweist, dass das Enzym eine überschießende Aktivierung dieser Zellen dämpft. Es blieb unklar, ob die Hyperreaktivität NSM2-defizienter T-Zellen durch eine deregulierte Stoffwechselaktivität unterstützt wird. Diese Arbeit zeigt, dass NSM2-Insuffizienz den Metabolismus ruhender CD4+-T-Zellen beeinflusst: Diese akkumulieren ATP in Mitochondrien und zeigen eine erhöhte basale glykolytische Aktivität, die auf einer erhöhten Glukoseaufnahme und Expression des GLUT1-Rezeptors beruht und mit einer Erhöhung intrazellulärer ATP-Werte und gesteigerten Zellrespiration einhergeht. Aufgrund ihrer bereits erhöhten basalen metabolische Aktivität zeigen NSM2 defiziente T Zellen eine im Vergleich zu Kontrollzellen schnellere, effizientere Aktivierung nach Kostimulation, die sich in Phosphorylierung von S6, eines mTORC1 Targets, sowie erhöhtem ATP Spiegel manifestiert. Dies kann jedoch nicht aufrechterhalten werden:Die mTORC1-Aktivierung, die die Größe der Mitochondrien, die oxidative Phosphorylierung und die ATP-Produktion reguliert, unter stationären Bedingungen in NSM2-defizienten CD4+-T-Zellen erhöht ist, ist nach 24-stündiger Kostimulation beeinträchtigt. Insgesamt scheint die NSM2-Aktivität wesentlich für die Regulation der basalen metabolischen Aktivität ruhender T-Zellen und der Vermeidung überschiessender Antworten nach Kostimulation zu sein, jedoch ebenso wichtig für die dauerhafte Aufrechterhaltung des Aktivierungssignals zu sein.

Un important réseau de cytokines serait en grande partie responsable de la formation des lésions psoriasiques. La présence de cellules immunitaires dans des biopsies de peaux psoriasiques suggère une implication majeure de ces dernières dans la maladie. La présente étude tente d'explorer la communication à caractère pro-inflammatoire entre lymphocytes T et kératinocytes psoriasiques. Notre modèle in vitro consiste en une monocouche de kératinocytes sains ou psoriasiques mis en co-culture avec des lymphocytes T sains isolés du sang humain. Les interactions cellulaires ont été étudiées suivant l'analyse de la production de 22 cytokines/chimiokines (technologie du Luminex xMap) et complétées par ELISA. Nos résultats démontrent que les kératinocytes psoriasiques avec les lymphocytes T augmentent leur production de TNF-a, de GM-CSF, de MCP-1, d'IL-6 et d'IL-8 ainsi que celle de lTFN-y, du sIL-2 Ra et de la Fractalkine lorsque 1TL-2 est présente comparativement à la co-culture kératinocytes sains-lymphocytes T. Des tests additionnels avec le neutrophile montrent que la production de cytokines inflammatoires est suffisante pour influencer ce dernier. Ces résultats démontrent l'interaction fonctionnelle entre les kératinocytes et les lymphocytes T ainsi que la persistance des caractéristiques pathologiques des kératinocytes psoriasiques in vitro. Ce modèle expérimental de coculture sera intéressant pour comprendre en profondeur des désordres cutanés auto-immuns comme le psoriasis.