Cytotoxic T lymphocytes (CTL) are major effector cells in the adaptive immune response against intracellular pathogens and cancers, killing targets with high precision. Precision is achieved through the specificity of the clonally expressed T cell receptor (TCR). TCRs recognise a specific peptide chain loaded into a major-histocompatability complex, triggering signalling, inducing the CTL to attach and kill target cells. Key stages in this attack are the initial conjugation followed by polarisation and docking of the centrosome to the junction of the two cells, the immune synapse (IS). This focuses secretion of the cytolytic components, perforin and granzyme, from modified lysosomes to kill the target cell. My PhD has utilised amino acid substitutions in the target peptide to alter its signal strength and shown this alters the subsequent killing efficiency of a target population. I developed new imaging and analysis techniques to investigate the effect of TCR signal strength at each step of the killing process. I show the first step, conjugation, is reduced for a percentage of cells with dwell times decreasing as TCR signal strength decreased. The next key step of centrosome polarisation and docking at the IS was also impaired for an increasing proportion of cells as TCR signalling reduced. Impaired centrosome docking reduced efficient granule recruitment to the IS, necessary for target killing. Centrosome docking was linked with the TCR-induced intracellular calcium flux, the duration of which increases with the strength of TCR signalling. This demonstrates how the process of CTL killing can be fine-tuned by the quality of antigen.

Lytic effector function of Natural Killer (NK) cells and CD8+ T cells occurs through discrete and regulated cell biological steps triggered by recognition of diseased cells. Recent studies of the NK cell synapse support the idea that dynamic nanoscale rearrangements in cortical filamentous (F)-actin are a critical cell biological checkpoint for lytic granule access to NK cell membrane. Loss of function mutations in the LYST gene, a well-characterised cause of Chediak- Hegashi syndrome (CHS), result in the formation of giant lysosomal organelles including lytic granules. Here, we report a mismatch between the extent of cortical F-actin remodelling and enlarged lytic granules that limits the functionality of LYST- deficient NK cells in a human model of CHS. Using super-resolution stimulated emission depletion (STED) microscopy we found that LYST-deficient NK cells had nanoscale rearrangements in the organisation of cortical actin filaments that were indistinguishable from control cells- despite a 2.5-fold increase in the size of polarised granules. Importantly, treatment of LYST-deficient NK cells with actin depolymerising drugs increased the formation of small secretory domains at the synapse and restored their ability to lyse target cells. These data establish that sub-synaptic F-actin is the major factor limiting the release of enlarged lytic granules from CHS NK cells, and reveal a novel target for therapeutic interventions. While the importance of cortical actin filaments in NK cell cytotoxicity have been established, its persistence at the early stages of T cell synapse formation is disputed. We studied the organisation of cortical actin filaments in synapses formed by primary human T cells using STED microscopy and detected intact cortical actin filaments in key T cell effector subsets including memory CD8+ T cells as early as 5-minutes post-activation. Quantitative analysis revealed that activation specific rearrangements in cortical actin filaments at both CD4+ and CD8+ T cell synapses serve to increase the space between filaments. Additionally, comparison of cytolytic T cells with freshly isolated and IL-2 activated primary NK cells revealed that rapid maturation of the cortical actin meshwork is a specific feature of CD8+ T cell lytic synapses. Using chemical inhibition of actin nucleators, we show that increased cortical relaxation is mediated primarily by the activity of actin related proteins (Arp) -2/3. Taken together, these data establish the critical requirement for dynamic rearrangements in cortical actin filaments at lytic synapses but underscore cell-specific differences in its regulation.

Lors d’une réponse immune, les lymphocytes T et les cellules présentatrices d’antigènes (CPA) interagissent entre elles. La synapse immunologique (SI), interface de contact entre les deux cellules, est le site où une cascade de signalisation se met en place. Les lymphocytes T subissent alors un profond réarrangement au niveau de la membrane plasmique et du cytoplasme : les protéines impliquées dans cette signalisation sont alors recrutées à la synapse immunologique. Nous nous intéressons à une nouvelle structure appelée “l’antisynapse” qui se localise au pôle opposé à celui de la synapse immunologique. L’objectif de notre étude est de déterminer la composition de cette nouvelle structure et sa cinétique d’apparition et de disparition. Afin d’étudier cette structure, nous faisons des conjugués in vitro entre des CPA et des lymphocytes T et nous observons la formation de ces contacts sur cellules vivantes ou cellules fixés. L’antisynapse est composée de molécules de signalisations que l’on retrouve classiquement à la synapse immunologique, tels que LAT, CD3, lck ou la PI3K. Grâce à la sonde fluorescente ROZA récemment développée au laboratoire1, nous avons montré que la kinase ZAP-70 est activée à l’antisynapse. Ces observations sont cohérentes avec le fait que nous avons déjà observé la présence de protéines avec des tyrosines phosphorylées à ce pôle. Cette structure précoce et transitoire s’observe fréquemment et apparaît très souvent avant que la synapse ne puisse être détectée. Son apparition est indépendante de la signalisation en aval du TCR et peut être déclenchée par un signal d’adhésion. D’autre part, le cytosquelette de microtubules semble jouer un rôle majeur dans sa disparition. Le rôle de l’antisynapse est toujours en cours d’étude mais nous avons déjà pu montrer qu’elle constituait un point de stockage pour les protéines destinées à former la synapse immunologique au moment de sa formation. Grâce à cette structure nous essayons de mieux comprendre comment le signalosome s’assemble dans la cellule T. Nous voulons également comprendre comment une telle structure peut apparaître aussi rapidement et quelles sont les voies de signalisation mises en jeu dans sa formation. / During the immune response, T lymphocytes and antigen presenting cells (APC) are known to develop strong interactions. The immunological synapse (IS), structure established at the interface between the two cells, is the site where a cascade of signaling events is initiated and may lead to a physiological response. T lymphocyte undergoes a profound rearrangement in the plasma membrane and in the cytoplasm: proteins which are involved in the signaling are recruited to the immunological synapse. We have recently described a new structure that we have called antisynapse (ASI), located at the cell pole opposite to the synapse1. The purpose of this work is to characterize the components of this new structure and their kinetic of appearance and disappearance. To study this structure, we made in vitro contact between APC and T lymphocytes and we observed these conjugates either in live or fixed conditions. Surprisingly, the antisynapse contains most of the signaling molecules classically reported as components of the immunological synapse such as LAT, CD3, Lck or PI3K. By using the fluorescent probe ROZA that we recently developed1, we have shown that ZAP-70 is activated at the antisynapse. This observation is consistent with the fact that we have also observed the presence of tyrosine-phosphorylated proteins at the ASI. Interestingly, we have observed that LFA-1, a protein involved in the adherence, is also found at the ASI. Our results indicate that this transient structure develops frequently and appears rapidly after the contact between the T cell (around one minute) and the APC. Surprisingly, antisynapse formation is independent on TCR signaling but is triggered by adhesion. Furthermore, it disappears using the microtubule network. The role of the antisynapse is currently under investigation but we have shown that it constitutes a stock of proteins ready to go to the forming immune synapse. We currently try to take advantage of this structure to better understand how the T cell signalosome may assemble and to find out if, functionally, the T cell takes advantage of this structure. We also try to understand how this paradoxical structure can appear so rapidly and what are the signaling pathways involved in its establishment.

No description available.

Soft x-ray microscopes routinely perform high-resolution 3D imaging of biological cells in their near-native environment with short exposure times at synchrotron radiation facilities. Some laboratory-sized microscopes are aiming to make this imaging technique more accessible to a wider scientific community. However, these systems have been hampered by source instabilities hindering routine imaging of biological samples with short exposure times. This Thesis presents work performed on the Stockholm laboratory x-ray microscope. A novel heat control system has been implemented, improving the stability of the laser-produced plasma source. In combination with recent upgrades to the imaging system and an improved cryofixation method, the microscope now has the capability to routinely produce images with 10-second exposure time of cryofixed biological samples. This has allowed for tomographic imaging of cell autophagy and cell-cell interactions. Furthermore, a numerical 3D image formation model is presented as well as a novel reconstruction approach dealing with the limited depth of focus in x-ray microscopes. / <p>QC 20170505</p>

CD4 co-receptor of main T cell receptor (TCR) is essential for proper development of T lymphocytes and their function in adaptive immune responses. It is believed that CD4 stabilizes the interaction of TCR with antigenic ligand, peptide-MHC, and thereby improves T cell-dependent responses during immune reaction. CD4 is transmembrane glycoprotein with a number of structural motifs in its intracellular domain which do not dramatically affect its sorting to the plasma membrane but can influence its local organization at nanoscale. CD4 was shown to transiently accumulate in the immunological synapse formed between T cell and antigen-presenting cell. Such accumulation is rapidly followed by its internalization and/or delocalization outside the synapse. This is in contrast with TCR which accumulates strongly in the immunological synapse and is later found enriched in the central area of this structure. It is therefore unclear how TCR and its CD4 co-receptor function together when binding to their common ligand during the initiation of signaling in T cells. We aim to study localization of CD4 at nanoscale using advanced fluorescence microscopy techniques achieving significant improvements in resolution. In this work, CD4 and its mutant variants, potentially causing its different localization at the...