Abstract: -killer cells eliminate infected and cancerous cells with precision by positioning their centrosome near the interface (immunological synapse) with the target cell. The mechanism of centrosome positioning has remained controversial, in particular the role of microtubule dynamics in it. We re-examined the issue in the experimental model of Jurkat cells presented with a T cell receptor-binding artificial substrate, which permits controlled stimulation and reproducible measurements. Neither 1-µM taxol nor 100-nM nocodazole inhibited the centrosome positioning at the “synapse” with the biomimetic substrate. At the same time, in micromolar taxol but not in nanomolar nocodazole the centrosome adopted a distinct peripheral rather than the normally central position within the synapse. This effect was reproduced in a computational energy-minimization model that assumed no microtubule dynamics, but only a taxol-induced increase in the length of the microtubules. Together, the experimental and computational results indicate that microtubule dynamics are not essential for the centrosome positioning, but that the fit of the microtubule array in the deformed body of the conjugated T cell is a major factor. The possibility of modulating the T-cell centrosome position with well-studied drugs and of predicting their effects in silico appears attractive for designing anti-cancer and antiviral therapies.
Abstract: T cells of the immune system target infected and tumor cells in crowded tissues with high precision by coming into direct contact with the intended target and orienting the intracellular Golgi apparatus and the associated organelles to the area of the cell-cell contact. The mechanism of this orientation remains largely unknown. To further elucidate it we used three-dimensional microscopy of living T cells presented with an artificial substrate mimicking the target cell surface. The data indicate that long, finger-like processes emanate from the T cell surface next to the intracellular Golgi apparatus. These processes come in contact with the substrate and retract. The retraction accompanies the reorientation of the T cell body which brings the Golgi apparatus closer to the stimulatory substrate. Numerical modeling indicates that considering the forces involved the retraction of a process attached with one end to the cell body near the Golgi apparatus and with the other end to the substrate can bring the Golgi apparatus to the substrate by moving the entire cell body. The dynamic scenarios that are predicted by the quantitative model explain features of the reorientation movements that we measured but could not explain previously. We propose that retraction of the surface processes is a force-generating mechanism contributing to the functional orientation of T lymphocytes.
Abstract: Orientation of organelles inside T cells (TC) toward antigen-presenting cells (APC) ensures that the immune response is properly directed, but the orientation mechanisms remain largely unknown. Structural dynamics of TC are coupled to dynamics of T-cell receptor (TCR), which recognizes antigen on the APC surface. Engagement of the TCR triggers its internalization followed by delayed polarized recycling to the plasma membrane through the submembrane recycling compartment (RC), which organelle shares intracellular location with the TC effector apparatus. TCR engagement also triggers TC-APC interface expansion enabling further receptor engagement. To analyze the interplay of the cell-cell contact and receptor dynamics, we constructed a new numerical model. The new model displays the experimentally observed selective stabilization of the contact initiated next to the RC, and only transient formation of contact diametrically opposed to the RC. In the general case wherein the TC-APC contact is initiated in an arbitrary orientation to the RC, the modeling predicts that the contact dynamics and receptor recycling can interact, resulting effectively in migration of the contact to the TC surface domain adjacent to the submembrane RC. Using three-dimensional live-cell confocal microscopy, we obtain data consistent with this unexpected behavior. We conclude that a TC can stabilize its contact with an APC by aligning it with the polarized intracellular traffic of TCR. The results also suggest that the orientation of TC organelles, such as the RC and the effector apparatus, toward the APC can be achieved without any intracellular translocation of the organelles.
Abstract: Activation of T cells of the immune system involves recognition of the antigen by the T cell receptor and subsequent internalization and recycling of this receptor. We present a numerical model for this process that accounts for the polarity of the intracellular traffic determined by the polarization of the microtubule-organizing center to the immunological synapse. Unexpectedly, the model explains the observed accumulation of receptors at the immunological synapse mainly as dynamic maintenance of the receptor density there, while the surface receptors everywhere else are depleted, even though the internalization occurs primarily at the synapse. In the case of an unsuccessful polarization of the microtubule-organizing center, which alters the polarity of the receptor trafficking, the model explains the absence of receptor accumulation as a dynamic downregulation at the synapse. The experiment shows that in this case the interaction of the T cell with its target is aborted. Disruption of recycling leads in the experiment to accumulation of the incompletely polarized cells. We propose that receptor recycling is a mechanism whereby the cell can sense its internal structure and detect polarity errors, analogous to checkpoint signaling mechanisms that ensure fidelity of cell division.
Abstract: Directed secretion of cytotoxins or cytokines by T cells during immune response depends on migration of the centrosome in the T cell to the interface with the target cell. The mechanism of the centrosome translocation has been elusive. The presented computational analysis demonstrates that the centrosome should be positioned at the interface if the T cell attempts simultaneously (a) to minimize its surface area, (b) to maximize the interface area, (c) to maintain the cell volume and (d) to straighten the microtubules. Live three-dimensional microscopy and measurements show that the optimal position of the centrosome is achieved in large part (by about 40%) via rolling of the entire T cell body on the target surface; this movement appears to entrain the centrosome. The theoretical and experimental results draw attention to the previously unrecognized role of the whole-cell structure and whole-cell movements in the T cell polarization.
Abstract: A complex study of the effects of Acilact on the immune and interferon status, phagocyte defense, and cytokine balance in sickly children showed that Acilact had a positive effect on the immune system in these patients. The preparation is able to normalize abnormal immune parameters, and does not influence healthy immune system. In some ways Acilact has advantages over IRS 19 vaccine. Simultaneous administration of these two preparations is appropriate in some cases.
Abstract: BACKGROUND: Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution. METHODS: Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison. RESULTS: Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution. CONCLUSIONS: This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.
Abstract: Using scoring of survival of irradiated Drosophila embryos the moderate effects of fs-laser irradiation on embryogenesis and indirect evidence of possible induction of DNA repair mechanisms are demonstrated.
