The impact of negative selection on thymocyte migration in the medulla

Developing thymocytes are screened for self-reactivity before they exit the thymus, but how thymocytes scan the medulla for self antigens is unclear. Using two-photon microscopy, we observed that medullary thymocytes migrated rapidly and made frequent, transient contacts with dendritic cells. In the presence of a negative selecting ligand, thymocytes slowed, became confined to areas of approximately 30 mum in diameter and had increased contact with dendritic cells surrounding confinement zones. One third of polyclonal medullary thymocytes also showed confined, slower migration and may correspond to autoreactive thymocytes. Our data suggest that many autoreactive thymocytes do not undergo immediate arrest and death after encountering a negative selecting ligand but instead adopt an altered migration program while remaining in the medullary microenvironment

Thymocyte-dendritic cell interactions near sources of CCR7 ligands in the thymic cortex

Little is known about the dynamics of the interactions between thymocytes and other cell types, as well as the spatiotemporal distribution of thymocytes during positive selection in the microenvironment of the cortex. We used two-photon laser scanning microscopy of the mouse thymus to visualize thymocytes and dendritic cells (DCs) and to characterize their interactions in the cortex. We show that thymocytes make frequent contacts with DCs in the thymic cortex and that these associations increase when thymocytes express T cell receptors that mediate positive selection. We also show that cortical DCs and the chemokine CCL21 expression are closely associated with capillaries throughout the cortex. The overexpression of the chemokine receptor CCR7 in thymocytes results in an increase in DC-thymocyte interactions, while the loss of CCR7 in the background of a positive-selecting TCR reduces the extent of DC-thymocyte interactions. These observations identify a vasculature-associated microenvironment within the thymic cortex that promotes interactions between DCs and thymocytes that are receiving positive selection signals

A novel chemotaxis assay in 3-d collagen gels by time-lapse microscopy

Contains fulltext : 111018.pdf (publisher's version ) (Open Access)The directional cell response to chemical gradients, referred to as chemotaxis, plays an important role in physiological and pathological processes including development, immune response and tumor cell invasion. Despite such implications, chemotaxis remains a challenging process to study under physiologically-relevant conditions in-vitro, mainly due to difficulties in generating a well characterized and sustained gradient in substrata mimicking the in-vivo environment while allowing dynamic cell imaging. Here, we describe a novel chemotaxis assay in 3D collagen gels, based on a reusable direct-viewing chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane. The diffusion process has been analysed by monitoring the concentration of FITC-labelled dextran through epifluorescence microscopy and by comparing experimental data with theoretical and numerical predictions based on Fick's law. Cell migration towards chemoattractant gradients has been followed by time-lapse microscopy and quantified by cell tracking based on image analysis techniques. The results are expressed in terms of chemotactic index (I) and average cell velocity. The assay has been tested by comparing the migration of human neutrophils in isotropic conditions and in the presence of an Interleukin-8 (IL-8) gradient. In the absence of IL-8 stimulation, 80% of the cells showed a velocity ranging from 0 to 1 microm/min. However, in the presence of an IL-8 gradient, 60% of the cells showed an increase in velocity reaching values between 2 and 7 microm/min. Furthermore, after IL-8 addition, I increased from 0 to 0.25 and 0.25 to 0.5, respectively, for the two donors examined. These data indicate a pronounced directional migration of neutrophils towards the IL-8 gradient in 3D collagen matrix. The chemotaxis assay described here can be adapted to other cell types and may serve as a physiologically relevant method to study the directed locomotion of cells in a 3D environment in response to different chemoattractants

Rapid and accurate generation of various concentration gradients using polydimethylsiloxane-sealed hydrogel device

We report a microfluidic device that can rapidly and accurately generate various concentration gradients in a controllable manner for the chemotaxis study of motile bacterial cells by integrating hydrogel into polydimethylsiloxane (PDMS) microchannels. We performed numerical simulations for both the PDMS-sealed hydrogel hybrid device and a representative conventional hydrogel-based device to theoretically compare their characteristics. In addition, we experimentally demonstrated that the PDMS-sealed hydrogel device not only produces various linear and nonlinear concentration gradients without flow-induced shear stresses on motile bacterial cells but also exhibits remarkable advantages over conventional hydrogel-based devices. For example, the PDMS-sealed hydrogel device can be used for fast and accurate generation of various concentration gradients, prevents dehydration of hydrogel and evaporation of solutions, directs diffusion of chemicals such as chemoattractants, exhibits long-term durability, and is easy to handle. Because the hydrogel used is biocompatible and arbitrary concentration profiles can be easily designed and produced on a chip, we believe that not only the PDMS-sealed hydrogel fabrication method but also the versatile concentration gradient generation device can be used for various studies on interaction between chemicals and cells including bacterial chemotaxis assays.close0