Naive T lymphocytes traffic through the organism in search for antigen, alternating between blood and secondary lymphoid organs. Lymphocyte homing to lymph nodes relies on CCL21 chemokine sensing by CCR7 receptors, while exit into efferent lymphatics relies on sphingolipid S1P sensing by S1PR1 receptors. While both molecules are claimed chemotactic, a quantitative analysis of naive T lymphocyte migration along defined gradients is missing. Here, we used a reductionist approach to study the real-time single-cell response of naive T lymphocytes to CCL21 and serum rich in bioactive S1P. Using microfluidic and micropatterning ad hoc tools, we show that CCL21 triggers stable polarization and long-range chemotaxis of cells, whereas S1P-rich serum triggers a transient polarization only and no significant displacement, potentially representing a brief transmigration step through exit portals. Our in vitro data thus suggest that naive T lymphocyte chemotax long distances to CCL21 but not toward a source of bioactive S1P.

https://doi.org/10.1016/j.isci.2023.107695

Non-exhaustive model of naive T lymphocyte traffic after accessing lymph nodes. Cells are gently attracted by long-range CCL19 and CCL21 gradients toward the central parenchyma, where they encounter higher and homogeneous concentrations of CCL21 that allow their random walk throughout the T cell zone. Moreover, S1P-rich serum triggers a transient polarization only and no significant displacement, potentially representing a brief transmigration step through exit portals.

Optical tweezers are a light-based technique for micromanipulating objects. It allows to move objects such as microbeads and cells, and to record minute forces down to a few pN, which makes it a technique very well adapted to mechanical measurements on living cells (Gennerich, 2017). We are interested in the mechanotransduction properties of lymphocytes. We seek to dissect the effect of forces and cell mechanics on the cellular response, in the context of the immune system. T cell mechanotransduction has been recently demonstrated to be instrumental in the finesse and accuracy of the response of the latter Puech & Bongrand (2021)]. In addition, cells can exert forces when performing their action, e.g. cytotoxic T cells are using forces to kill target infected cells (Basu et al., 2016).
Using optical tweezers and specifically decorated beads as handles, we pull membrane nano- tubes from gently adhered living lymphocytes (Sadoun et al., 2020). Such nanotubes are usually used to probe the tension of adherent cells (Diz-Muñoz et al., 2010). By varying the antibodies that are used to decorate the beads, we select the molecule type we specifically pull on, and we then explore the molecules which are characteristic of the immune synapse, which is one of the key organizational structures that have profound implications in T cell recognition and action (Baldari & Dustin, 2017).
Using this approach, we probe not only the forces of recognition of the given antibody to its target molecule, but also, by using strong extracellular bridges, we probe the cytosolic link of the probed molecule to the cytoskeleton. Such a link has been proposed to be instrumental in the way T cells can apply or feel forces through the molecule. A theoretical model has been built and has been recently reported in a dedicated article (Manca et al., 2023). Furthermore, we will demonstrate the application of the software on full data.

The experimentally obtained data consists of force signal as a function of time (among other parameters), in the three directions of space, obtained in large quantities (at least 10 per cell / bead couple, and up to 20 couples tested per sample), containing rich and detailed features that can relate to molecular and/or cellular mechanics that our model explores. It is therefore needed to standardize and semi-automate data analysis to help the experimentalist, often a biologist, to extract relevant features from the experimental data sets.

https://doi.org/10.21105/joss.04877

The role of force application in immune cell recognition is now well established, the force being transmitted between the actin cytoskeleton to the anchoring ligands through receptors such as integrins. In this chain, the mechanics of the cytoskeleton to receptor link, though clearly crucial, remains poorly understood. To probe this link, we combine mechanical extraction of membrane tubes from T cells using optical tweezers, and fitting of the resulting force curves with a viscoelastic model taking into account the cell and relevant molecules. We solicit this link using four different antibodies against various membrane bound receptors: antiCD3 to target the T Cell Receptor (TCR) complex, antiCD45 for the long sugar CD45, and two clones of antiCD11 targeting open or closed conformation of LFA1 integrins. Upon disruption of the cytoskeleton, the stiffness of the link changes for two of the receptors, exposing the existence of a receptor to cytoskeleton link—namely TCR-complex and open LFA1, and does not change for the other two where a weaker link was expected. Our integrated approach allows us to probe, for the first time, the mechanics of the intracellular receptor–cytoskeleton link in immune cells.

https://doi.org/10.1038/s41598-023-42599-9