Studying novel forms of interleukin receptor signaling

Interleukins are important signaling proteins that are secreted by cells of the immune system in order to communicate with neighboring cells. One of these interleukin proteins, interleukin-2, is in the focus of our project. Interleukin-2 gets released by T helper cells and, as a so-called autocrine hormone, also has an effect on the releasing cell itself. This signal is processed by the T-cell in parallel to many other input signals and is therefore a determining factor for the outcome of the immune response. Interleukin-2 is bound by the interleukin-2 receptor, a membrane protein of the T cell. Until now it was assumed that the binding occurs mainly on the plasma membrane of the T cell. However, data from our Hungarian collaboration partner suggests that the binding occurs mainly in intracellular organelles such as the endoplasmic reticulum and the Golgi apparatus, but these intracellular interactions have not yet been confirmed by microscopy due to insufficient resolution of the imaging processes. In this project we try to observe the two proteins - i.e. the interleukin-2 and the interleukin-2 receptor - at the same time using high-resolution microscopy. We will use single-molecule localization microscopy for this, which is based on localizing individual dye molecules very precisely in the cellular context. The method can achieve a resolution in the range of less than ten nanometers, which corresponds to an approximately ten-fold improvement compared to conventional light microscopy. The two proteins are specifically labeled with dyes using antibodies and examined using two-color microscopy. In addition, we will try to capture the diffusional dynamics of the proteins in a cellular context. For this purpose, we will record the 3-dimensional movement of individual dye-labeled receptor proteins within the cell using high-resolution microscopy. Taken together, our project aims to shed new light on the spatial-temporal sequence of interleukin-2 binding and thus on a process that is essential for the immune response.

Interleukin-2 (IL-2) and -15 (IL-15) are important regulators of activation, homeostasis, proliferation and survival of T cells, which play a central role in adaptive immune responses against pathogens and cancer. The receptors for IL-2 and IL-15 each consist of 3 subunits, 2 of which are identical. Therefore, IL-2 and -15 have several common functions, such as promoting T cell proliferation; interestingly, they play opposing roles in immunological memory. Immunological memory relies on the long-term survival of T cells so that the immune system can provide a prompt response upon repeated encounters with the same pathogen. IL-15 is the main survival factor of memory T cells, acting via transpresentation (TP), which relies on the interaction between an antigen-presenting cell and the T cell. In this project, we aimed at using high-resolution, single-molecule microscopy to study the organization of IL-2 and I-15 receptors in cellular membranes. The project was carried out in close cooperation with our collaboration partner György Vamosi from the University of Debrecen, Hungary. We pursued 2 main directions: 1) We used single-molecule tracking to compare the movement of IL-2 in native cells and so-called membrane blebs. Blebs do not have an actin-based cytoskeleton, therefore different diffusion behavior should give insight into the influence of the cytoskeleton on protein motility. The experiments showed that the movement of IL-2 in blebs is massively accelerated compared to the cellular plasma membrane. Together with the results of our partners from Debrecen we could show that on the molecular level the complexes of IL-2/15 receptors (as well as MHC) are present in blebs as well as in native membranes, but lose contact to the actin cytoskeleton. We observed an organization into larger microdomains of these receptor clusters in the plasma membrane, but it largely disappeared in blebs. 2) Many years ago we developed a method called TOCCSL to quantitatively study co-diffusion of proteins in cell membranes. Here we wanted to apply this method to the interaction study between IL-2 and IL-15 receptors. Before we could start this experiment, however, it was necessary to examine the method itself more closely for possible deviations in the results obtained. We therefore carried out very precise Monte Carlo simulations in which we simulated the method on the computer and quantified exactly how various experimental parameters influence the results obtained. These results will serve as a valid basis for future TOCCSL experiments.