Biophysical aspects of mild heat stress in T- cell signaling

Most animals raise their body temperature above its normal set point to face infection. Accumulating evidence indicates that hyperthermia affects cellular stress resistance and the immune system as well. While fever is beneficial in most various diseases it should be carefully controlled to avoid undesired side effects especially in cases of extreme inflammation. The reactions that help the organisms to adapt to stressful conditions generally follow a programmed series of events termed stress response. Investigation of the cellular stress response is of great importance to our understanding of how cells respond and adapt to various changes in their environment especially in disease states. The two laboratories collaborating in the current project aim to identify mild fever-like heating induced alterations of the cellular membrane that surrounds the cells interior which plays an important role in both the immune and stress response. By combining our complement expertise on the field of molecular stress biology (Szeged), membrane biophysics (Vienna and Szeged), single molecule microscopy in the immunological synapse (Vienna) we could shed light on how immune system feels the heat. Zooming in on membrane nano-organization engaged in hyperthermia-induced stress tolerance and enhanced immune response could help us to understand why hyperthermia could have both pro- and anti- inflammatory effects which could help the development of novel therapies.

Is there a change on the molecular length scale in our adaptive immune system when we have a fever-for instance, when our body is affected by an infection? We investigated this question using murine T-cells, which we can specifically activate in the lab with a known pathogen. In this setup, we replace the cellular interaction partner of the T-cell, which typically presents the pathogen on its surface in our body, with an artificial cell system. This allows us to precisely control the quantity and other properties of the activating proteins. Together with our collaborators in Szeged, Hungary, we examined potential changes using highly sensitive fluorescence microscopy. Specifically, we were interested in whether the initial recognition of the pathogen and the subsequent activation of the T-cell are influenced. To simulate febrile temperatures, the temperature in the observation chamber, where the artificial cell system interacts with the T-cells, was raised to 40C. The results were then compared to experiments conducted at physiological temperature (37C). Unsurprisingly, it was observed that all proteins-both those on the artificial cell system and the T-cell receptors we monitored-moved faster at higher temperatures. However, we also demonstrated that the artificial cell system is highly sensitive to temperature changes. For instance, the density of proteins decreases more rapidly than expected, as these proteins detach more easily from the membrane at elevated temperatures. Additionally, complete immobilizations of proteins on the glass surface used in the setup occurred, which significantly distorted the interaction experiments with the T-cells. Therefore, in an initial step, new experimental conditions had to be identified to prevent these detachment and immobilization processes. By modifying the composition of the buffer solution used in the experiments, these effects were successfully counteracted. Given the widespread use of this artificial cell system, the related publication currently in progress is likely to attract considerable interest. After optimizing the experimental system, the single-molecule fluorescence microscopy method was further evaluated through computer simulations to identify parameters that could distort the measurement results. A publication on this work has appeared in the journal Biophysical Journal. Under these optimized conditions, first experiments revealed that the sensitivity of T-cells increases at higher temperatures. In other words, a lower amount of pathogens is required to trigger an immune response at fever-like temperatures. This implies that fever has a significant-positive in this case-impact at the molecular level. The exact molecular mechanisms responsible for this heightened sensitivity are now being investigated in a follow-up study.