A generic system for adhering cells to supported lipid bilayers

Abstract

One of the main contributors in the immune system are T cells. The exact molecular mechanisms behind T cell activation are not yet fully understood but it starts through the contact between the T cell and an infected cell. In order to study such interaction, supported lipid bilayers (SLBs) are used to mimic cell membranes and to reduce its complexity but still retaining the fundamental property of lateral mobility. The project was based on two different systems for adhering T cells to SLBs which do not make use of specific adhesion proteins that are not easily available commercially: (i) SLBs with RGD moiety and (ii) SLBs with biotinylated antibodies bound to the SLB via biotin-streptavidin binding.
Jurkat E6-1 cells transfected with the rat CD48 were found to not bind the SLB with RGD moiety because of low levels of integrin expression. This system, however, could be used for the adhesion of different cell lines, as the mobility of the lipids ensured the formation of a homogeneous and fluid SLB that mimicked the cell membrane. For SLB with antibodies, the immobile fraction of streptavidin (SA) in the SLB increased from 15 to 80% upon addition of antibodies suggesting the binding and a possible cross-linking of SA with antibodies. Using an SLB with SA and anti-rCD48, Jurkat’s adhesion fraction was 75% when a concentration of 800ng/mL of SA was used and 41% with a lower concentration (150 ng/mL). With the latter concentration, it was also possible to visualize the recruitment of anti-rCD48 within the contact area.
This system was not able to activate T-cells significantly, yielding a suitable platform for T cell adhesion without employing not purified protein ligands.

Popular Abstract

We are continually exposed to pathogens, such as bacteria and virus which can causes diseases. Our body’s defense mechanism is called immune system which consists of different cells that can distinguish between healthy cells and infected cells, killing the latter or activating mechanisms that lead to their elimination. One specific type of immune cells is called T cells. Their surface presents an important receptor (TCR), molecule that recognize and bind specific molecules, and other proteins that play a fundamental rule in the formation of a cell-cell contact. T cells are activated when TCRs recognize and bind molecules of pathogens that are presented by a second receptor called major histocompatibility complex (MHC) on other cells called antigen presenting cells (APC).
The molecular mechanisms of T cell activation are complex and not yet fully understood. The initial process involves phenomena occurring on the surface of the T cell and the infected cell; through a series of cascading processes, the activation of the cell occurs. With the purpose of facilitating such studies that this work focused on build generic system that had the ability to adhere T cells onto a supported lipid bilayer. Supported lipid bilayers (SLBs) are models used to mimic the cell membrane; they consist of two layers of lipids linked together through particular interactions that are deposited onto a solid support (e.g. glass). To mimic cell membranes, the lipids that make up the SLB should be mobile, that is, they should move laterally within the bilayer. Therefore, for all systems devised, the mobility of the SLB was analyzed.
For adhesion to occur, the SLB must contain molecules that can be recognized by other T-cell molecules, for example, by TCR, adhesion proteins or other receptors.
Therefore, the first system devised saw an SLB containing 3 amino acids that are recognized and bound by integrins, proteins found on the surface of T cells. This type of system did not lead to T- cell adhesion on the SLB because of the small amount of integrins present on the surface of the cells I studied.
Another system was an SLB decorated with antibodies, molecules that have the ability to specifically bind other targets. Hence antibodies with the ability to bind TCR and other T-cell proteins were used, resulting in a good percentage of adhered cells.
For the latter system, its ability to activate cells had also been analyzed. The system composed of antibodies specific for a protein not involved in activation had a low ability to activate cells. For this reason, the system could be a suitable platform for adhesion of T cell and used for future studies on activation.

• author: De Carlo, Emanuela
• supervisor: Peter Jönsson
• organization: Department of Chemistry
• course: KEMP30 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: T-cells, supported lipid bilayers, biochemistry
• language: English
• id: 9101260
• date added to LUP: 2022-10-04 11:37:54
• date last changed: 2022-10-04 11:37:54