Studies on the costimulatory function of CD63

In order to become fully activated a T cell requires two distinct signals during its interaction with Antigen presenting cells (APC): T cell receptors have to recognize MHC-bound antigenic peptide ligands ("signal 1") and costimulatory receptors need to be engaged by their cognate ligands ("signal 2"). By sustaining and amplifying the antigen specific signals, costimulatory signals are initiating and promoting expansion and differentiation of antigen specific T cells. There are numerous receptor-ligand interactions that can costimulate T cell responses but engagement of the CD28 receptor is generally accepted to generate the most potent costimulatory signals. We have previously demonstrated that an immobilized antibody to the tetraspan molecule CD63 can match costimulatory effects triggered by CD28 antibodies. In the presence of APC however, CD63 antibodies interfere with T cell responses, indicating that CD63 is a component of a novel and potent T cell costimulatory pathway. In this study we want to perform a detailed analysis of this pathway to reveal similarities and differences between human T cells costimulated via CD28 and CD63. Importantly we want to elucidate the mechanisms underlying the ability of CD63 to transduce potent activating signals into human T cells. Molecular interaction partners of CD63 that are involved in this process should be identified using proteomic-based approaches as well as ligand screening methods. Furthermore we propose to identify molecules that connect CD63 to the intracellular signalling machinery in human T cells and analyse the signalling pathways that are engaged upon CD63 costimulation. A particularly interesting feature of T cells activated via CD63 is their ability to show elevated responses to secondary stimulation indicating that this pathway may trigger a unique activation program in human T cells. Using genome-wide expression analysis we will try to identify genes that are specifically regulated by engagement of CD63 and thus could encode candidate molecules that promote secondary T cell responses.

T cells have essential tasks in immunological processes. This project has dealt with molecules that have a decisive role in the regulation of T cell responses the so-called costimulatory molecules. They can act activating or inhibitory and thus are able to enhance or down modulate T cell responses. In addition to the well-characterized primary T-cell costimulatory molecules (CD28; CTLA-4; CD80 and CD86) there is a number of so-called alternative costimulatory molecules that have also an important role in T cell responses. However, the function of several of these molecules is not well understood. In previous work we could show that CD63 a molecule, which has not been thoroughly investigated in the context of T cell activation can act as a very potent T cell costimulator. Within this project we have investigate the role of this molecule but also the function of other alternative costimulatory molecules and pathways in T cell activation processes. For this we have developed a cellular system, which is excellently suited to investigate the role of alternative costimulatory molecules during the activation of human T cells. With this tool, which we have designated T cell stimulators, we were able to gain important information regarding the function of alternative costimulatory molecules in T cell responses. Moreover, T cell stimulator cells will be an invaluable tool to tackle additional vital issues relating to the regulation of T cell responses in the future. CD28-negative T cells are especially interesting in the context of alternative costimulators since the efficient activation of these cells critically depends on such molecules. In elderly the majority of CD8 T cells are deficient in CD28 but also younger individuals have a significant subset of CD28 negative CD8 cells. Importantly these cells contribute to the impairment of immune function that is associated with old age but also with chronic virus infection and autoimmunity. We have used our T cell stimulators to investigate the role of alternative costimulatory molecules regarding their ability to contribute to the activation of CD28 negative T cells. Our results demonstrate a prominent role for the CD2 molecule in costimulating CD28 negative T cells, whereas all other molecules that were investigated had a limited capacity to activate this T cell subset. This indicates that CD2 is essential for the function of these cells but also that this costimulator might contribute to the expansion of CD28 deficient cells that is observed in elderly and chronically infected individuals. Therefore CD2 might be a therapeutic target to counteract the impairment of immune function during aging or persistent virus infection. In order to investigate the role of CD63 in T cell activation processes we have developed a T cell reporter line that allows measuring transcription factors that play a major role in T cell activation simultaneously. We were able to show that antibodies against CD63 induce an activation program that is highly reminiscent of the one that is induced upon engagement of the primary T cell costimulator CD28. Thus the CD63 molecule might exert its costimulatory function through CD28.