Functional characterization of B7-H3

According to the 2 signal concept of lymphocyte activation, T cells are efficiently activated by receiving antigen- specific signals and additional signals that result from the interaction of costimulatory molecules with receptors on T cells. The B7 molecules, which are the prototype costimulatory molecules on APC, can activate T cells via CD28 but by interacting with CTLA-4 they can also convey inhibitory signals to these cells. More recently five additional members of this family - B7 homologs - have been identified and were shown to play important roles in the regulation of immune responses. Stimulatory as well as inhibitory functions of these molecules in the context of T cell activation have been described. One member of this family, B7-H3, is of special interest for several reasons: The functional role of B7-H3 is not clear since contradictory results have been reported regarding its effects on the stimulation of T-cells. Furthermore, although evidence for receptors on activated T cells had been reported, no such ligands have been identified to date. Since in addition to APC, B7-H3 is broadly expressed in peripheral tissue as well as tumour cell lines and primary tumours, the consequences of B7-H3- interaction with these unknown ligands might have important implications on the regulation of immune responses. In a previous study we could not find evidence for a significant effect of B7-H3 on the activation of primary T cells. It is however possible that - as described for other B7 homologs, the relevance of B7-H3 lies in its function on already activated T cells. In this project we will therefore focus on the role of human B7-H3 during secondary responses. We will use our antibodies, which are the only B7-H3 specific mAbs described to date, to analyse the effects of this molecule on the capacity of dendritic cells to re-stimulate previously activated T cells. In addition, we will also investigate the effects of B7-H3 on T cell activation in alternative, less complex experimental systems. Using recombinant B7-H3-Ig fusion proteins we will study the interactions of B7-H3 with activated T cells. Finally we propose to identify ligands for B7-H3 by a classical expression cloning approach.

According to the 2 signal concept of lymphocyte activation, T cells are efficiently activated by receiving antigen- specific signals and additional signals that result from the interaction of costimulatory molecules with receptors on T cells. The B7 molecules, which are the prototype costimulatory molecules on APC, can activate T cells via CD28 but by interacting with CTLA-4 they can also convey inhibitory signals to these cells. More recently five additional members of this family - B7 homologs - have been identified and were shown to play important roles in the regulation of immune responses. Stimulatory as well as inhibitory functions of these molecules in the context of T cell activation have been described. One member of this family, B7-H3, is of special interest for several reasons: The functional role of B7-H3 is not clear since contradictory results have been reported regarding its effects on the stimulation of T-cells. Furthermore, although evidence for receptors on activated T cells had been reported, no such ligands have been identified to date. Since in addition to APC, B7-H3 is broadly expressed in peripheral tissue as well as tumour cell lines and primary tumours, the consequences of B7-H3- interaction with these unknown ligands might have important implications on the regulation of immune responses. In a previous study we could not find evidence for a significant effect of B7-H3 on the activation of primary T cells. It is however possible that - as described for other B7 homologs, the relevance of B7-H3 lies in its function on already activated T cells. In this project we will therefore focus on the role of human B7-H3 during secondary responses. We will use our antibodies, which are the only B7-H3 specific mAbs described to date, to analyse the effects of this molecule on the capacity of dendritic cells to re-stimulate previously activated T cells. In addition, we will also investigate the effects of B7-H3 on T cell activation in alternative, less complex experimental systems. Using recombinant B7-H3-Ig fusion proteins we will study the interactions of B7-H3 with activated T cells. Finally we propose to identify ligands for B7-H3 by a classical expression cloning approach.