The T cell transmembrane molecule CTLA-4 negatively regulates antigen-mediated T cell signals. The cytoplasmic signal transduction cascade that activates the transcription factor NF-kB is a crucial target of CTLA-4 inhibition. We propose that CTLA-4 blocks antigen-mediated activation of NF-kB through two distinct mechanisms:
1. Physical displacement of the costimulatory molecule, CD28, from the central T cell/APC interaction zone by direct competition with CTLA-4 for B7 ligands on the antigen-presenting cell (APC).
2. Delivery of an inhibitory signal that interferes with cytoplasmic signal transduction events in antigen activation of NF-kB.
In spite of many studies of CTLA-4 activity, the mechanism of CTLA-4 inhibition of NF-kB activation remains largely undefined. We will employ direct microscopic observation to examine sub-cellular protein redistribution events that are crucial to the function of CTLA-4. These experiments will reveal to what extent CTLA-4 inhibition of NF-kB activation is the result of physical blockade of CD28 interaction with B7 at the cell surface vs. the generation of inhibitory signals within the cytoplasm. Ultimately, this work will identify candidate target molecules against which small molecule inhibitors of CTLA-4 activity could be designed. Such drugs would likely have substantial therapeutic benefit in combating human cancer.
In a second approach, we are examining the effects of CTLA-4 expression on NF-kB activation in the secondary lymphoid organs of living mice. These experiments will demonstrate to what extent CTLA-4 expression down modulates NF-kB activation in vivo. In addition, we expect these experiments to show whether CTLA-4 alters the kinetics of in vivo antigen-driven NF-kB activation.
To attempt to define the molecular mechanisms of CTLA-4 inhibition of T cell activation, we will examine sub-cellular protein redistribution events that are crucial to the function of CTLA-4. We will use fluorescence microscopy for advanced, 3-dimensional live cell imaging of the redistribution of fluorescently tagged T cell signaling proteins during the process of T cell activation. We will also use the technique of fluorescence resonance energy transfer (FRET) to define close molecular interactions that are of critical importance for this inhibitory phenomenon.
For in vivo studies of CTLA-4 regulation of NF-kB activation, we are developing a novel in vivo imaging method that combines detection of bioluminescence and quantum dot fluorescence, in order to detect functional, antigen-driven interactions between T cells and antigen presenting cells.