In the past years the human JSL1 T cell line has become an invaluable tool to investigate signal induced alternative splicing during T cell activation, as many aspects of splicing regulation observed in primary human T cells are recapitulated in this cell line. Microarray and deep sequencing approaches have revealed genes whose splicing pattern changes robustly upon activation, but functional implications have not been investigated. We are using a variety of techniques, among them transfection of Morpholino oligomers to selectively manipulate isoform ratios, to characterize functional consequences of selected splice events during T cell activation. In addition to Jsl1 cells we are using primary human and mouse T cells to confirm our findings in a more physiological setting.
One of the targets we are working on is the adaptor protein Traf3, which has been described as a negative regulator of the non-canonical (nc) NFkB pathway. We were able to show that T cell specific and activation-dependent Traf3 alternative splicing forms a Traf3 variant that, in contrast to the full length protein, activates the non-canonical NFkB pathway upon T cell activation. This signaling pathway induces the expression of several cytokines with a potential role in regulating T cell dependent adaptive immunity.
Effector molecules, such as cytokines, that are produced during T cell activation need to be secreted in order to be biologically active. Protein secretion during T cell activation is a highly regulated process, but previous research has almost exclusively focused on post-Golgi compartments. We have identified Sec16, a protein involved in an earlier step in protein secretion, to be alternatively spliced during T cell activation. This led to our interest in the COPII machinery and its regulation during T cell activation. We are using confocal microscopy, protein export assays as well as protein-protein interaction studies and FACS analyses to characterize the activity of different Sec16 isoforms in regulating the shape and functionality of the COPII machinery in naive and activated T cells.
With our work we could show that two essential processes during T cell activation, production and secretion of effector molecules, are controlled by alternative splicing. These results suggest that alternative splicing has a much stronger impact on the functionality of activated T cells than previously anticipated and we assume that our continued analyses will uncover more splice events involved in controlling different aspects of T cell biology.