INHIBITION OF T CELL ACTIVATION THROUGH INTERRUPTION OF ADAPTOR PROTEIN ASSOCIATIONS

Abstract

P1006 Aims: Crucial for the integration of biochemical signals in multiple hematopoietic lineages, adaptor molecules seem a logical therapeutic target. SLP-76 is an adaptor that links proximal and distal T cell receptor (TCR) signaling events through its function as a molecular scaffold in the assembly of multi-molecular signaling clusters. Previously, we have provided data that supports a model in which Gads, a Grb2 family member constitutively bound to SLP-76, is recruited to the cell membrane through inducible association with the adaptor LAT following TCR engagement. This shuttles SLP-76 and its inducible binding partners to the immune synapse allowing for signaling complex assembly with subsequent activation of second messenger pathways. We propose that through specific interruption of the SLP-76/Gads association it may be possible to attenuate T cell responsiveness while preserving other essential SLP-76 functions. Methods: SLP-76−/− Jurkat T cells were reconstituted with GFP-tagged SLP-76 plus either a dominant negative Gads Binding Fragment (GBF), a cDNA encoding the Gads binding domain of SLP-76 amino-terminally tagged with the fluorescent molecule DSRed2, or DSRed2 alone. SLP-76 recruitment following TCR engagement was visualized using live confocal microscopy. We measured calcium flux, CD69 upregulation, and activation of NF-AT to assess function of the reconstituted Jurkat cells. In addition, we created bone marrow chimeras expressing the GBF through retroviral transduction and assessed its effect on thymocyte development and on the responsiveness of both central and peripheral T cell pools. Results: Jurkat cells expressing GFP/SLP-76 and DSRed2 demonstrated initial clustering of SLP-76 at the periphery of the T cell as it contacted a stimulatory surface with subsequent movement to the central point of contact. In those cells expressing the GFP/SLP-76 and DSRed2/GBF fusion proteins, no clustering of SLP-76 was observed. In the absence of SLP-76 recruitment, in cells expressing GFP/SLP-76 and the DSRed2/GBF, we found a marked decrease in Ca2+ mobilization, upregulation of CD69, and activation of NF-AT following TCR stimulation relative to those coexpressing GFP/SLP-76 and DSRed2. Bone marrow chimeras expressing the GBF through retroviral transduction demonstrated impaired development of single positive (SP) thymocytes, a decreased CD4SP to CD8SP ratio, and diminished thymocyte function. The peripheral T cell compartment is similarly impaired. Conclusions: Here we show that interruption of the SLP-76/Gads association results in attenuation of T cell development and responsiveness and suggest this may provide a novel strategy for immune modulation in the settings of alloreactivity and autoimmunity.