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Molecular imaging analysis of the function of the cytoskeletal regulator DOCK2 during physiological T cell activation

Final Report Summary - 2PM T CELL IMAGING (Molecular imaging analysis of the function of the cytoskeletal regulator DOCK2 during physiological T cell activation)

During the ERG funding period, we have made overall good progress in the project, with only minor deviations. We have made use of our twophoton microscopy (2PM) imaging platform to address the roles for T cell trafficking in lymph nodes of live, anesthetized mice. First, we have identified the role of cytoskeletal regulators (which include small GTPases such as Rac and Rho, as well as their regulators such as DOCK proteins) on T cell migration in vivo. Furthermore, we performed in vivo imaging of dendritic cells lacking DOCK8, which is also mutated in a rare human genetic disease. DOCK8-deficient humans suffer from severe skin infections and a hyper IgE syndrome, yet the underlying cause of the pathology is less well known. We showed that dendritic cells, critical regulators of the adaptive immune system, showed migration defects in the absence of DOCK8, in particular in dense tissue such as skin. The role of the DOCK2-binding HIV-1 protein Nef was analyzed in retrovirally transduced T cells. We report that Nef interferes with T cell migration through confined environments, which sheds light on the pathophysiology of this factor. Finally, we have made use of 2PM imaging to explore B lymphocyte motility in lymphoid tissue. As a follow up, we are currently preparing a manuscript showing that DOCK2 regulates B cell migration in this site (manuscript in preparation). Our proposed search for DOCK2-binding partners, on the other hand, could not be completed owing to technical difficulties. The company, to which we had outsourced the yeast two hybrid screening, was not able to produce high enough levels of “bait” expression for a meaningful screen. In the meantime, we have employed 2PM imaging to analyze interactions of T cell receptor (TCR) - transgenic mice lacking DOCK2, an activator for the small GTPase Rac, with dendritic cells. Our in vivo results show that DOCK2 plays a dual role during T cell activation. On the one hand, in abundance of dendritic cells, T cells lacking DOCK2 are still able to undergo productive interactions with dendritic cells, although their activation is impaired owing to reduced TCR signaling. On the other hand, when dendritic cell levels are low, DOCK2 is absolutely required for productive T cell encounters with these cells. As a consequence, T cell activation is completely abolished under these conditions. In sum, DOCK2 is a potential drug target since it can be regulated in a dose-dependent manner. We are currently writing a manuscript describing these findings for submission to a renowned peer-reviewed journal.