The immune system's ability to adjust the potency of its response to an external threat is exploited by most immunotherapies. Targeting the inhibitory receptors PD1 or CTLA4 to modulate the activity and function of T cells has been an extremely successful strategy for treating cancer. These checkpoint therapies block the extracellular domains of cell surface receptors with antibodies. However, antibodies often have inadequate pharmacokinetics and cannot penetrate relevant tissues. Alternative ways of inhibiting these trans-membrane receptors by targeting downstream effectors are currently not available, as the molecular mechanisms of signal transduction are not fully understood, and the identified intracellular signaling molecules are important in a wide range of cell types, signaling pathways, and cellular compartments. Thus, it is important to characterize signal transduction mechanisms that are specific to T cells. The recruitment and activation of downstream kinases and phosphatases by transmembrane receptors is one way that this specificity is achieved. This study investigates the mechanism by which the SHP2 phosphatase is recruited to and activated by the inhibitory receptor PD1 in T cells. To this end, a multidisciplinary approach will be employed that utilizes biochemical, structural, and biophysical approaches, as well as single molecule imaging, namely super resolution fluorescence microscopy and single particle tracking. First, the effects of SHP2 phosphorylation and PD1 binding on SHP2 conformation and phosphatase activity will be examined. Hydrogen-Deuterium Exchange ? Mass spectrometry analyses of wild-type and mutant versions of SHP2 (in their phosphorylated and/or PD1 bound forms) will determine the nature and locations of conformational changes in SHP2. This information will be correlated to changes in activity to identify structure-function relationships. Second, interaction dynamics between SHP2 and PD1 will be analyzed in vivo and in vitro. Microscopy approaches will be used to determine recruitment kinetics of SHP2 to PD1 and correlate them to changes in SHP2 binding affinities. Mutant analyses will explore the molecular underpinning of these interactions and determine whether they can be altered to modulate T cell responses, as well as affect disease onset and progression in mouse models of melanoma and diabetes. Third, the spatio-temporal relations between SHP2, PD1, and components of the T cell receptor signaling pathway will be investigated using cutting edge single molecule imaging technologies. These approaches will also utilize wild-type and mutant versions of SHP2 to determine whether the membrane dynamics and distribution of SHP2 can be altered to change T cell immune responses. In conclusion, the suggested research will uncover mechanisms unique to the activation of SHP2 through the PD1 pathway in activated T cells. These mechanisms are potential targets for allosteric and small molecule inhibitors, thereby providing a viable alternative to current immunotherapies.
Studying spatio-temporal mechanisms rooted in the formation of multi-protein complexes and the architecture of the plasma membrane provides novel insights into the molecular underpinnings of membrane signaling and the causes for a large number of diseases that are due to defects in membrane signaling. How these mechanisms are utilized during T cell functions will determine if we can exploit them to modulate immune responses for the development of new therapeutic strategies. The proposal uses multidisciplinary approaches to determine how protein modifications, complex formations and plasma membrane compartmentalization control and restrict enzyme activities to specific pathways.
