Spatiotemporal Dynamics of Assembly and Activation of Class II Cytokine Receptors

Class II cytokine receptors are important pleiotropic regulators of the immune system that play a central role in pathogen defense, tumor surveillance and immune system homeostasis. Most of these activities are very promising for biomedical applications, which, however, have so far failed to succeed due to severe undesired side effects resulting from the pleiotropic nature of these cytokine receptors. Controlling the functional plasticity of class I/II cytokine receptor signaling by engineered cytokines has recently emerged as a promising approach to selectively reduce such side effects. In this context, systematic studies on the IFNalpha/beta receptor and other systems have identified that the binding kinetics of the ligand-receptor interaction play an important role in defining signaling specificity. This has been explained by altered equilibrium and dynamics of the signaling complex in the plasma membrane. In this work, I have investigated how the spatiotemporal organization and dynamics of signaling complexes regulate activation and signaling specificity of other members of the class II cytokine receptors. I focused on the type II IFN and IL-10 systems that supposedly form hexameric ligand-receptor signaling complexes in the plasma membrane. To this end, we developed an orthogonal multicolor anti-GFP nanobody-based labeling strategy, that allowed imaging of up to four different class II cytokine receptor subunits simultaneously. Using this labeling strategy, I investigated the spatiotemporal dynamics of IFNGR and IL-10R complex assembly by co-localization and co-tracking of single receptor subunits. Thereby, I did show that unliganded receptor subunits of IFNGR and IL-10R remain monomeric at the cell surface, whereas binding of the ligand led to fast and efficient receptor homo- and hetero-dimerization, verifying a ligand-induced receptor complex assembly model for both cytokine receptors. Moreover, I verified the hexameric ligand-receptor complex structure in cellulo. Analysis of single molecule trajectories and co-trajectories revealed a decrease in mobility and diffusion of IFNGR and IL-10R subunits upon ligand stimulation indicating receptor confinement and endocytosis. In this context, I identified an abnormal diffusion behavior of IL-10R2 that was dependent on the length of its transmembrane helix. We used partial agonists for both receptor complexes to systematically alter receptor binding stoichiometry and complex stability in the plasma membrane and correlated these with downstream signaling responses. Our analysis revealed a minor contribution of the second low affinity receptor subunit and its associated kinase to the overall signaling activity. However, the second high affinity binding subunit was indispensable to acquire full signaling potential. We managed to obtained decoupling of gene expression for both hexameric class II cytokine receptors by utilizing engineered ligands with altered receptor binding affinities. Our findings could pave the way for new biomedical approaches with engineered IFNgamma and IL-10 in the future. Furthermore, we uncovered pathogenic mechanisms behind the IFNGR2-T168N mutant and auto-IFNgamma antibodies, both of which prominently cause the Mendelian Susceptibility to Mycobacteria Disease (MSMD) syndrome, showing that both interfere with IFNGR activation by preventing recruitment of IFNGR2 into receptor complexes