Single-Molecule Imaging Reveals Chemokine Receptor Contributions to the T Cell Immunological Synapse

Abstract

The immunological synapse (IS) is a highly dynamic yet tightly organised platform for information transfer between T cells and a number of different antigen-presenting cell (APC) types. It is essential for the correct activation of T cells in response to infection, the prevention of autoimmunity, and licensing of APCs for anti-microbial processes. Chemokine receptors are G-protein coupled receptors that recognise inflammatory and homeostatic chemokines, mediating leukocyte migration towards sites of infection or within specialised tissues of the immune system. In recent years it has become apparent that several chemokine receptors, particularly CXCR4, also contribute to the stability and longevity of the IS, and do so through mechanisms distinct from those involved in cell migration. Nonetheless, their spatiotemporal organisation within the complex architecture of the IS is unstudied, and hence integration with the known signalling events of T cell activation is not understood. Here we use total-internal reflection fluorescence (TIRF) microscopy to examine CXCR4 and other chemokine receptors within primary human CD4+ T cells during the process of IS formation on supported lipid bilayers. Single-molecule tracking reveals initial exclusion of receptors followed by active recruitment into the centre of the contact, driven by cytoskeletal reorganisation. Receptor mutants with aberrant organisation are able to undermine the correct formation of the IS and tip the signalling balance in favour of migratory responses, highlighting the importance of this process for correct T cell function. Combining TIRF with super-resolution structured illumination microscopy reveals that chemokine receptors segregate from the T cell receptor at the nanoscale, contrary to the presumed model of receptor recruitment to the IS. These observations shed light on the mechanisms by which chemokine receptors contribute to IS stability, and hence how its formation is regulated during a coordinated immune response.