Clustering and Transport of Costimulatory Receptors at the Helper T Cell Immunological Synapse

PMThe Helper T cell (TH) immunological synapse (IS) is the patterned cell-cell junction between a TH cell and an antigen-presenting cell (APC) mediating the self/non-self discrimination that leads to the adaptive immune response. It consists of the canonical receptor-ligand pairs, T cell antigen receptor---peptide-loaded major histocompatibility complex (TCR:pMHC) and lymphocyte function-associated antigen 1---intracellular adhesion molecule 1 (LFA-1:ICAM-1), along with a host of other costimulatory molecules, among them CD28 and its ligands B7-1 and B7-2. Upon cell-cell contact, CD28:B7-1 complexes, like those of TCR:pMHC, form microclusters and are concentrated at the junction center to form a central supramolecular activation cluster (cSMAC), while LFA-1:ICAM-1 forms a peripheral SMAC (pSMAC) surrounding the central assembly. While CD28:B7-1 microclusters colocalize with those of TCR:pMHC early in junction formation, they antilocalize with one another at the mature cSMAC. The mechanisms responsible for this dynamic shift in associative behavior are currently unknown. The work described in this dissertation addresses the organization of B7-1 microclusters with respect to the canonical IS receptor-ligand pairs during synapse formation. The specific system studied is the hybrid live cell---supported bilayer synapse, in which the APC is replaced with a supported lipid bilayer displaying relevant surface ligands attached through a histidine-nickel linkage. With this new system, time- and position-independent antilocalization is observed between B7-1 and ICAM-1, while prior research results showing a dynamic shift in B7-1:TCR association from early, peripheral colocalization to later antilocalization in the cSMAC are reproduced. The latter phenomenon is probed by patterning diffusion barriers onto the bilayer support, which trap microclusters in the pSMAC at late time points. These trapped microclusters do not display the antilocalization observed in a mature cSMAC. Finally, substantial though partial actin remodeling is observed as a consequence of B7-1 stimulation in the absence of TCR signaling, resulting in central accumulation of B7-1 that may be abolished by drugging of the actin cytoskeleton. Together, the results illustrate the complex and crowded molecular environment of the IS, both at the membrane and in the cytosol, and the role of costimulation in augmenting TCR-derived signaling to effect a robust immune respons