T-cell synaptic ectosomes relay signals through microcluster transfer

Abstract

Background: Extracellular vesicles (EV) are proposed to transfer information between cells. In the immunological synapse T cell receptor (TCR) interaction with pMHC drives microcluster formation and signalling that is terminated in parts through sorting of TCR into EVs that bud into the synapse, synaptic ectosomes (SE). Previously, we used correlative light and electron microscopy to characterize SEs. However, this approach has some limitations such as the poor resolution of fluorescent signals and the lack of information on receptor organization in individual SE. Methods: SE released by CD4 T cells were captured on planar supported lipid bilayer (PSLB) containing either ICAM1, ICAM1 and aCD3 or ICAM1, aCD3, CD40 and ICOSL. SEs were stained with WGA to visualize the membrane and with directly conjugated antibodies against TCR, CD40L, ICOS, BST2 and imaged by multicolour dSTORM. To assess functionality of released SEs, DCs maturation after incubation with SEs was measured by cytokine array.