Development of an exosome-based tagging method to label CD8+ T effector cells that immune surveil arteriolar endothelium for transcriptional characterization

Abstract

T cell function is highly regulated during acute viral infection to ensure an effective immune response. Upon antigen exposure, T cells experience a unidirectional differentiation that can be studied based on the increased expression of CX3CR1. Previous work from our group has identified a subset of CX3CR1hi CD8+ T effector cells that patrol along vessel walls, mostly on arterioles, enabling a rapid response to endothelial viral infections. In an attempt to isolate and characterize patrolling CD8+ effector T cells, we used Kaede (a photoconvertible protein) to specifically label intravascular T cells interacting with the endothelium at a given time point. However, this method can neither distinguish patrolling cells interacting with the endothelial wall for different periods of time nor the signal strength they receive, thus producing a heterogenous outcome with cells at various stages of patrolling. To further study patrolling T cells, we have developed a complementary approach using endothelium-derived fluorescent exosomes to tag T cells actively interacting with arterioles. This technique allows us to obtain a sizable amount of better synchronized T effectors for in depth characterization. In this regard, bulk RNA-seq unveiled a proliferating transcriptional gene signature for patrolling CD8+ T cells. This result is in agreement with the scRNA-seq data obtained with the Kaede model. Moreover, we also detected multiple Trav, Traj, Trbv and Trbj genes specifically upregulated in either patrolling or circulating T cells in both transcriptional analyses. However, by analyzing the VJ combinations and CDR regions of individual cells detected by TCR clonotyping, minor differences were identified. Finally, using a predictive software for transcriptional regulators (BART, Binding Analysis for Regulation of Transcription), we found a striking amount of epigenetic modulators upregulated preferentially in patrolling CD8+ effector T cells, indicating that these effector cells might be experiencing active epigenetic regulation. These findings suggest that CD8+ effector T cells interacting with arteriolar endothelium during the anti-viral immune response receive signals that shape them at transcriptional and epigenetic level.