SRA

Traumatic injuries to the central nervous system (CNS) affect millions of people worldwide yet lack an effective treatment. These injuries contain infiltrating immune cells that can promote tissue repair and could be exploited for therapeutic benefit. Here, using single-cell RNA-sequencing of T cells infiltrating the injured CNS we demonstrate their clonal expansion and antigen specificity towards CNS derived self-peptides. We confirm the beneficial effect of these injury-associated autoimmune CD4+ T cells in murine models of optic nerve and spinal cord injury. Subsequently, using mRNA-based transient T cell receptor (TCR) reconstitution, we demonstrate a therapeutic T cell strategy to alleviate CNS injury. Treatment of CNS-injured mice with this therapy improved locomotion and alleviated histological signs of damage, through regulation of myeloid cells, without detrimental autoimmune side effects. This strategy provides a means of developing custom-designed T cell therapies for CNS injury, and possibly for other neurodegenerative disorders. Overall design: To FACS sort T cells, mice were given a lethal dose of anesthetics by i.p. euthasol (10% v/v) and transcardially perfused with ice-cold 0.025% (w/v) heparin in PBS. Before perfusion, peripheral blood was collected by retro-orbital bleeding (200-300ul) and diluted 1:1 with PBS containing heparin. Red blood cells were lysed twice by resuspension in ACK lysis buffer for 5 min at RT. Cells were washed with FACS buffer (2 mM EDTA, 25 mM HEPES, 1% BSA in 1X PBS) before being re-suspended for immunostaining. After perfusion, meninges and diaphragm were digested for 20 and 30 min, respectively, at 37 °C with 1.4 U/mL of Collagenase VIII (Sigma Aldrich) and 35 U/mL of DNAse I (Sigma Aldrich) in IMDM (Sigma Aldrich) media. Following the digestion step, the tissues were gently pressed through 70 µm nylon mesh cell strainers with a sterile plastic plunger. Cells were then centrifuged at 450g at 4°C for 4:30 min. The cell pellets were resuspended in ice-cold FACS buffer and stained at 1:300 dilution. The single-cell suspensions were immunostained with Live/Dead Fixable Aqua Dead (Biolegend; #Cat: 423102), anti-CD45 APC (BD Bioscience; Clone: 30-F11; #Cat: 559864), anti-TCR FITC (Biolegend; Clone: H57-597; #Cat: 109206), anti-CD4 PE (BD Bioscience; Clone: RM4-5; #Cat: 553049), and anti-CD8a PE (eBioscience; Clone: 53-6.7; #Cat: 12-0081-81) using the BD Influx Cell Sorter (BD Biosciences). CD4+ and CD8+ T cells were combined into a single tube (CD45+TCR +CD4+ and CD45+TCR +CD8+, respectively). Cells were sequenced in 4 separate runs to improve sequencing depth via 5' chemistry.