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: The injured portion of spinal cord (injury site ±0.5 mm) was collected for T cell isolation 2 weeks after injury. Mice were injected with a lethal dose of Euthasol (10% vol/vol) and transcardially perfused with PBS containing 0.025% heparin. For spinal cord isolation, the spine was cut on both sides and the spinal cord was flushed out using a PBS filled syringe with 18-gauge needle. Spinal cord was chopped using a surgical blade and digested in digestion buffer (RPMI-1640 medium supplemented with 2% FBS, 1 mg mL-1 Collagenase VIII, and 0.5 mg mL-1 of DNase I) at 37 °C for 30 minutes, triturated with a 1 mL pipette, and digested for another 15 minutes. After digestion, enzymes were neutralized with RPMI with 10% FBS and tissue samples were mechanically homogenized and filtered through 70 µm cell strainer. Cells were then centrifuged at 450 g for 4 minutes and the supernatant was removed. Myelin was removed by resuspending cells with 30% percoll or 10-15% BSA in PBS and centrifuged at 800 g for 10 minutes with a slow brake. After centrifugation, the upper myelin-containing layer and supernatant were removed, and the cell pellet was resuspended with RPMI containing 10% FBS. Red blood cells were lysed with ammonium-chloridepotassium (ACK) lysis buffer for 1 minute at room temperature and neutralized with the same volume of RPMI containing 10% of FBS. Cells were then centrifuged and cell pellets were resuspended with RPMI containing 10% FBS and kept on ice until use. Single-cell suspensions were washed with PBS. Fc receptors were blocked for 5 min with the anti-CD16/CD32 antibody cocktail and cells were incubated with primary antibody cocktails for 10 minutes at room temperature for surface staining. Antibodies were all diluted in FACS buffer (2% BSA, 1 mM EDTA, 20 mM HEPES in PBS). After staining, samples were washed, resuspended with FACS buffer (with DAPI if needed), filtered with 70 µm cell strainer and sorted using a BD Biosciences FACSAria II or BD Influx cell sorter. Viable T cells (DAPI- CD45+ Thy1.2+ CD4+ or CD8+) were sorted into a 1.5mL Eppendorf tube with RPMI medium containing 10% FBS. CD4+ and CD8+ T cells were combined. T cells were washed, counted, and resuspended with PBS containing 0.04% BSA. T cell samples were loaded onto a 10X Genomics Chromium platform for Gel Bead-in Emulsions (GEMs) and Mouse T cell Chromium Single Cell V(D)J Enrichment Kit and Chromium Single Cell 5' Library & Gel Bead Kit were used separately to generate a cDNA library targeting the TCR and for transcriptome expression.