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| Status |
Public on Jul 23, 2024 |
| Title |
Engineered T cell therapy for central nervous system injury [CD45+ scRNA-seq] |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
Mice were induced with spinal cord injury followed immediately by injection of Cp-T cells or PBS as control. The injured portion of spinal cord (injury site ±0.5 mm) was collected for cell isolation 7 days 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. CD45 Microbeads UltraPure (130-052-301, Miltenyi Biotec) and MACS LS columns (Miltenyi Biotec) were used as per manufacturer’s instructions to isolate the CD45+ population. Isolated CD45+ cells were resuspended in PBS containing 0.04% BSA. Cell number and viability were determined using Viastain AOPI Staining Solution (CS2-0106, Nexcelom Bioscience) and CellDrop FL Fluorescence Cell Counter (DeNovix). Sample loading and library construction were performed using the 10X Genomics Chromium platform and Chromium Single Cell 3’ Library & Gel Bead Kit version 3.
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| Web link |
https://www.nature.com/articles/s41586-024-07906-y
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| Contributor(s) |
Gao W, Dykstra T, Kipnis J |
| Citation(s) |
39232158 |
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| Submission date |
Oct 23, 2022 |
| Last update date |
Oct 22, 2024 |
| Contact name |
Jonathan Kipnis |
| Organization name |
Washington University in Saint Louis
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| Department |
Pathology and Immunology
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| Lab |
Kipnis Lab
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| Street address |
4515 McKinley Ave
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (2) |
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| This SubSeries is part of SuperSeries: |
| GSE216391 |
Engineered T cell therapy for central nervous system injury |
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| Relations |
| BioProject |
PRJNA893395 |
