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| Status |
Public on Jul 23, 2024 |
| Title |
Engineered T cell therapy for central nervous system injury [FACS-Tcells] |
| 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 |
CD4+ T cells with retrovirus-based TCR-Cp and GFP expression were injected intravenously into the mice after injury. The injured part of spinal cord (injury site ±0.5 mm) and spleen 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. Spleen were directly collected using forceps. 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. Spleen was directly minced to get single lymphocyte. 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. Cp-T cells (DAPI- CD45+ CD11b- TCRβ+ CD4+ CD8- GFP+) from the injured spinal cord and spleen were sorted into individual wells of a 96-well plate with 10X lysis buffer (635013, TaKaRa) and 5% RNase inhibitor (03335399001, Millipore Sigma). Plates was sealed with adhesive plate seal, frozen rapidly over dry ice after sorting, and stored at -80 °C until sequencing.
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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 (192)
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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 |
PRJNA893393 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE216388_all.gene_counts.csv.gz |
974.3 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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