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| Status |
Public on Mar 11, 2024 |
| Title |
Kidins220 and Aiolos promote thymic iNKT cell development by reducing TCR signals |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
Development of T cells is controlled by the signal strength of the TCR. The scaffold protein Kinase D-interacting substrate of 220 kDa (Kidins220) binds to the TCR; however, its role in T cell development was unknown. Here, we show that T cell-specific Kidins220 knock-out (T-KO) mice have strongly reduced invariant natural killer T (iNKT) cell numbers and modest decreases in conventional T cells. Enhanced apoptosis due to increased TCR signaling in T-KO iNKT thymocytes of developmental stage 2 and 3 shows that Kidins220 downregulates TCR signaling at these stages. scRNAseq indicated that the transcription factor Aiolos is downregulated in Kidins220-deficient iNKT cells. Analysis of an Aiolos KO demonstrated that Aiolos is a downstream effector of Kidins220 during iNKT cell development. In the periphery, T-KO iNKT cells show reduced TCR signaling upon stimulation with α-galactosylceramide, suggesting that Kidins220 promotes TCR signaling in peripheral iNKT cells. Thus, Kidins220 reduces or promotes signaling dependent on the iNKT cell developmental stage.
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| Overall design |
To perform the single-cell RNA sequencing experiments, cells of 4 thymi of Ctrl mice were pooled and the same was done with 4 thymi of T-KO mice. Cells were stained with CD1d tetramers, anti-TCRβ, anti-NK1.1, anti-CD44 and anti-CD24 antibodies on ice. Cells were sorted by flow cytometry for TCRβ+, CD1dt+, CD24+, NK1.1- cells to obtain stage 0 iNKT cells, and for TCRβ+, CD1dt+, NK1.1+, CD24-, CD44+ cells to obtain stage 3 iNKT cells. This was done twice in parallel, to have two replicas each. After sorting, each cell population was barcoded by hash-tagged antibodies (TotalSeqC format, anti-mouse Hashtag 1 to 8; clone M1/42, 30-F11, BioLegend). The antibody concentrations used were 1 ug per million cells. After staining, cells were washed three times in PBS containing 2% BSA and 0.01% Tween 20, followed by centrifugation (300 x g 5 min at 4 °C) and supernatant exchange. Then all 8 populations were pooled (2x stage 0 Ctrl, 2x stage 0 T-KO, 2x stage 3 Ctrl, 2x stage 3 T-KO) for single-cell RNA sequencing.
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| Contributor(s) |
Sagar S |
| Citation(s) |
38489359 |
| Submission date |
Jan 13, 2024 |
| Last update date |
Mar 19, 2024 |
| Contact name |
Sagar - |
| E-mail(s) |
sagar@uniklinik-freiburg.de
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| Organization name |
University Medical Center Freiburg
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| Department |
Department of Internal Medicine II
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| Lab |
Sagar
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| Street address |
Hugstetter Straße 55
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| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (2) |
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| Relations |
| BioProject |
PRJNA1064406 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE253196_barcodes.tsv.gz |
74.6 Kb |
(ftp)(http) |
TSV |
| GSE253196_features.tsv.gz |
284.2 Kb |
(ftp)(http) |
TSV |
| GSE253196_matrix.mtx.gz |
113.3 Mb |
(ftp)(http) |
MTX |
| GSE253196_nkt_stage0_final.rds.gz |
13.9 Mb |
(ftp)(http) |
RDS |
| GSE253196_nkt_stage3_final.rds.gz |
52.8 Mb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
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