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| Status |
Public on Apr 16, 2021 |
| Title |
Coupled analysis of transcriptional states and somatic hypermutation in germinal center B cells reveals role for oxidative phosphorylation in positive selection |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
In this work we illustrate the power of coupling single cell analysis of transcriptional states with somatic hypermutation of germinal center B cells to reveal unanticipated roles of metabolic programs in the positive selection. We use massively parallel 5'-end scRNA-seq to simultaneously capture transcriptome states and mutation profiles of IgH variable genes in individual cells. Our computational analyses reveal that the OXPHOS module is enhanced in GC B cells undergoing positive selection in response to different antigens. Through deleting the Cox10 gene, which encodes a cytochrome oxidase assembly factor, specifically in GC B cells, we demonstrate that OXPHOS activity is required for cell division and positive selection. Furthermore, we show that chemical augmentation of OXPHOS activity facilitates the positive selection of GC B cells in vivo. Our findings therefore reveal that tuning of OXPHOS activity by increased affinity BCRs is critical for clonal expansion and positive selection.
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| Overall design |
Six to eight weeks old C56BL/6J (Jax 000664) mice were immunized intraperitoneally with 100 μg NP(23)-KLH (Biosearch Technologies) or OVA (Sigma) mixed with 50% (v/v) Alum (ThermoFisher Scientific) and 1 μg LPS (Sigma). NP-specific GC B cells were sorted as 7AAD–B220+FashiGL-7hiNP+ on day 13 post immunization using MA900 (Sony) with 100 μm Chip at 4 °C. For the immunization with OVA, total GC B cells were sorted as 7AAD–B220+FashiGL-7hi for scRNA-seq. Single cells were captured via the GemCode Single Cell Platform using the GemCode Gel Bead, Chip and 5’-end Library Kits (10X Genomics), according to the manufacturer’s protocol. Libraries were sequenced on a NovaSeq (Illumina). Additionally, OVA-binding GC B cells were sorted for bulk BCR library generation and sequenced on Miseq (Illumina).
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| Web link |
https://doi.org/10.1038/s41590-021-00936-y
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| Contributor(s) |
Chen D, Wang Y, Fu S, Singh H, Xu H |
| Citation missing |
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| |
| Submission date |
Jul 17, 2020 |
| Last update date |
May 25, 2021 |
| Contact name |
Heping Xu |
| E-mail(s) |
xuheping@westlake.edu.cn
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| Organization name |
Westlake University
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| Street address |
18 Shilongshan Road
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| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310024 |
| Country |
China |
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| Platforms (2) |
| GPL16417 |
Illumina MiSeq (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (22)
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| Relations |
| BioProject |
PRJNA646919 |
| SRA |
SRP272363 |
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