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Status |
Public on Feb 21, 2020 |
Title |
Peyer’s Patch Germinal Centers Select Innate-like BCRs |
Organism |
Mus musculus |
Experiment type |
Expression profiling by high throughput sequencing Other
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Summary |
Primary B cell receptor (BCR)/antibody variable region exons are generated by V(D)J recombination with junctional diversification creating immensely diverse antigen contact-encoding CDR3s. While most antigen-driven germinal centers (GCs) are transient, gut microbiota-dependent intestinal Peyer’s patch (PP) GCs are chronic. The nature of chronic PP GC BCR repertoires and somatic hyper-mutation (SHM) patterns has remained enigmatic. To elucidate the physiological repertoire of PP GC BCRs, we developed a high throughput antibody repertoire and SHM assay. Remarkably, PP GCs from different mice expanded public clonotypes, each often with identical IgH CDR3. These CDR3s represent innate-like BCRs that appear much more frequently than expected in naïve B cell repertoire by IGoR modeling, but not frequently enough to enter PP GCs at the observed recurrence without cellular selection. Consistently, some public clonotypes are gut microbiota-dependent and encode antibodies reactive to bacteria glycans, while others are not. SPF fecal transfer to germ-free (GF) mice restored two germ-dependent clonotypes, providing direct evidence for BCR selection. In support of this, we identified recurrently selected SHMs in four of the public clonotypes, demonstrating affinity maturation in chronic PP GCs. Our findings suggest that persistent gut antigens select for innate-like BCR clonotypes to seed chronic PP GCs.
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Overall design |
We validated our primer design for LAM-HTGTS-Rep-SHM-Seq with mixed JH or JL bait compared with VH or VL bait, with 3 replicates each. We included NP-CGG and SRBCs IP immunized splenic GC and naïve samples as controls for our LAM-HTGTS-Rep-SHM-Seq method to follow BCR selection. We did LAM-HTGTS-Rep-SHM-Seq on PP GC and naïve samples from 18 SPF mice, 10x3 GF mice, 5 GF+SFB mice, 10 GF+SPF mice, 15 AID-deficient SPF mice. We also included single cell RNA sequencing data from 4 of the PP GC samples from SPF mice.
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Contributor(s) |
Chen H, Zhang Y, Ye AY, Du Z, Xu M, Lee C, Hwang JK, Kyritsis N, Ba Z, Littman DR, Alt FW |
Citation(s) |
32499646 |
Submission date |
Nov 21, 2019 |
Last update date |
Jun 10, 2020 |
Contact name |
Frederick W Alt |
Organization name |
Boston Children's Hospital / Harvard Medical School
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Department |
Program in Cellular and Molecular Medicine (PCMM)
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Lab |
Frederick Alt
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Street address |
1 Blackfan Circle
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City |
Boston |
State/province |
MA |
ZIP/Postal code |
02115 |
Country |
USA |
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Platforms (2) |
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Samples (225)
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Relations |
BioProject |
PRJNA591005 |
SRA |
SRP231174 |
Supplementary file |
Size |
Download |
File type/resource |
GSE140795_RAW.tar |
1.3 Gb |
(http)(custom) |
TAR (of VLOUPE, XLS) |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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