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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 04, 2024 |
| Title |
Anitbody affinity birth through somatic hypermutation |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
B cell somatic hypermutation (SHM) and selection in germinal center (GC)s enhance antibody affinity to antigen. Conventional understanding holds that SHM enhances pre-existing antibody specificities, limiting the scope of SHM-based antibody evolution to those established in the primary repertoire through V(D)J recombination. By tracking pre-defined non-specific B cells, we observed consistent SHM in non-cognate antibody genes after immunization in settings of diverse, polyclonal B and T cells. Removing B cell competition enabled these non-specific yet somatically mutating antibodies to develop entirely new specificities to diverse antigens. Our findings introduce an updated model, where SHM drives antibody diversification without requiring initial antigen specificity. This reveals that B cell competition, rather than a necessity for specific affinity, limits the emergence of new affinities (termed here as affinity birth) by SHM and highlights the mammalian adaptive immune system’s capacity to explore antibody-antigen interactions beyond the epitopes targeted by the V(D)J-dependent primary antibody repertoire.
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| Overall design |
Different tissues (spleen, mesenteric lymph node and peyer’s patches) of unimmunized mice were harvested, processed and cells were counted. 1-2 x 106 cells, whenever possible, were aliquoted and stained with 0.5 ug of cell hashing antibodies (BioLegend Total-Seq C antibodies, Hashtag 1-9, different hashtag antibodies per tissue sample) after blocking with TruStain FcX™ PLUS (anti-mouse CD16/32, BioLegend, Cat# 156604) according to manufacturer’s protocol. After incubating with hashing antibodies, cells were washed and stained with FACS staining panel as mentioned above - DAPI, Dump (CD4, CD8, F4/80 and Gr1), B220. Total B cells were sorted (upto 50,000 cells per tissue and cells from all three tissues of one mouse were pooled) and proceeded for scRNA-seq library preparation using Chromium GEM-X Single Cell 5' Reagent Kits v3 (10X Genomics) according to the manufacturer’s protocol. Briefly, sorted B cells were pooled, counted, and 33,000 to 50,000 cells were loaded on the GEM-X Chip to run on Chromium X for gel-beads in emulsion (GEMs) generation. Thereafter RT-PCR, cDNA amplification, fragmentation, end repair, A-tailing and sample indexing were performed. Libraries were quantitated and analysed at Tape station (Agilent Bioanalyzer) during intermediate steps of library preparation. Final libraries were quantified by qPCR (KAPA Library Quantification Kit Illumina® Platforms, Kapa Biosystems # KR0405), diluted to 2 nM and pooled. 650 pM of pooled libraries were mixed with 15% of PhiX control library and loaded on NextSeq™ 2000 P4 XLEAP-SBS™ Reagent Kit (100 Cycles, Illumina # 20100994. Libraries were sequenced on Illumina NextSeq™ 2000 sequencer with paired-end reads for 28 cycles of read 1, 10 cycles of i7 index, 10 cycles of i5 index, and 90 cycles of read 2, targeting a median depth of 10,000 reads per cell (5,000 reads per cell for BCR libraries and 5,000 reads per cell for hashtag libraries).
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| Contributor(s) |
Zuo T, Gautam A, Saghaei S, Khobragade SN, Ahmed R, Mahdavinia A, Zarghami M, Pacheco GA, Green K, Travers M, Garcia N, Rao V, Allahyari Z, Kumar S, Novak R, Hwang JK, Wesemann DR |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Nov 28, 2024 |
| Last update date |
Dec 04, 2024 |
| Contact name |
Duane R. Wesemann |
| E-mail(s) |
DWESEMANN@BWH.HARVARD.EDU
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| Organization name |
Brigham and Women's Hospital
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| Department |
Medicine, Division of Allergy and Clinical Immunology
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| Street address |
77 Avenue Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platforms (1) |
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| Samples (8)
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| GSM8655927 |
B cells, B220+, BCR_HAuMT_Set1, BCR |
| GSM8655928 |
B cells, B220+, CSP_B18383_Set2, CSP |
| GSM8655929 |
B cells, B220+, BCR_B18383_Set2, BCR |
| GSM8655930 |
B cells, B220+, CSP_HAuMT_Set2, CSP |
| GSM8655931 |
B cells, B220+, BCR_HAuMT_Set2, BCR |
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| Relations |
| BioProject |
PRJNA1191854 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE283094_RAW.tar |
23.3 Mb |
(http)(custom) |
TAR (of CSV, FASTA, MTX, TSV) |
| GSE283094_WL-46-CSP_fea.csv.gz |
228 b |
(ftp)(http) |
CSV |
| GSE283094_WL-48-CSP_fea.csv.gz |
226 b |
(ftp)(http) |
CSV |
| GSE283094_WL-52-CSP_fea.csv.gz |
228 b |
(ftp)(http) |
CSV |
| GSE283094_WL-54-CSP_fea.csv.gz |
226 b |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
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