5' Rapid Amplification of cDNA Ends and Illumina MiSeq Reveals B Cell Receptor Features in Healthy Adults, Adults With Chronic HIV-1 Infection, Cord Blood, and Humanized Mice.
Details
Serval ID
serval:BIB_B2E36376710D
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
5' Rapid Amplification of cDNA Ends and Illumina MiSeq Reveals B Cell Receptor Features in Healthy Adults, Adults With Chronic HIV-1 Infection, Cord Blood, and Humanized Mice.
Journal
Frontiers in immunology
ISSN
1664-3224 (Print)
ISSN-L
1664-3224
Publication state
Published
Issued date
2018
Peer-reviewed
Oui
Volume
9
Pages
628
Language
english
Notes
Publication types: Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
Publication Status: epublish
Publication Status: epublish
Abstract
Using 5' rapid amplification of cDNA ends, Illumina MiSeq, and basic flow cytometry, we systematically analyzed the expressed B cell receptor (BCR) repertoire in 14 healthy adult PBMCs, 5 HIV-1+ adult PBMCs, 5 cord blood samples, and 3 HIS-CD4/B mice, examining the full-length variable region of μ, γ, α, κ, and λ chains for V-gene usage, somatic hypermutation (SHM), and CDR3 length. Adding to the known repertoire of healthy adults, Illumina MiSeq consistently detected small fractions of reads with high mutation frequencies including hypermutated μ reads, and reads with long CDR3s. Additionally, the less studied IgA repertoire displayed similar characteristics to that of IgG. Compared to healthy adults, the five HIV-1 chronically infected adults displayed elevated mutation frequencies for all μ, γ, α, κ, and λ chains examined and slightly longer CDR3 lengths for γ, α, and λ. To evaluate the reconstituted human BCR sequences in a humanized mouse model, we analyzed cord blood and HIS-CD4/B mice, which all lacked the typical SHM seen in the adult reference. Furthermore, MiSeq revealed identical unmutated IgM sequences derived from separate cell aliquots, thus for the first time demonstrating rare clonal members of unmutated IgM B cells by sequencing.
Keywords
5' Flanking Region/genetics, Adult, Animals, B-Lymphocytes/immunology, Chronic Disease, Clone Cells, Computational Biology, Fetal Blood/physiology, HIV Infections/immunology, HIV-1/physiology, Healthy Volunteers, High-Throughput Nucleotide Sequencing/methods, Humans, Immunoglobulin M/genetics, Mice, Mice, SCID, Mutation/genetics, Receptors, Antigen, B-Cell/genetics, B cell repertoire, HIV, cord blood, deep sequencing, humanized mouse
Pubmed
Web of science
Publisher's website
Open Access
Yes
Create date
09/05/2023 13:59
Last modification date
29/11/2024 18:02