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BCR repertoire analysis of wild-type and Cic-null B-1a cells

ABSTRACT: Purpose: B-1a cells have a distinct BCR repertoire compared with that of B-2 cells. To examine whether CIC loss affects the BCR repertoire in B-1a cells, we analyzed mRNA sequences of immunoglobulin heavy (Igh) and light (Igk and Igl) chain genes in B-1a cells from 12-week-old control and Cicf/f;Cd19-Cre mice. Methods: Peritoneal cavity B-1a cells (IgM+, CD19+, CD5+, CD43+) were sorted by a MoFlo-XDP (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. Long Read iR-Profile Reagent System (iRepertoire) was used to generate NGS libraries covering BCR chains including Igh, Igk, and Igl. Briefly, nested inside and outside primers selectively amplified all V- and C- regions and incorporated communal adaptors. Following clean up, only target amplicons, which contain 5’ and 3’ communal adaptors, were exponentially amplified. Amplified libraries were multiplexed for sequencing on the Illumina Miseq platform. Sequence reads were de-multiplexed according to the barcode sequences. Results: Trimmed reads were mapped to germline V, D and J reference sequences downloaded from the IMGT database. IgH diversity and the usage of variable (V) segments in heavy (Ighv) chain and light (Igkv and Iglv) chain genes were comparable between control and Cic-null B-1a cells. Analysis of non-templated (N)-nucleotide addition at V(D)J junctions revealed that Cic-null B-1a cells have a higher proportion of zero to two N-nucleotides-containing-BCRs than control cells. Conclusions: Our study presents the first comparative BCR repertoire analysis of wild-type and Cic-null B-1a cells. We concluded that CIC deficiency does not dramatically alter the BCR repertoire in B-1a cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE164147 | GEO | 2021/12/16

REPOSITORIES: GEO

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Project description:Developing B lymphocytes undergo V(D)J recombination to assemble germline V, D, and J gene segments into exons that encode the antigen-binding variable region of immunoglobulin (Ig) heavy (H) and light (L) chains. IgH and IgL chains associate to form the B cell receptor (BCR), which upon antigen binding activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. Ability of V(D)J recombination to generate vast primary B cell repertoires results from combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as HTGTS repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or non-productive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions and also for following antibody affinity maturation processes. We employed high-throughput genome-wide translocation sequencing adapted repertoire sequencing (HTGTS-Rep-seq) to study antibody repertoires. For HTGTS-Rep-seq libraries, we utilize bait coding ends of J segments to identify, in unbiased fashion, mouse IgH DJH repertoires [processed tlx files] along with both productive and non-productive IgH V(D)J repertoires from both pro-B and peripheral B cells [processed xls files of samples 1-18, 21-51]. Similarly, we also identify mouse productive and non-productive Igk repertoires from peripheral B cells [processed xls files of samples 19,20,52-57].

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BCR repertoire analysis of wild-type and Cic-null B-1a cells

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