AID is also required for V exon somatic hypermutation (SHM), which typically occurs in activated germinal center (GC) B cells ( 5, 6). DNA cleavage and repair events can result in IgH class switch recombination (CSR), where removal of C H region DNA positions alternative C Hs (e.g., Cγ, Cε, Cα) downstream of the V exon. Upon activation, B cells can undergo two other forms of diversification, both initiated by activation-induced cytidine deaminase (AID). Mature naïve B cells express both IgM and IgD due to alternative C H splicing of C µ and C δ. Productive assembly of both IgH and IgL chains results in IgM expression on the surface of immature B cells, forming the antigen-binding part of the BCR. Non-productive Ig sequences that appear in sequence data sets can be identified as such in the data processing stage. Although the IgH and IgL alleles that assemble non-productively do not produce protein, they are transcribed to contribute to the mRNA pool of the cell. This process results in B cells monoallelically expressing one B cell receptor (BCR) specificity, although rare cells expressing IgH from two alleles, as well as both Igκ and Igλ, have been observed as well ( 3, 4). In this regard, if a V exon assembly attempt does not result in a productive reading frame, a subsequent attempt occurs on the sister allele. V(D)J recombination usually occurs in an allelically ordered way. V(D)J recombination is dependent upon Rag1 and Rag2, occurs at the IgH locus before the IgL loci, and Igκ is usually attempted before Igλ assembly. Non-templated (N) and palindromic (P) nucleotides are added to inter-segment junctions, further adding to the diversity. There are two IgL loci-namely, Igκ and Igλ-which have their own pools of tandemly arranged V L and J L gene segments that are assembled by VJ recombination in precursor (pre-) B cells after productive IgH assembly ( 1, 2). The IgH locus contains many related, but distinct V H, D H, and J H gene segments, which are genomically organized in tandem and selected in a semi-random process for somatic V(D)J assembly in bone marrow progenitor (pro-) B cells.
The IgH V-region is encoded by an exon that is generated somatically from assembly of three gene segments, named variable (also abbreviated as V, not to be confused with the V segment-containing V exon), diversity (D), and joining (J) gene segments. Antibodies are composed of a combination of two identical heavy (H) and two identical light (L) immunoglobulin (Ig) chains, each with variable (V) and constant (C) regions. Because BCR analysis harbors additional complexities, such as immunoglobulin (Ig) (i.e., antibody) gene somatic hypermutation and class switch recombination, the emphasis of this review is on Ig/BCR sequence analysis.Īnalysis and interpretation of antibody repertoire data require an understanding of the complex processes of somatic antigen receptor gene dynamics. We discuss the general steps in the process of immune repertoire generation including sample preparation, platforms available for sequencing, processing of sequencing data, measurable features of the immune repertoire, and the statistical tools that can be used for analysis and interpretation of the data. Here, we review current methods and challenges of library preparation, sequencing and statistical analysis of lymphocyte receptor repertoire studies. In this regard, sample preparation and sequence analysis techniques, which have largely been borrowed and adapted from other fields, continue to evolve. With these opportunities have come significant challenges in understanding the analysis techniques that most accurately reflect underlying biological phenomena. The advent of efficient high-throughput sequencing of lymphocyte antigen receptor genes has recently generated unprecedented opportunities for exploration of adaptive immune responses.
Somatic assembly of T cell receptor and B cell receptor (BCR) genes produces a vast diversity of lymphocyte antigen recognition capacity. Division of Rheumatology, Department of Medicine, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States.
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