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Strategies for B-cell receptor repertoire analysis in primary immunodeficiencies: from severe combined immunodeficiency to common variable immunodeficiency.

IJspeert H, Wentink M, van Zessen D, Driessen GJ, Dalm VA, van Hagen MP, Pico-Knijnenburg I, Simons EJ, van Dongen JJ, Stubbs AP, van der Burg M - Front Immunol (2015)

Bottom Line: Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before.Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection.In this perspective, we describe strategies and considerations for analysis of the naive and antigen-selected B-cell repertoires in primary immunodeficiency patients with a focus on severe combined immunodeficiency and common variable immunodeficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam , Rotterdam , Netherlands.

ABSTRACT
The antigen receptor repertoires of B- and T-cells form the basis of the adaptive immune response. The repertoires should be sufficiently diverse to recognize all possible pathogens. However, careful selection is needed to prevent responses to self or harmless antigens. Limited antigen receptor repertoire diversity leads to immunodeficiency, whereas unselected or misdirected repertoires can result in autoimmunity. The antigen receptor repertoire harbors information about abnormalities in many immunological disorders. Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before. Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection. In this perspective, we describe strategies and considerations for analysis of the naive and antigen-selected B-cell repertoires in primary immunodeficiency patients with a focus on severe combined immunodeficiency and common variable immunodeficiency.

No MeSH data available.


Related in: MedlinePlus

V(D)J recombination and B-cell development. (A) Schematic overview of V(D)J recombination. DNA double strand breaks are introduced by the RAG proteins, subsequently the DNA is processed and ligated by DNA repair proteins from the NHEJ pathway. (B) Examples of V(D)J junctions showing junctional diversity by nucleotides that are removed, non-templated (N) nucleotides (blue) that are added, or presence of palindromic (P) nucleotides (red). (C) The B-cell repertoire in peripheral blood can be divided into the naive repertoire and the antigen-selected repertoire.
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Figure 1: V(D)J recombination and B-cell development. (A) Schematic overview of V(D)J recombination. DNA double strand breaks are introduced by the RAG proteins, subsequently the DNA is processed and ligated by DNA repair proteins from the NHEJ pathway. (B) Examples of V(D)J junctions showing junctional diversity by nucleotides that are removed, non-templated (N) nucleotides (blue) that are added, or presence of palindromic (P) nucleotides (red). (C) The B-cell repertoire in peripheral blood can be divided into the naive repertoire and the antigen-selected repertoire.

Mentions: The antigen receptor repertoire is defined as the total set of different B-cell (BR) or T-cell receptors (TRs). The loci encoding these receptors consist of multiple variable (V), diversity (D), and joining (J) genes, which can be recombined via V(D)J recombination to ensure the enormous diversity of the antigen receptors. V(D)J recombination starts with induction of DNA double strand breaks (DSBs) by the recombination-activating gene products RAG1 and RAG2 between the coding element and the recombination signal sequence (Figure 1A) (1). The DNA ends that contain the recombination signal sequence, the so-called signal ends, are blunt DNA ends, which can be ligated directly to form the signal joint. The other ends (called the coding ends because they contain the coding sequence of the Ig or TR gene) are blocked by a covalent phosphodiester bond between the top and the bottom strand of the DNA. These DNA hairpins are recognized, processed, and repaired by the non-homologous end joining pathway (NHEJ) (Figure 1A). First, the Ku70/80 heterodimer forms a ring around the DNA end that can migrate into the DNA after initial binding. Ku70/80 bound to a DNA end can then attract the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), which acquires protein kinase activity upon DNA end binding. DNA-PKCS autophosphorylation induces a conformational change in the DNA-bound complex of Ku70/80 and DNA-PKCS, collectively called the DNA-PK complex (2, 3). After this conformational change, Artemis opens the DNA hairpins (4, 5). If the ends are compatible, they can be ligated by ligase IV, which forms a stable complex with XRCC4. XLF (XRCC4-like factor), which has also been called “Cernunnos” (6). Before ligation, non-templated (N) nucleotides can be inserted by terminal deoxynucleotidyl transferase (TdT) or deleted via exonuclease activity (Figure 1B) (7, 8). In addition to the NHEJ components, several other factors are required to ensure efficient ligation of so-called “difficult breaks” or “complex DNA damage.” Extensive analysis of DSB repair kinetics revealed that these DSBs are mainly localized to heterochromatin and require opening of the closed chromatin structure in order to be repaired by NHEJ during the G1 phase of the cell cycle or HR in the G2 phase. Chromatin opening probably requires the initial phosphorylation of histone H2AX, the ATM kinase, the MRE11/RAD50/Nijmegen breakage syndrome (NBS)1 complex, and several enzymes necessary for ubiquitin addition near the DSB, including RNF8 and RNF168 (9–11).


Strategies for B-cell receptor repertoire analysis in primary immunodeficiencies: from severe combined immunodeficiency to common variable immunodeficiency.

IJspeert H, Wentink M, van Zessen D, Driessen GJ, Dalm VA, van Hagen MP, Pico-Knijnenburg I, Simons EJ, van Dongen JJ, Stubbs AP, van der Burg M - Front Immunol (2015)

V(D)J recombination and B-cell development. (A) Schematic overview of V(D)J recombination. DNA double strand breaks are introduced by the RAG proteins, subsequently the DNA is processed and ligated by DNA repair proteins from the NHEJ pathway. (B) Examples of V(D)J junctions showing junctional diversity by nucleotides that are removed, non-templated (N) nucleotides (blue) that are added, or presence of palindromic (P) nucleotides (red). (C) The B-cell repertoire in peripheral blood can be divided into the naive repertoire and the antigen-selected repertoire.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389565&req=5

Figure 1: V(D)J recombination and B-cell development. (A) Schematic overview of V(D)J recombination. DNA double strand breaks are introduced by the RAG proteins, subsequently the DNA is processed and ligated by DNA repair proteins from the NHEJ pathway. (B) Examples of V(D)J junctions showing junctional diversity by nucleotides that are removed, non-templated (N) nucleotides (blue) that are added, or presence of palindromic (P) nucleotides (red). (C) The B-cell repertoire in peripheral blood can be divided into the naive repertoire and the antigen-selected repertoire.
Mentions: The antigen receptor repertoire is defined as the total set of different B-cell (BR) or T-cell receptors (TRs). The loci encoding these receptors consist of multiple variable (V), diversity (D), and joining (J) genes, which can be recombined via V(D)J recombination to ensure the enormous diversity of the antigen receptors. V(D)J recombination starts with induction of DNA double strand breaks (DSBs) by the recombination-activating gene products RAG1 and RAG2 between the coding element and the recombination signal sequence (Figure 1A) (1). The DNA ends that contain the recombination signal sequence, the so-called signal ends, are blunt DNA ends, which can be ligated directly to form the signal joint. The other ends (called the coding ends because they contain the coding sequence of the Ig or TR gene) are blocked by a covalent phosphodiester bond between the top and the bottom strand of the DNA. These DNA hairpins are recognized, processed, and repaired by the non-homologous end joining pathway (NHEJ) (Figure 1A). First, the Ku70/80 heterodimer forms a ring around the DNA end that can migrate into the DNA after initial binding. Ku70/80 bound to a DNA end can then attract the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), which acquires protein kinase activity upon DNA end binding. DNA-PKCS autophosphorylation induces a conformational change in the DNA-bound complex of Ku70/80 and DNA-PKCS, collectively called the DNA-PK complex (2, 3). After this conformational change, Artemis opens the DNA hairpins (4, 5). If the ends are compatible, they can be ligated by ligase IV, which forms a stable complex with XRCC4. XLF (XRCC4-like factor), which has also been called “Cernunnos” (6). Before ligation, non-templated (N) nucleotides can be inserted by terminal deoxynucleotidyl transferase (TdT) or deleted via exonuclease activity (Figure 1B) (7, 8). In addition to the NHEJ components, several other factors are required to ensure efficient ligation of so-called “difficult breaks” or “complex DNA damage.” Extensive analysis of DSB repair kinetics revealed that these DSBs are mainly localized to heterochromatin and require opening of the closed chromatin structure in order to be repaired by NHEJ during the G1 phase of the cell cycle or HR in the G2 phase. Chromatin opening probably requires the initial phosphorylation of histone H2AX, the ATM kinase, the MRE11/RAD50/Nijmegen breakage syndrome (NBS)1 complex, and several enzymes necessary for ubiquitin addition near the DSB, including RNF8 and RNF168 (9–11).

Bottom Line: Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before.Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection.In this perspective, we describe strategies and considerations for analysis of the naive and antigen-selected B-cell repertoires in primary immunodeficiency patients with a focus on severe combined immunodeficiency and common variable immunodeficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Erasmus MC, University Medical Center Rotterdam , Rotterdam , Netherlands.

ABSTRACT
The antigen receptor repertoires of B- and T-cells form the basis of the adaptive immune response. The repertoires should be sufficiently diverse to recognize all possible pathogens. However, careful selection is needed to prevent responses to self or harmless antigens. Limited antigen receptor repertoire diversity leads to immunodeficiency, whereas unselected or misdirected repertoires can result in autoimmunity. The antigen receptor repertoire harbors information about abnormalities in many immunological disorders. Recent developments in next generation sequencing allow the analysis of the antigen receptor repertoire in much greater detail than ever before. Analyzing the antigen receptor repertoire in patients with mutations in genes responsible for the generation of the antigen receptor repertoire will give new insights into repertoire formation and selection. In this perspective, we describe strategies and considerations for analysis of the naive and antigen-selected B-cell repertoires in primary immunodeficiency patients with a focus on severe combined immunodeficiency and common variable immunodeficiency.

No MeSH data available.


Related in: MedlinePlus