Limits...
The analysis of clonal expansions in normal and autoimmune B cell repertoires.

Hershberg U, Luning Prak ET - Philos. Trans. R. Soc. Lond., B, Biol. Sci. (2015)

Bottom Line: But how are individual clones defined?Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development.We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Engineering, Science and Health Systems, Drexel University, Bossone 7-711, 3141 Chestnut Street, Philadelphia, PA 19104, USA Department of Immunology and Microbiology, College of Medicine, Drexel University, Bossone 7-711, 3141 Chestnut Street, Philadelphia, PA 19104, USA.

ABSTRACT
Clones are the fundamental building blocks of immune repertoires. The number of different clones relates to the diversity of the repertoire, whereas their size and sequence diversity are linked to selective pressures. Selective pressures act both between clones and within different sequence variants of a clone. Understanding how clonal selection shapes the immune repertoire is one of the most basic questions in all of immunology. But how are individual clones defined? Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development. Next, we discuss how clones are defined using different experimental methods. We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape. We discuss methods that visualize sequence variants within the same clone and allow us to consider collections of shared mutations to determine which sequences share a common ancestry. Finally, we comment on features of frequently encountered expanded B cell clones that may be of particular interest in the setting of autoimmunity and other chronic conditions.

No MeSH data available.


Related in: MedlinePlus

Schematic of antibody H and L chain gene rearrangements. The full range of rearrangement products that can be generated at the H chain and L chain loci is shown. In the case of the heavy chain locus (IgH), there are three kinds of rearrangements: D to J rearrangements, V to DJ rearrangements and VH replacements (VHR). Note that all H chain rearrangements are deletional and that once a complete VDJ rearrangement has taken place, all of the D gene segments are consumed. During VH replacement, an upstream VH gene can invade into a pre-existing VDJ rearrangement via a cryptic heptamer (white triangle) that is located in the 3′ end of most VH genes. VH replacement has the potential to elongate the CDR3, because the 3′ end of the preceding VH gene is usually retained in the rearrangement. For the L chain loci (IgL), the kappa locus can undergo primary Vκ–Jκ rearrangement, leapfrogging rearrangement or recombining sequence (RS) deletional rearrangement. In the case of the leapfrogging rearrangement shown, rearrangement of an upstream Vκ gene to a downstream Jκ gene occurred by inversion. Inversional rearrangement retains the original Vκ–Jκ rearrangement on the chromosome in an inverted orientation. This remnant rearrangement is referred to as a reciprocal product. The κ locus can also undergo deletion by rearrangement to RS, a non-coding sequence that is approximately 25 kb downstream of Cκ. RS rearrangement can occur via the cryptic heptamer in the JC intron (i-RS) or by deletional rearrangement of a Vκ gene to RS. Both types of rearrangements inactivate the κ locus by deletion of the constant region exon, Cκ. Finally, lambda (λ) L chain rearrangement can occur. Most λ-expressing B cells have undergone RS deletion on one or both κ alleles. All of these rearrangements can be tracked and used to evaluate clonality, particularly in hybridoma studies (see text). Squares indicate exons, triangles recombination signal sequences, fused triangles represent signal joins and fused boxes indicate coding joins. Dashed lines indicate regions where recombining gene segments come together.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4528416&req=5

RSTB20140239F2: Schematic of antibody H and L chain gene rearrangements. The full range of rearrangement products that can be generated at the H chain and L chain loci is shown. In the case of the heavy chain locus (IgH), there are three kinds of rearrangements: D to J rearrangements, V to DJ rearrangements and VH replacements (VHR). Note that all H chain rearrangements are deletional and that once a complete VDJ rearrangement has taken place, all of the D gene segments are consumed. During VH replacement, an upstream VH gene can invade into a pre-existing VDJ rearrangement via a cryptic heptamer (white triangle) that is located in the 3′ end of most VH genes. VH replacement has the potential to elongate the CDR3, because the 3′ end of the preceding VH gene is usually retained in the rearrangement. For the L chain loci (IgL), the kappa locus can undergo primary Vκ–Jκ rearrangement, leapfrogging rearrangement or recombining sequence (RS) deletional rearrangement. In the case of the leapfrogging rearrangement shown, rearrangement of an upstream Vκ gene to a downstream Jκ gene occurred by inversion. Inversional rearrangement retains the original Vκ–Jκ rearrangement on the chromosome in an inverted orientation. This remnant rearrangement is referred to as a reciprocal product. The κ locus can also undergo deletion by rearrangement to RS, a non-coding sequence that is approximately 25 kb downstream of Cκ. RS rearrangement can occur via the cryptic heptamer in the JC intron (i-RS) or by deletional rearrangement of a Vκ gene to RS. Both types of rearrangements inactivate the κ locus by deletion of the constant region exon, Cκ. Finally, lambda (λ) L chain rearrangement can occur. Most λ-expressing B cells have undergone RS deletion on one or both κ alleles. All of these rearrangements can be tracked and used to evaluate clonality, particularly in hybridoma studies (see text). Squares indicate exons, triangles recombination signal sequences, fused triangles represent signal joins and fused boxes indicate coding joins. Dashed lines indicate regions where recombining gene segments come together.

Mentions: Hybridomas are non-secretory myeloma cells fused to normal B cells [20]. They provide virtually unlimited amounts of genetic material, and because they secrete the antibody of the normal B cell, they provide a facile means of studying that antibody. Because of the abundance of available genetic material, the gene rearrangements at all of the antibody loci can be studied in hybridomas. The variety of antibody gene rearrangements that can be used to identify and track expanded B cell clones are shown in figure 2. To define clonally related hybridomas, one can readily generate full-length sequences of the expressed H and L chains. The analysis of these sequences can yield information about the hypervariable third complementarity-determining region (CDR3), the H + L chain pair, and even about SHMs within the variable region genes of the H and L chains. The sizes of the DNA fragments that contain various gene rearrangements by Southern blotting can be used as another clone-specific genetic feature of hybridomas. One can also perform polymerase chain reactions (PCRs) to genotype the expressed H and L chain rearrangements, and the non-productive or deletional rearrangements (such as non-productive VDJ rearrangements on the non-expressed H chain allele or RS rearrangements at the kappa locus). One can also characterize reciprocal products, such as Vκ–Jκ rearrangements that are retained on the chromosome when a subsequent inversional rearrangement occurs between an upstream Vκ and a downstream Jκ gene segment. Although nearly all of these other rearrangement products are not as diverse as the H chain CDR3, finding concordance among them in two different hybridomas is a powerful indication that the hybridomas are clonally related [21].Figure 2.


The analysis of clonal expansions in normal and autoimmune B cell repertoires.

Hershberg U, Luning Prak ET - Philos. Trans. R. Soc. Lond., B, Biol. Sci. (2015)

Schematic of antibody H and L chain gene rearrangements. The full range of rearrangement products that can be generated at the H chain and L chain loci is shown. In the case of the heavy chain locus (IgH), there are three kinds of rearrangements: D to J rearrangements, V to DJ rearrangements and VH replacements (VHR). Note that all H chain rearrangements are deletional and that once a complete VDJ rearrangement has taken place, all of the D gene segments are consumed. During VH replacement, an upstream VH gene can invade into a pre-existing VDJ rearrangement via a cryptic heptamer (white triangle) that is located in the 3′ end of most VH genes. VH replacement has the potential to elongate the CDR3, because the 3′ end of the preceding VH gene is usually retained in the rearrangement. For the L chain loci (IgL), the kappa locus can undergo primary Vκ–Jκ rearrangement, leapfrogging rearrangement or recombining sequence (RS) deletional rearrangement. In the case of the leapfrogging rearrangement shown, rearrangement of an upstream Vκ gene to a downstream Jκ gene occurred by inversion. Inversional rearrangement retains the original Vκ–Jκ rearrangement on the chromosome in an inverted orientation. This remnant rearrangement is referred to as a reciprocal product. The κ locus can also undergo deletion by rearrangement to RS, a non-coding sequence that is approximately 25 kb downstream of Cκ. RS rearrangement can occur via the cryptic heptamer in the JC intron (i-RS) or by deletional rearrangement of a Vκ gene to RS. Both types of rearrangements inactivate the κ locus by deletion of the constant region exon, Cκ. Finally, lambda (λ) L chain rearrangement can occur. Most λ-expressing B cells have undergone RS deletion on one or both κ alleles. All of these rearrangements can be tracked and used to evaluate clonality, particularly in hybridoma studies (see text). Squares indicate exons, triangles recombination signal sequences, fused triangles represent signal joins and fused boxes indicate coding joins. Dashed lines indicate regions where recombining gene segments come together.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4528416&req=5

RSTB20140239F2: Schematic of antibody H and L chain gene rearrangements. The full range of rearrangement products that can be generated at the H chain and L chain loci is shown. In the case of the heavy chain locus (IgH), there are three kinds of rearrangements: D to J rearrangements, V to DJ rearrangements and VH replacements (VHR). Note that all H chain rearrangements are deletional and that once a complete VDJ rearrangement has taken place, all of the D gene segments are consumed. During VH replacement, an upstream VH gene can invade into a pre-existing VDJ rearrangement via a cryptic heptamer (white triangle) that is located in the 3′ end of most VH genes. VH replacement has the potential to elongate the CDR3, because the 3′ end of the preceding VH gene is usually retained in the rearrangement. For the L chain loci (IgL), the kappa locus can undergo primary Vκ–Jκ rearrangement, leapfrogging rearrangement or recombining sequence (RS) deletional rearrangement. In the case of the leapfrogging rearrangement shown, rearrangement of an upstream Vκ gene to a downstream Jκ gene occurred by inversion. Inversional rearrangement retains the original Vκ–Jκ rearrangement on the chromosome in an inverted orientation. This remnant rearrangement is referred to as a reciprocal product. The κ locus can also undergo deletion by rearrangement to RS, a non-coding sequence that is approximately 25 kb downstream of Cκ. RS rearrangement can occur via the cryptic heptamer in the JC intron (i-RS) or by deletional rearrangement of a Vκ gene to RS. Both types of rearrangements inactivate the κ locus by deletion of the constant region exon, Cκ. Finally, lambda (λ) L chain rearrangement can occur. Most λ-expressing B cells have undergone RS deletion on one or both κ alleles. All of these rearrangements can be tracked and used to evaluate clonality, particularly in hybridoma studies (see text). Squares indicate exons, triangles recombination signal sequences, fused triangles represent signal joins and fused boxes indicate coding joins. Dashed lines indicate regions where recombining gene segments come together.
Mentions: Hybridomas are non-secretory myeloma cells fused to normal B cells [20]. They provide virtually unlimited amounts of genetic material, and because they secrete the antibody of the normal B cell, they provide a facile means of studying that antibody. Because of the abundance of available genetic material, the gene rearrangements at all of the antibody loci can be studied in hybridomas. The variety of antibody gene rearrangements that can be used to identify and track expanded B cell clones are shown in figure 2. To define clonally related hybridomas, one can readily generate full-length sequences of the expressed H and L chains. The analysis of these sequences can yield information about the hypervariable third complementarity-determining region (CDR3), the H + L chain pair, and even about SHMs within the variable region genes of the H and L chains. The sizes of the DNA fragments that contain various gene rearrangements by Southern blotting can be used as another clone-specific genetic feature of hybridomas. One can also perform polymerase chain reactions (PCRs) to genotype the expressed H and L chain rearrangements, and the non-productive or deletional rearrangements (such as non-productive VDJ rearrangements on the non-expressed H chain allele or RS rearrangements at the kappa locus). One can also characterize reciprocal products, such as Vκ–Jκ rearrangements that are retained on the chromosome when a subsequent inversional rearrangement occurs between an upstream Vκ and a downstream Jκ gene segment. Although nearly all of these other rearrangement products are not as diverse as the H chain CDR3, finding concordance among them in two different hybridomas is a powerful indication that the hybridomas are clonally related [21].Figure 2.

Bottom Line: But how are individual clones defined?Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development.We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Engineering, Science and Health Systems, Drexel University, Bossone 7-711, 3141 Chestnut Street, Philadelphia, PA 19104, USA Department of Immunology and Microbiology, College of Medicine, Drexel University, Bossone 7-711, 3141 Chestnut Street, Philadelphia, PA 19104, USA.

ABSTRACT
Clones are the fundamental building blocks of immune repertoires. The number of different clones relates to the diversity of the repertoire, whereas their size and sequence diversity are linked to selective pressures. Selective pressures act both between clones and within different sequence variants of a clone. Understanding how clonal selection shapes the immune repertoire is one of the most basic questions in all of immunology. But how are individual clones defined? Here we discuss different approaches for defining clones, starting with how antibodies are diversified during different stages of B cell development. Next, we discuss how clones are defined using different experimental methods. We focus on high-throughput sequencing datasets, and the computational challenges and opportunities that these data have for mining the antibody repertoire landscape. We discuss methods that visualize sequence variants within the same clone and allow us to consider collections of shared mutations to determine which sequences share a common ancestry. Finally, we comment on features of frequently encountered expanded B cell clones that may be of particular interest in the setting of autoimmunity and other chronic conditions.

No MeSH data available.


Related in: MedlinePlus