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VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma
Published on: December 28, 2015
Four subclones with distinct immunoglobulin light chain phenotypes. (kappa+lambda+, kappa+, lambda+ and
Y Matsuo1, S Nakamura, T Ariyasu
1Fujisaki Cell Center, Hayashibara Biochemical Labs Inc, Okayama, Japan.
Insights
A new human acute lymphoblastic leukemia (ALL) cell line, BALM-9, exhibits simultaneous kappa and lambda light chain expression. Subclones derived from BALM-9 provide a model for studying B cell maturation and double light chain expression.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Acute lymphoblastic leukemia (ALL) is a heterogeneous hematologic malignancy.
- B cell maturation involves complex immunoglobulin (Ig) expression.
- Simultaneous expression of kappa and lambda light chains is a rare phenomenon in B cells.
Purpose of the Study:
- To establish and characterize a novel human ALL cell line, BALM-9.
- To investigate the mechanism of simultaneous kappa and lambda light chain expression.
- To develop a model system for studying B cell maturation stages.
Main Methods:
- Establishment of the BALM-9 cell line from peripheral blood.
- Two-color flow cytometry for immunophenotyping.
- Limiting dilution for subclone isolation.
- Western blotting for protein analysis.
- Cytogenetic and Southern blotting for genetic analysis.
Main Results:
- BALM-9 cells exhibited a mixed immunoglobin (Ig) phenotype with kappa and lambda light chain expression.
- Distinct subclones (BALM-9KL, BALM-9K, BALM-9N) were isolated, representing different light chain expression patterns.
- Cytogenetic and Southern blotting confirmed shared ancestry and a common 14q32 chromosomal rearrangement.
Conclusions:
- The BALM-9 cell line and its subclones offer a valuable model for studying B cell development.
- The findings suggest that kappa lambda double positive B cells may represent a transient stage in normal B cell maturation.
- This model can aid in understanding the mechanisms underlying double light chain expression.
Abstract:
A human acute lymphoblastic leukemia (ALL) cell line, BALM-9, was established from the peripheral blood specimen of a immunoglobin (lg) phenotype, the established BALM-9 cell line expressed both kappa and lambda light (L) chains simultaneously in a range of 30-80%. Two-color flow cytometric analysis demonstrated that there was a distinct population of kappa lambda double positive cells as well as kappa single, lambda single, and double negative populations. Therefore, subclones were obtained from each population by limiting dilution and were designated BALM-9KL (kappa+lambda+), BALM-9K (kappa+lambda-), BALM 9N (kappa-lambda-). Western blotting confirmed the results of the immunofluorescence test at the protein level. In BALM-9N, L chains were absent even in the cytoplasm as demonstrated by Western blotting. Evidence that the subclones have the same ancestry was provided both by cytogenetic analysis and by Southern blotting, which revealed the 14q32 chromosomal rearrangement as a common abnormality and the same IgH gene arrangement among the subclones. The existence of a kappa lambda positive B cell population suggests a transient stage of normal B cell maturation. These subclones might represent such a stage and thus provide a useful means of analyzing the mechanism of this double light chain expression.
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