ABSTRACT
A heteromobility duplex tracking assay was developed to analyze B-cell clonality. The assay was based on the genetic variability of B-cell immunoglobulin (Ig) sequences. Binding of amplified (Ig) sequences to a single-stranded radiolabeled Ig DNA probe resulted in the formation of heteroduplexes. The mobilities of these heteroduplexes helped to distinguish clonal B cells.
Abstract
The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rearrangements that occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was provided by the identification of a human acute lymphoblastic leukemic cell line that undergoes spontaneous differentiation from pro-B cell to the pre-B and B-cell stages with concomitant changes in the gene expression profile that normally occur during B-cell differentiation. After confirming the clonality of the progressively differentiating cells, an analysis of immunoglobulin genes and transcripts indicated that pro-B cell members marked by the same DJ rearrangement generated daughter B cells with multiple VH and VL gene segment rearrangements. These findings validate the principle of intraclonal V(D)J diversification during B-cell generation and define a manipulable model of human B-cell differentiation.
The ageing human B cell repertoire: a failure of selection?