Studying the Replication History of Human B Lymphocytes by Real-Time Quantitative (RQ-)PCR

The cells of the adaptive immune system, B and T lymphocytes, each generate a unique antigen receptor through V(D)J recombination of their immunoglobulin (Ig) and T-cell receptor (TCR) loci, respectively. Such rearrangements join coding elements to form a coding joint and delete the intervening DNA as circular excision products containing the signal joint. These excision circles are stable structures that cannot replicate and have no function in the cell. Since the coding joint in the genome is replicated with each cell division, the ratio between coding joints and signal joints in a population of B cells can be used as a measure for proliferation. This chapter describes a real-time quantitative (RQ-)PCR-based approach to quantify proliferation through calculating the ratio between coding joints and signal joints of the frequently occurring intronRSS-Kde rearrangements in the IGK light chain locus. Besides its use in normal B-cell biology, quantification of B-cell replication can inform on abnormal proliferation in human diseases and in B-cell neogenesis following depletion therapy.