Analysis of the B-cell progenitor compartment at the level of single cells

Andreas Ehlich, Verena Martin, Werner Müller, Klaus Rajewsky

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Background: During B-cell development in the mouse, the VH, DH and JH elements of the immunoglobulin heavy chain (IgH) locus are rearranged, firstly by DH-JH joining, and then by VH-DHJH joining. In-frame ('productive') VHDHJH joints and DHJH joints in reading frame 2 (one of the three possible DH reading frames) allow the expression of μ and truncated μ chains (Dμ proteins), respectively. The expression of such molecules from one of the two IgH loci of a cell is thought to interfere with VH-DHJH recombination on the other IgH locus, and to guide the cells through further development. Results: We have developed a gene amplification assay that permits the examination of rearranged immunoglobulin genes in single cells. Using this assay, we monitored cells bearing DHJH and/or VHDHJH joints at early stages of development: in CD43+ B-cell progenitors, subdivided into fractions A, B, C and C′ by flow cytometry, and in CD43- pre-B cells (fraction D). Fraction C was enriched for cells with two non-productive VHDHJH joints. Cells containing both a DHJH joint in DH reading frame 2 and a VHDHJH joint were not seen in any fraction. All fraction D cells harbored an in-frame VHDHJH joint. Cells with two productive VHDHJH joints appear to be selected against throughout development. Conclusions: Cells expressing Dμ proteins appear to be arrested in development as a result of inhibited VH-DHJH joining. Expression of the μ chain is required for maturation into CD43- pre-B cells; accordingly, cells carrying two non-productive VHDHJH joints accumulate in the CD43+ compartment. Such a developmental arrest may also affect cells that express self-reactive VHDHJH antibody domains. Our results indicate further that allelic exclusion at the IgH locus is already established at the pre-B cell stage.
    Original languageEnglish
    Pages (from-to)573-583
    Number of pages10
    JournalCurrent Biology
    Volume4
    Issue number7
    Publication statusPublished - 1994

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