ANALYSIS OF THE B-CELL PROGENITOR COMPARTMENT AT THE LEVEL OF SINGLE CELLS

Background: During B-cell development in the mouse, the V-H, D-H and J (H) elements of the immunoglobulin heavy chain (IgH) locus are rearran ged, firstly by D-H-J(H) joining, and then by V-H-D(H)J(H) joining. In -frame ('productive') V(H)D(H)J(H) joints and D(H)J(H) joints in readi ng frame 2 (one of the three possible D-H reading frames) allow the ex pression of mu and truncated mu chains (D mu proteins), respectively. The expression of such molecules from one of the two IgH loci of a cel l is thought to interfere with V-H-D(H)J(H) recombination on the other IgH locus, and to guide the cells through further development. Result s: We have developed a gene amplification assay that permits the exami nation of rearranged immunoglobulin genes in single cells. Using this assay, we monitored cells bearing D(H)J(H) and/or V(H)D(H)J(H) joints at early stages of development: in CD43(+) B-cell progenitors, subdivi ded into fractions A, B, C and C' by flow cytometry, and in CD43(-) pr e-B cells (fraction D). Fraction C was enriched for cells with two non -productive V(H)D(H)J(H) joints. Cells containing both a D(H)J(H) join t in D-H reading frame 2 and a V(H)D(H)J(H) joint were not seen in any fraction. All fraction D cells harbored an in-frame V(H)D(H)J(H) join t. Cells with two productive V(H)D(H)J(H) joints appear to be selected against throughout development. Conclusions: Cells expressing D mu pr oteins appear to be arrested in development as a result of inhibited V -H-D(H)J(H) joining. Expression of the mu chain is required for matura tion into CD43(-) pre-B cells; accordingly, cells carrying two non-pro ductive V(H)D(H)J(H) joints accumulate in the CD43(+) compartment. Suc h a developmental arrest may also affect cells that express self-react ive V(H)D(H)J(H) antibody domains. Our results indicate further that a llelic exclusion at the IgH locus is already established at the pre-B cell stage.