Molecular characterization of transgene-induced immunodeficiency in B-less mice using a novel quantitative limiting dilution polymerase chain reaction method

Abstract

B-less mice express a human immunoglobulin (Ig)λ transgene that induces a severe deficiency of both immature pre-B and mature B lymphocytes. To understand this perturbation in B lymphopoiesis, we have devised a sensitive limiting dilution polymerase chain reaction assay that quantitates specific Ig rearrangements and thus quantitates B lineage cells at various stages of differentiation within unfractionated bone marrow. We find that there are significantly reduced frequencies of both VH-to-DJH and VK-to-JK rearrangements in the transgenic strain, whereas the frequency of D-to-JH rearrangements approximates that of wild type. Since Ig gene rearrangements occur in a stepwise fashion in which D-to-JH joining precedes that of VH-to-DJH and VK-to-JK, these results indicate that the major block of B lymphocyte development in the B-less strain occurs after D-to-JH rearrangement. Interestingly, sequence analysis of residual VHDJH junctions from transgenic pre-B lymphocytes reveals that an abnormally high proportion of these are out of frame and therefore nonproductive. Taken together, these data suggest that early expression of the transgenic λ protein specifically prevents the development of a normal-sized population of precursor B lymphocytes coexpressing functional IgH. The transgene-induced immunodeficiency appears to arise by a precocious maturation process in which precursors bypass a developmental stage associated with cellular expansion.