We previously described a check-point for allelic exclusion that occurs at the pre-B to immature B cell transition and is dependent upon IgH intronic enhancer Eμ. combinations with superior signaling properties can overcome the signaling defect associated with low Igμ-chain and eliminate the selective advantage of “double-producers” that accomplish higherIgμ-chain levels through expression of a second IgH allele. Finally we found that “double-producers” in Eμ-deficient mice include a subpopulation with autoreactive BCRs. We infer that BCRs with IgH chain from your Eμ-deficient allele are ignored during unfavorable selection due to their comparatively low density. In summary these studies show Eμ’s effect on IgH levels at the pre-B to immature B cell transition strongly influences allelic exclusion the breadth of the mature BCR repertoire and the emergence of autoimmune B cells. INTRODUCTION B lymphocytes develop from progenitor cells in mouse bone marrow (BM) through sequential rearrangements of immunoglobulin heavy (locus has been shown to be essential for efficient heavy chain variable region (VH) gene assembly and also enhances the transcription of IgH genes (26 27 In previous studies we circumvented the need for G-749 Eμ in VHgene assembly to study its functions after this process (28 29 To do this we produced an Eμ-deficient allele with a pre-assembled heavy chain variable region gene (B1-8VH) knocked into the endogenous locus (VHΔa Fig. 1). We found that in pre-B cells this allele was expressed at half the level of an identical but Eμ-intact allele (VHEμa) resulting in ~? normal cytoplasmic Igμ levels (28). We proposed that this reduction in Igμ expression caused a decrease in the surface density of newly emerging BCRs thereby reducing BCR-mediated signals and the likelihood of transition to the immature B cell stage. Supporting this hypothesis was our finding that mature splenic B cells expressing Igμ from only the Eμ-deficient IgH allele (VHΔa single-producers) experienced undergone unusually considerable light-chain editing the process that has been described previously as a means by which an emerging B cell replaces its autoreactive receptor with an innocuous one(28 30 We suggested that in this case however light-chain editing was occurring because of weak BCR signals (low Igμ) that were insufficient to indicate formation of a functional BCR and thus turn off the recombination machinery (the recombination-activating genes RAG-1 and RAG-2). Only when a light chain was found that could combine with the B1-8Igμ-chain and somehow increase the BCR transmission beyond the threshold for positive selection would an individual pre-B cell transit to the immature B cell stage. Three Rabbit polyclonal to AMPK gamma1. predictions of this hypothesis are that B1-8 H-chainBCR signals in developing pre-B cells of VHΔa/WTb mice are of lower mean strength than their counterparts in VHEμa/WTb animals that this results in less efficient generation of the immature B cell pool and that the rate of the pre-B to immature B cell transition in these Eμ-deficient animals should be responsive to changes in Ig light-chain availability and structure. We test these predictions in the current study. Physique 1 Diagram of wild-type (WTb) and B1-8VH knock-in loci with and without Eμ (designated VHEμa and VHΔa respectively) Another striking feature of mice heterozygous for the Eμ-deficient allele (VHΔa/WTb) was a defect in allelic exclusion in both the immature and mature B cell pools(28). Approximately 20% splenic B cells expressed Igμ from both the VHΔa knock-in allele G-749 and a functionally-rearranged WTb allele (“double-producers”). We found that the B1-8VH knock-in G-749 around the Eμ-deficient allele to the same extent (28). Rare pre-B cells that circumvented this inhibition were present in both VHΔa/WTb and VHEμa/WTb animals. However thesegave rise to a significant quantity of “double-producers” immature B cells only in the VHΔa/WTb mice but not in the VHEμa/WTb animals exposing an Eμ-dependent “check-point” for IgH allelic exclusion at the pre-B to immature B cell transition. We suggest that this in fact is usually another manifestation of the inability of the VHΔa allele to produce Igμ and therefore BCR at levels sufficient for positive selection. Since the functionally put together IgH gene around the WTb allele retains Eμ it allows for BCR levels sufficient for positive selection putting such double-producers at a selective advantage over single-producers (no Eμ) in VHΔa/WTb mice. In the G-749 current study we have tested this hypothesis by asking whether genetic manipulations that augment the pre-B to immature B.