Block in Development at the Pre-B-II to Immature B Cell Stage in Mice Without Igκ and Igλ Light Chain

Abstract

Silencing individual C (constant region) genes in a؊/؊ background reduces mature B cell levels, and L chain-deficient ( ؊/؊ ؊/؊ ) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal. (1). L chain genes are encoded by two different loci, and in the mouse there are an extensive number of V gene segments upstream of five J and one C gene (2). The mouse L chain locus contains within an ϳ200-kb region 2 sets of V, J, and C genes that can independently rearrange: V2-Vx-J2-C2-J4-C4 and V1-J3-C3-J1-C1 (3). C1 appears to be predominantly expressed, followed by C2 and then C3, which are less frequently expressed (4), while C4 is not found to be expressed due to a lack of functional splice sites (5). Although the locus is Ͼ10 times larger than the locus, with Ͼ100 V genes, this extensive complexity is not regarded as the reason why most mouse Abs carry a L chain. It may be that the mouse locus is simply more efficient in DNA rearrangement, which is supported by the finding that in the majority of cells with rearranged V the locus is still in germline configuration, while in most cells expressing L chain the locus is either nonproductively rearranged or deleted (6).Several mouse strains with silenced L chain locus have been described. They were generated by homologous integration of a selectable marker gene in C or targeted removal of C or J (7-11). Silencing expression of L chain shed light on isotype exclusion and L chain activation, and it was concluded that and expression are separate and independent events. Although homozygous Ϫ/Ϫ mice compensate for the deficiency with increased production, their splenic B cells and ϩ cells in the bone marrow are reduced compared with those in normal mice (7, 9), which may suggest that L chain rearrangement and expression are relatively inefficient. However, despite the lack of L chain, these mice are healthy and can mount an efficient immune response (11).During B cell development gene segments encoding Ig H chains rearrange first by D (diversity) to J H recombination at the pro-B cell stage. This is followed by V H to D-J H recombination at the pre-B-I stage; if a H chain can pair with a surrogate L chain, consisting of VpreB and 5 protein, this forms a surface expressed pre-B cell receptor (pre-BCR) 3 at the pre-B-II differentiation stage. Ordered rearrangement of Ig genes appears to direct B cell development and provides important checkpoints to establish ...

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain

“…However, it is thought that it may have wider and more fundamental roles, 29 and discussions of its importance have addressed 3 main areas: the phylogenetic pressure retaining LC, 30 developmental regulation, 23 and preserving structural integrity of antibodies by HC and LC association. 31 In early B-cell development, LC pairing with HC displaces the BiP chaperone, which results in cell surface or membrane Ig expression 12,13 essential for B-cell survival and differentiation.…”

“…The derivation of LC-deficient (L Ϫ/Ϫ ), 23 C truncation (knock-in NR), 24 NRL Ϫ/Ϫ , 25 and C H deletion (C⌬) 26 mice has been described. Animals were housed in the Babraham barrier facility, and procedures were carried out under project license PPL 80/1872, approved by the United Kingdom Home Office.…”

Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse

“…Unfortunately, there is no information about pre-B cell development in camelids or whether an H chain without C H 1 can associate with a surrogate L chain to form the pre-BCR necessary to progress B cell development. However, from gene targeting studies in the mouse, it is clear that B cell development without surrogate L chain can progress (42), whereas B cell development without L chain is blocked after H chain expression and maturation up to the immature B cell stage (4).…”

“…Developmental progression is blocked at the pre-B-I cell stage when H chain expression is prevented, although H and/or L chain transcripts may be found (2,3). Likewise, silencing of both -and -L chain loci blocks B cell development, but at the somewhat later pre-B-II stage, which allows normal development up to pre-BCR expression (4). With the lack of L chain, -H chain is retained in the cytoplasm of immature bone marrow B cells, and their further development, with subsequent migration and colonization of the spleen, is prevented (4).…”

“…Likewise, silencing of both -and -L chain loci blocks B cell development, but at the somewhat later pre-B-II stage, which allows normal development up to pre-BCR expression (4). With the lack of L chain, -H chain is retained in the cytoplasm of immature bone marrow B cells, and their further development, with subsequent migration and colonization of the spleen, is prevented (4). Targeted modification of the IgH locus has permitted expression of truncated Ig polypeptides (5), and introduction of Ig transgenes consisting of shorter chains or removed domains has allowed single chain expression (6 -8).…”