Expression of a targeted lambda 1 light chain gene is developmentally regulated and independent of Ig kappa rearrangements

Abstract

Immunoglobulin light chain (IgL) rearrangements occur more frequently at Ig kappa than at Ig lambda. Previous results suggested that the unrearranged Ig kappa locus negatively regulates Ig lambda transcription and/or rearrangement. Here, we demonstrate that expression of a VJ lambda 1-joint inserted into its physiological position in the Ig lambda locus is independent of Ig kappa rearrangements. Expression of the inserted VJ lambda 1 gene segment is developmentally controlled like that of a VJ kappa-joint inserted into the Ig kappa locus and furthermore coincides developmentally with the occurrence of Ig kappa rearrangements in wild-type mice. We conclude that developmentally controlled transcription of a gene rearrangement in the Ig lambda locus occurs in the presence of an unrearranged Ig kappa locus and is therefore not negatively regulated by the latter. Our data also indicate light chain editing in approximately 30% of lambda 1 expressing B cell progenitors.

Figure 1.

Targeted insertion of a prerearranged VJλ1 gene segment into the germline of the Igλ locus. (a) Overview of the genomic organization of the Igλ germline locus (reference 37). The Igλ locus is composed of three functional Jλ-Cλ clusters (JCλ1–3) and one pseudo Jλ-Cλ cluster (JCλ4). Three Vλ gene segments have been identified; Vλ1, Vλ2, and Vλx. Constant region (C) exons are depicted as hatched boxes, V segments as open boxes, J segments as closed boxes. Arrows indicate DNAse hypersensitive sites (reference 22). Numbers indicate distances between selected exons in kb (reference 38). (b) Partial restriction endonuclease map of the Igλ germline (Igλ GL) locus, the mutated allele after homologous recombination (VJλ1i-ACN) and the mutated allele after Cre-loxP mediated deletion of the neo^R gene containing ACN cassette (VJλ1i). Arms of homology are shown in bold in VJλ1i-ACN. V, J, and C region gene segments are indicated as described in panel a, loxP sites are shown as open triangles. Double headed arrows and associated numbers depict the indicative restriction fragments and their respective sizes as revealed by either an external probe (5′V1) or an internal probe (3′C1). B, BamHI; R, EcoRI. (c) Southern blot analysis of one injected ES cell clone (VJλ1i-ACN), a heterozygous mouse mutant (VJλ1i/+), and a WT littermate (+/+). ES cell or thymic genomic DNA was digested with EcoRI and hybridized with 3′C1.

Figure 2.

All mature B cells of VJλ1i/+ mice express the inserted λ1 light chain. (a) Representative staining for κ and λ1 on CD19⁺ splenocytes from VJλ1i/+ and WT mice. Numbers indicate the percentage of cells per quadrant. (b) Semiquantitative RT-PCR analysis of sorted κ⁺, λ1⁺, and κ/λ1⁺ splenic B cells from WT mice and VJλ1i/+ mice. Testis RNA from WT mice served as negative control. 1:5 serially diluted cDNA was analyzed for reverse-transcribed λ1 and κ light chain message by PCR. A β-actin PCR was performed as internal control.

Figure 3.

Intracellular light chain expression in CD25⁺ pro-B cells and CD25⁻ pre-B cells of light chain insertion and WT mice. Bone marrow lymphocytes were stained for surface expression of B220, IgM, and CD25 and for intracellular light chain expression. Flow cytometric analyses of intracellular κ and λ expression in CD25⁻, B220⁺, IgM⁻ pro-B cells and CD25⁺, B220⁺, IgM⁻ pre-B cells are shown for WT, VJλ1i, D23κi, and LN1κ mice. Light chain (LC) expression is plotted against cell size (forward scatter, FSC). Numbers indicate the percentage of light chain expressing cells. Similar results were obtained in three or more independent experiments.

Figure 4.

Only low levels of Igκ gene rearrangements are detected in λ1⁺ B cells of VJλ1i/+ mice. (a) Partial restriction endonuclease map of the germline Igκ locus (not drawn to scale). V, J, and C gene segments, the 5′Jκ probe and the RS-probe are shown as boxes, the asterisk indicates a pseudo-J segment. The internal κ enhancer (iEκ) is shown as open circle, triangles depict RS recombination sites. The arrows below the V gene segments indicate their transcriptional orientation. The sizes of the germline EcoIRCI fragment and the germline EcoRI fragment are indicated as revealed by probes 5′Jκ and RS, respectively. Grey arrows visualize Vκ→Jκ and RS recombination events. E, EcoIRCI; R, EcoRI. (b and c) Genomic DNA of sorted κ⁺, κ/λ1⁺, and λ1⁺ CD19⁺ B cells from VJλ1i/+ and WT mice was analyzed by Southern blotting. To detect Vκ→Jκ rearrangements, DNA was digested with EcoIRCI and hybridized to 5′Jκ probe (b). RS-rearrangements were analyzed using an EcoRI digest and RS probe (c). Rehybridization with an IL-4 gene-specific probe served as internal control. The percentage of alleles retaining either the Igκ germline (Igκ GL) or the RS germline (RS GL) fragment is shown for each lane. Asterisks indicate fragments that originate from VJ recombination by inversion.

Figure 5.

BrdU incorporation in immature B cells of VJλ1i/+, D23κi/+, and WT mice. Mice were intraperitoneally injected with BrdU and analyzed at different time points thereafter. Immature B cells were defined as B220^low/λ1⁺ and were subdivided according to κ expression. Panel a shows representative histograms from a mouse analyzed 30 h after BrdU injection. The percentage of BrdU⁺ cells in κ⁻ (bottom left panel) and κ⁺ (bottom right panel) B cell subpopulations was determined as shown. (b and c) The percentage of BrdU⁺ immature B cells is plotted against the time after BrdU injection. VJλ1i/+ mice (circles) were compared to either WT mice (squares) (b) or D23κi/+ mice (diamonds) (c). Open symbols correspond to λ1⁺ immature B cells, closed symbols depict κ⁺ immature B cells in WT and D23κi/+ mice or κ/λ1+ immature B cells in VJλ1i/+ mice. Each pair of symbols represents one animal.