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. 2008 Feb;28(2):161-70.
doi: 10.1016/j.immuni.2007.12.011. Epub 2008 Feb 7.

Rearrangement of mouse immunoglobulin kappa deleting element recombining sequence promotes immune tolerance and lambda B cell production

José Luis Vela  1 , Djemel Aït-Azzouzene, Bao Hoa Duong, Takayuki Ota, David Nemazee
Affiliations

Rearrangement of mouse immunoglobulin kappa deleting element recombining sequence promotes immune tolerance and lambda B cell production

José Luis Vela et al. Immunity. 2008 Feb.

Abstract

The recombining sequence (RS) of mouse and its human equivalent, the immunoglobulin (Ig) kappa deleting element (IGKDE), are sequences found at the 3' end of the Ig kappa locus (Igk) that rearrange to inactivate Igk in developing B cells. RS recombination correlates with Ig lambda (Iglambda) light (L) chain expression and likely plays a role in receptor editing by eliminating Igk genes encoding autoantibodies. A mouse strain was generated in which the recombination signal of RS was removed, blocking RS-mediated Igk inactivation. In RS mutant mice, receptor editing and self-tolerance were impaired, in some cases leading to autoantibody formation. Surprisingly, mutant mice also made fewer B cells expressing lambda chain, whereas lambda versus kappa isotype exclusion was only modestly affected. These results provide insight into the mechanism of L chain isotype exclusion and indicate that RS has a physiological role in promoting the formation of lambda L chain-expressing B cells.

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Figures

Figure 1
Figure 1
Evaluation of RS recombination in spleen and BM B220+ cells of RS−/− and littermate RS+/+ mice. Four-fold serial dilutions of the indicated DNA samples were subjected to PCR reactions to detect the indicated DNA rearrangements. PCR products were electrophoresed on agarose gels, blotted, and abundance quantitated by southern blot using specific probes. A) Upper panels, PCR detection of RS recombination to JCκ intronic sites (RS to κ intron) and Vκ sites (RS to Vκ). Below is shown recombinations between Vκ and Jκ (Vκ-Jκ) and excision products of Vλ1 to Jλ1 recombination (λ excision product). Similar results were obtained in at least two additional independent experiments. B) Quantitation of λ excision product levels in a sample of 3 mice/group.
Figure 2
Figure 2
Evaluation of reduced λ B cell frequencies in RS mutant mice. B cells from the BM and spleens of RS−/− and RS+/+ mice were stained with antibodies to CD45R (B220), Igκ, and Igλ1–3and analyzed by multicolor flow cytometry. A) Frequencies of cells carrying Igλ 1–3 in the spleen and in newly formed (B220intermediate) and mature (B220high) BM B cells. B) Upper plot shows costaining for κ and λ in BMs of the indicated mice. Lower panels, gating used in A to distinguish newly formed from recirculating sIg+ B cells in BM.
Figure 3
Figure 3
Analysis of anti-dsDNA antibody in sera of the RS mutant and wild type mice. A) Anti-dsDNA titers in seven month old RS+/+ and RS−/− mice. B) Anti-dsDNA titers in 5 month old Bcl2 Tg:RS+/+ and Bcl2 Tg;RS−/− mice. Each dot represents the value obtained from a different individual mouse.
Figure 4
Figure 4
Analysis of IgL-chain expression of RS mutant B cells developing in the presence of a κ superantigen. A-D) Spleen or BM cells of the indicated mice were simultaneously analyzed by multicolor flow cytometry for surface expression of Igλ and cytoplasmic expression of κ (cIgκ). Analyzed cells were gated on lymphocytes unless otherwise indicated.
Figure 5
Figure 5
Analysis of the effect of RS mutation on B cell tolerance in apoptosis-resistant cells. BM chimeras were generated using donor BM from RS-sufficient or RS-mutant mice that also carried a Bcl2 transgene enforcing B lineage restricted expression. Irradiated recipient mice carried a ubiquitously expressed κ-macroself transgene (Aït-Azzouzene et al., 2005) to promote negative selection of κ+ cells. Radiation chimeras were analyzed at 10 weeks post reconstitution. A) Analysis of λ and κ cell frequencies in BM and spleens of the indicated mice. (Note that κ staining in this figure involves surface staining, rather than the cytoplasmic staining of permeabilized cells as shown in Fig. 4A.) B,C) Summary analysis of the κ and λ frequencies of total splenic cells, and newly formed (B220intermediate) and recirculating B cells (B220high) from the indicated chimeras. Note increased κ frequency and reduced λ frequency among BM and spleen cells of κ-macroself recipients of Bcl2 Tg;RS−/− bone marrow. D) Igκ and Igλ serum immunoglobulin levels measured in the indicated chimeric mice.
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