Receptor editing occurs frequently during normal B cell development

Abstract

Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that lambda light chain genes usually rearrange after kappa genes. This allowed us to analyze kappa loci in IgMlambda+ cells to determine how frequently in-frame kappa genes fail to suppress lambda gene rearrangements. To do this, we analyzed recombined VkappaJkappa genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the kappa locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged kappa loci, as nearly half (47%) of the RS-inactivated VkappaJkappa joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.

Figure 1

RS rearrangements inactivate and preserve VκJκ joins. A rearranged, potentially functional κ locus (A) can be silenced by two types of RS recombination: Vκ-RS (B) or VκJκ-intron-RS (C). Type C retains the prior VκJκ join, and the RS recombination event eliminates the known cis acting elements that are critical for efficient rearrangement and expression, thus freezing the locus from further VκJκ recombination. Also shown are the intronic recombination sequence 1 (IRS1) (32), the intronic (iE) and 3′ kappa (3′E) enhancers (35, 36), and the recombining sequence (RS) (27, 28) element. Probes IVS (1) and RS 0.8 (27, 28) are indicated by filled boxes.

Figure 2

Sequence analysis of (A) productive and (B) nonproductive VκJκ-intron-RS rearrangements from FACS^® sorted, IgM⁺λ⁺ splenic B cells. Vκ gene family and Jκ gene usage were assigned based on homologies to expressed VκJκ genes (52) or homology searches of Genbank and the Kabat Ig database. Translated Vκ FWR3 (codons 70–88), CDR3, and Jκ5 sequences to the conserved phenylalanine (F) are shown for productive rearrangements, whereas FWR3 and CDR3 sequences are shown for nonproductive rearrangements, with the asterisk (*) adjacent to CDR3 in B denoting an out of frame VκJκ join. The nucleotide sequences of the unrearranged Jκ intronic recombining sequence 1 (IRS1) and RS element (both of which contain a consensus heptamer sequence adjacent to the Δ symbol) are shown above the sequences of the IRS1-RS joins present in each PCR clone. The RS join sequence for clone 17 was not determined. Underlined nucleotides could have been donated by either the IRS1 or the RS sequence, and N region addition (bold) and P-encoded nucleotides are shown between the joins. Repeats denote the number of times a particular sequence was observed.

Figure 3

Sequence analysis of VκJκ- intron-RS rearrangements from IgMλ hybridomas. (A) Sequences of the VκJκ rearrangements. The first digit in the hybridoma name indicates the fusion experiment number. Myeloma fusion partners were either NSO-bcl2 (fusion 1) or SP2/0 (fusions 2–5). Vκ gene family and Jκ gene usage were assigned as described in Fig. 2. P and NP denote productive and nonproductive VκJκ rearrangements, respectively. Translated amino acid sequences of Vκ FWR, CDR, and Jκ sequences to the conserved phenylalanine (F) residue are shown for productive rearrangements, and Vκ FWR and CDR sequences, with * denoting an out of frame VκJκ join and # denoting an in-frame stop codon, are shown for nonproductive rearrangements. These sequence data are available from EMBL/Genbank/DDBJ under accession numbers AF087023–AF087034 and AF087460–AF087467. (B) Sequences of the RS rearrangements (as described in Fig. 2).

Figure 4

“Repair” of intron-RS recombination-silenced VκJκ genes by restoration of Cκ exon and surrounding elements reveals that silenced κL chains can pair with their original μ chain partner. The graph shows representative results from a μκ ELISA comparing several IgMκ transfectoma antibodies (Tfc) to their IgMλ parental hybridoma antibodies (Hyb). Antibodies in supernatants were captured on plastic using adsorbed anti-μ chain antibodies and revealed with anti-κ conjugates. Bars indicate the SD determined from antibodies assayed in triplicate. The concentrations of the hybridoma antibodies were at least 10-fold higher than those of the transfectoma antibodies based on comparison to a TEPC 183 (μ, κ) standard curve.