Conserved cryptic recombination signals in Vκ gene segments are cleaved in small pre-B cells

The cleavage of recombination signals (RS) at the boundaries of immunoglobulin V, D, and J gene segments initiates the somatic generation of the antigen receptor genes expressed by B lymphocytes. RS contain a conserved heptamer and nonamer motif separated by non-conserved spacers of 12 or 23 nucleotides. Under physiologic conditions, V(D)J recombination follows the "12/23 rule" to assemble functional antigen-receptor genes, i.e., cleavage and recombination occur only between RS with dissimilar spacer types. Functional, cryptic RS (cRS) have been identified in V_H gene segments; these V_H cRS were hypothesized to facilitate self-tolerance by mediating V_H → V_HDJ_H replacements. At the Igκ locus, however, secondary, de novo rearrangements can delete autoreactive VκJκ joins. Thus, under the hypothesis that V-embedded cRS are conserved to facilitate self-tolerance by mediating V-replacement rearrangements, there would be little selection for Vκ cRS. Recent studies have demonstrated that V_H cRS cleavage is only modestly more efficient than V(D)J recombination in violation of the 12/23 rule and first occurs in pro-B cells unable to interact with exogenous antigens. These results are inconsistent with a model of cRS cleavage during autoreactivity-induced V_H gene replacement.

To test the hypothesis that cRS are absent from Vκ gene segments, a corollary of the hypothesis that the need for tolerizing V_H replacements is responsible for the selection pressure to maintain V_H cRS, we searched for cRS in mouse Vκ gene segments using a statistical model of RS. Scans of 135 mouse Vκ gene segments revealed highly conserved cRS that were shown to be cleaved in the 103/BCL2 cell line and mouse bone marrow B cells. Analogous to results for V_H cRS, we find that Vκ cRS are conserved at multiple locations in Vκ gene segments and are cleaved in pre-B cells.

Our results, together with those for V_H cRS, support a model of cRS cleavage in which cleavage is independent of BCR-specificity. Our results are inconsistent with the hypothesis that cRS are conserved solely to support receptor editing. The extent to which these sequences are conserved, and their pattern of conservation, suggest that they may serve an as yet unidentified purpose.