Multiple, conserved cryptic recombination signals in VH gene segments: detection of cleavage products only in pro B cells

Abstract

Receptor editing is believed to play the major role in purging newly formed B cell compartments of autoreactivity by the induction of secondary V(D)J rearrangements. In the process of immunoglobulin heavy (H) chain editing, these secondary rearrangements are mediated by direct V(H)-to-J(H) joining or cryptic recombination signals (cRSs) within V(H) gene segments. Using a statistical model of RS, we have identified potential cRSs within V(H) gene segments at conserved sites flanking complementarity-determining regions 1 and 2. These cRSs are active in extrachromosomal recombination assays and cleaved during normal B cell development. Cleavage of multiple V(H) cRSs was observed in the bone marrow of C57BL/6 and RAG2:GFP and microMT congenic animals, and we determined that cRS cleavage efficiencies are 30-50-fold lower than a physiological RS. cRS signal ends are abundant in pro-B cells, including those recovered from microMT mice, but undetectable in pre- or immature B cells. Thus, V(H) cRS cleavage regularly occurs before the generation of functional preBCR and BCR. Conservation of cRSs distal from the 3' end of V(H) gene segments suggests a function for these cryptic signals other than V(H) gene replacement.

Figure 1.

The proportion of RS-length sequences with RIC scores above a given threshold. The number of RS-length sequences with RIC greater than the score indicated on the x axis was divided by the number of all RS-length segments begining with CA. The resulting proportion is indicated on the y axis. Results for 28-bp segments (top) and for 39-bp segments are shown (bottom). Filled circles (•) indicate the proportion for segments in the orientation of physiological RS (O1), and open triangles (▵) indicate the proportion in the opposite orientation (O2). Orientation was assigned arbitrarily for the chromosome 8 sequence. Solid lines indicate proportions computed on V_H gene segments; dashed lines indicate segments from chromosome 8.

Figure 2.

O2 cRSs are found at multiple locations within mouse V_H gene segments. RIC₁₂ was computed for all 28-bp segments embedded in mouse V_H gene segments. RIC₁₂ scores of potential cRSs (RIC ₁₂ > −55) are plotted against the segment's nucleotide position (IMGT numbering). Open circles (○) indicate potential 12-cRSs and filled circles (•) indicate those segments with RIC₁₂> −48.2, the lowest RIC₁₂ for which we have detected extrachromosomal recombination (18). Locations of CDR1, -2, and -3 are shown by the shaded areas of the graphs. Roman numerals indicate clusters of cRSs that are conserved across V_H gene families. Site I spans amino acid residues 18–22 (nt 54–63); site II spans amino acid residues 34–42 (nt 100–126); site III spans residues 50–62 (nt 148–184); site IV spans residues 64–69 (nt 190–205); and site V is amino acid residue 105 (nt 313).

Figure 3.

V_H cRS cleavage is detected only in pro–B cells from RAG2:GFP knock-in mice (21). (A) Sorted pro-, pre, and immature B cells were analyzed by RT- and LM-PCR. Mature, recirculating B cells (+IgD) were removed by incubation with anti-IgD (-IgD). RAG2:GFP fluorescence was detected in pro- and pre–B cells, but not in immature B cells. (B) RT-PCR for Rag1, Tdt, and GAPDH transcripts revealed RAG1 message in both pro–B and pre–B cells; Tdt expression was detected only in sorted pro–B cells. (C) LM-PCR for primary J_H, V_H, Jκ, and Dβ rearrangements demonstrate the lineage and developmental restriction of V_H cRS SEs, and confirms the purity of the sorted cell populations. V_H1, V_H2, and V_H5 cRS SEs were detected only in pro–B cells. CD14 PCR demonstrated the equivalence of genomic template.

Figure 4.

V_H cRS cleavage is dependent on Rag1. (A) BM B cells with a pro–B phenotype (B220^loCD43⁺IgM⁻Lin⁻) and a pre–B phenotype (B220^loCD43⁻IgM⁻Lin⁻) were sorted from sibling H50G^+/−Rag1^+/− (middle) or H50G^+/−Rag1^−/− (right) mice. Resolution of pro- and pre–B cells in H50G transgenic mice was not as complete as in RAG2:GFP mice (Fig. 3 A). Mature and immature B cells (+IgM) were excluded when anti-IgM was used as a negative marker (-IgM). (B) LM-PCR demonstrated SEs at both physiological RSs (J_H2, Jκ2) and cRSs (J558) were present only in RAG1/2-sufficient cells.

Figure 5.

V_H cRS SEs from C57BL/6 and μMT pro–B cells. (A) BM B cells were recovered from C57BL/6 and μMT mice and pro-, pre, and immature B cells (from C57BL/6), or pro–B cells (from μMT) mice were sorted as in Fig. 3. Genomic DNA was isolated and ligated to the BW linker. (B) LM-PCR on genomic DNA of sorted B cell populations from C57BL/6 or μMT mice was performed to detect V_H cRS SEs. cRS SEs were found only in cells with a pro–B phenotype. CD14 PCR was used to normalize template DNA.

Figure 6.

V_H1 cRSs are cleaved at 1–7% the efficiency of the J_H2 RS. (A) Titration of nested LM-PCR cycle numbers (15–35) to optimize comparison of J_H2 RS SE (top) and V_H1 cRS SE (bottom) products. LM-PCR of J_H2 RS SEs and V_H1 cRS SEs were resolved by gel electrophoresis and hybridized with gene-specific probes. (B) Densitometric analysis of the hybridized LM-PCR products showed that 25 cycles constitute the linear phase of amplification. Circles (•) and triangles (▴) indicate the densitometry of J_H2 RS SEs and V_H1 cRS SEs, respectively. (C) Serial threefold template dilution demonstrates that J_H2 RS SEs (top) are less than or equal to threefold more abundant than V_H1 cRS SEs (bottom). (D) Relative abundance of J_H2 RS SEs and V_H cRS SEs determined by real-time quantitative PCR. J_H2 RS SEs and V_H1 and V_H5 cRS SEs were amplified from the BM cells of C57BL/6 mice in a series of quantitative LM-PCR. cRS SE products were normalized to J_H2 RS SE product (the mean ± the SD) from the same sample by the comparative threshold cycle method. Subsequently, V_H cRS cleavage efficiencies were adjusted to template numbers (J_H2 = 1; V_H1 = 34; V_H5 = 7). The mean efficiency of V_H1 cRSs was determined to be 4.9 ± 0.2% of the J_H2 RS, whereas VH5 cRSs were 7.6 ± 0.3% as efficient.

Figure 7.

Rearrangement of site I–V V_H cRSs in pro–B cells could increase the IgH repertoire by V_H replacement or hybrid rearrangement. The primary VDJ germline configuration and the outcomes of secondary cRS rearrangements are depicted. V_H replacement (V→V or V→VDJ) is mediated by a cRS (gray triangle in V_H gene segment) to an upstream V_H RS (white triangle). (Intra-V)→VDJ rearrangement via a 59 cRS to a 59 cRSs in another V_H gene segment forms a hybrid rearrangement and creates a hybrid joint (between a CE and a SE) between two V_H gene segments. Note that hybrid rearrangements and V_H replacements depicted can increase the diversity of V_H gene segments by novel CDR combinations.