RECOMBINATION AND EXPRESSION OF ANTIGEN
RECEPTOR GENES

The antigen-binding (variable) regions of immunoglobulin (Ig) and T cell receptor (TCR) molecules are encoded by separate genetic elements (variable [V], diversity [D] and joining [J]). These elements recombine in the genome of developing B and T lymphocytes to form contiguous coding segments (V(D)J or VJ) for a diverse array of Ig and TCR variable regions. This process, which is a site-specific DNA recombination reaction, is known as V(D)J recombination. In B lymphocytes, the same VDJ coding segment can be expressed with different Ig constant (C) region genes (e.g., m, g, a); C region genes specify the class of Ig heavy (H) chain produced (e.g., IgM, IgG, IgA). The process that allows a given VDJH coding segment to be expressed with different CH regions is referred to as class switch recombination. Defects in V(D)J recombination or class switch recombination can result in severe immunodeficiency. A model for severe immuno-deficiency resulting from a defect in antigen receptor gene recombination is the scid mouse mutant. We continue to use scid and wild type mice for the study of antigen receptor gene recombination and expression.

Assembly of Ig and TCR genes from individual gene segments is essential in the development of lymphoid cells. The V(D)J recombination reaction, which mediates receptor assembly, is targeted by recombination signal sequences (RSS) that lie adjacent to each element of the three types of receptor gene segments, V, D, and J. The RSS consist of a conserved heptamer and nonamer motif separated by a spacer of conserved length (either 12 or 23 base pairs), but of non-conserved sequence. The first phase of V(D)J recombination consists of recognition of a pair of RSS with unlike spacers, and cleavage at the signal-coding borders of a given pair of coding segments. Both recognition and cleavage steps are mediated by the RAG1 and RAG2 proteins that are only expressed in lymphocytes. The cleavage step produces two types of broken molecules corresponding to V(D)J recombination intermediates: those with coding ends and those with signal ends. As a consequence of the cleavage mechanism, coding ends are covalently sealed to form a DNA hairpin. In contrast, signal ends are almost always blunt with no sequence loss or addition.

The second phase of V(D)J recombination involves end processing and joining, but is less well understood than the cleavage phase. Resolution of hairpin coding ends involves opening (nicking) the hairpins, a process that requires an endonuclease and DNA-dependent protein kinase (DNA-PK). The critical role of DNA-PK is clear from the phenotype of scid mice. In this mouse mutant, the catalytic subunit of DNA-PK is missing. This results in accumulation of hairpin coding ends and severely impaired V(D)J recombination. Studies of joined coding segments indicate that once hairpin coding ends are opened, the ends can be modified; modifications include the addition of non-templated nucleotides by terminal deoxy-nucleotidyl transferase and end-nibbling by unidentified nucleases.

Recently, we have identified broken molecules with open (non-hairpin) coding ends from the TCRd locus in wild type fetal thymocytes. We used the technique of ligation-mediated PCR (LMPCR) to recover broken Dd2 and Jd1 coding ends. In this technique, blunt 5' phosphorylated DNA ends are ligated with a partially double-stranded oligonucleotide, and then amplified by PCR using the ligation primer and a locus-specific primer. Quantitative analysis showed that recovery of coding ends was approximately 6-fold more efficient if the fetal thymus DNA was pretreated with T4 DNA polymerase before primer ligation, indicating that the majority of coding termini have single-strand overhangs rather than being blunt. To characterize the nonblunt coding ends further, the LMPCR analysis was repeated with blunt end linkers and with linkers designed to anneal to broken DNA with overhanging ends. The latter oligonucleotide linkers have degenerate 5'- or 3'-overhanging ends, 2 to 5 nucleotides, that will enable ligation to 5'- or 3'-overhanging ends of genomic DNA. Following linker ligation to DNA from fetal thymocytes or adult liver, PCR was performed using a locus specific primer mapping 200 base pairs 3' of Jd1. Figure 1 shows a Southern blot analysis of the LMPCR products hybridized to a Jd1 probe. The 1.1 kilobase (kb) product corresponds to a 3' Dd2 blunt signal end and is efficiently recovered using blunt end linker ligation (lane 1). The 0.2 kb product corresponding to Jd1 coding ends was recovered much more efficiently using linkers that can ligate directly to 3'-overhanging ends (lane 4) than with linkers that can ligate to 5'-overhanging ends (lane 2), or with blunt end linkers (lane 1). The amount of 200 base pair product recovered with the 3'-overhanging linker was almost equivalent to that amplified from T4 DNA polymerase pretreated thymus DNA (compare lanes 2 and 6). This result indicates that the majority of nonblunt coding ends have 3'-overhanging ends and that hairpin opening is asymmetric. One possible explanation for this result is that nicking by the hairpin endonuclease has a restricted orientation because of an asymmetric distribution of recombinase components in the synaptic complex.

STRUCTURE OF HYBRID JOINTS IN SCID MICE. RUETSCH

The usual outcome of a V(D)J reaction is that a pair of signal ends is joined to form a signal joint and a pair of coding ends is joined to form a coding joint. Sometimes, however, signal ends are joined to coding ends to form hybrid joints (Figure 2). Recent results obtained using a cell-free V(D)J recombination system indicate that hybrid joint formation can be mediated solely by the products of the recombination activation genes, RAG1 and RAG2 (Melek et al., Science 280:301, 1998). The reaction appears to be a chemical reversal of hairpin formation and results in precise, or nearly precise, joining of a signal and coding end. As scid mice are not deficient for RAG1 or RAG2, we were interested in testing for evidence of this reaction in these mice. Therefore, we looked at the structure of scid hybrid joints resulting from inversional rearrangement between JH1-4 and the Dfl16 or DQ52 elements. Representative results are shown in Figure 2.

Scid hybrid joints show extensive deletions of signal and coding nucleotides (Figure 2B). Also, approximately 20% of scid hybrid joints show nontemplated nucleotide additions. These properties indicate processing of the DNA ends before joining. Processing and joining of DNA ends following cleavage by RAG1 and RAG2 is known to involve different enzymes, including the catalytic subunit of DNA-PK (DNA-PKcs), which is defective in scid mice. The extensive loss of signal and coding nucleotides in scid hybrid joints is reminiscent of the extensive loss of coding nucleotides in scid coding joints. We conclude that scid hybrid joint formation does not reflect a reversal of hairpin formation, but rather a processing and joining of DNA ends using similar (or identical) machinery to that used in the formation of coding joints.

FIGURE 2.  (A)The scheme depicts formation of hybrid joints via inversional rearrangement. Solid and open triangles denote RSSs with either 23 bp or 12 bp spacer lengths, respectively. D and J coding regions are denoted as rectangles. (B) Structure of scid Dfl16.1-JH2 hybrid junctions. The numbers in parentheses denote the extent of nucleotide deletion into the signal and coding ends. The nucleotides in the middle denote non-templated N additions.

The extent to which the scid mutation may impair class switch recombination is unclear because scid B cell differentiation is arrested (at the pro-B cell stage) prior to the B cell stage at which class switch recombination normally occurs. This arrest reflects the inability of developing scid B lineage cells to rearrange their VH, DH and JH elements productively. However, if the requirement for VDJH rearrangement is circumvented by crossing a m H and k L chain transgene into the scid genome, then differentiation can proceed to the mature B cell stage. Therefore, to test whether scid B cells can undergo class switch recombination, we stimulated splenic B cells from m/k-transgene scid mice (3H9/Vk8 scid mice) with lipopolysaccharide and interleukin-4, two agents that induce recombination between the Sm and Sg1 switch regions. Our results show that approximately half of the stimulated spleen cultures from individual 3H9/Vk8 scid mice showed evidence of recombination between Sm and Sg1 switch regions. In 3H9/Vk8 scid/+ control mice, the percentage of spleen cell cultures with detectable Sm-Sg1 recombinant loci was approximately 80%. These preliminary results suggest that Sm-Sg1 switch recombination is not severely impaired by the scid mutation, consistent with our earlier results showing that B cell clones in leaky scid mice are able to produce multiple IgG isotypes (i.e., undergo class switch recombination).

Ig gene recombination (rearrangement) is ordered and occurs at distinct stages of B cell development. When a pro-B cell makes a productive H chain gene rearrangement, it ceases further H chain gene rearrangement and progresses to the pre-B cell stage. At the pre-B cell stage, the k and l L chain loci undergo rearrangement. Consistent with the above, in scid mice bearing the m transgene, 3H9, pro-B cells transit to the pre-B stage and then initiate k and l (detected as l1) rearrangement. However, in scid mice bearing the m transgene, M54, arising pre-B cells readily initiate k but not l1 rearrangement. Rearrangement at the l1 locus is strongly suppressed in M54 scid mice and also in M54 scid/+ mice (mice heterozygous for the scid mutation). These results indicate that l1 and k loci are differentially regulated. The mechanism for suppression of l1 rearrangement in M54 mice is currently being investigated.

CHANG, Y., BOSMA, M.J., BOSMA, G.C. Extended duration of DH-JH rearrangement in Ig H chain transgenic mice: Implications for regulation of allelic exclusion. J. Exp. Med. 189:1295-1305, 1999.

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998