Alternative V kappa gene rearrangements in a murine B cell lymphoma. An explantation for idiotypic heterogeneity.

Abstract

The diversity ofthe antigen binding portion ofthe Ig molecule is generated through the recombination of multiple discontinuous gene segments (1, 2). The heavy chain variable (V) region coding sequence is formed when one of hundreds of VN regions combines with one of 15-20 D regions and one of 4J . regions to create a complete VDJ segment (3, 4) . Light chain recombination proceeds in a similar fashion between V,, andJL segments (5). These events occur in a well-defined order with DJ . rearrangement preceding V.-to-DJ. recombination (6, 7), followed later by V,;to-J,, recombination. The exact site of recombination between these segments is imprecise, thus creating further diversity at the joints . Single nucleotides (N segments) may be added at the limits of the D region in heavy chain rearrangements (8, 9) . These mechanisms, along with the association of different heavy and light chains, result in an expected antibody repertoire of >1011 Ig molecules.

Secondary V(D)J Rearrangements and B Cell Receptor-Mediated Down-Regulation of Recombination Activating Gene-2 Expression in a Murine B Cell Line

“…It has previously been reported that Id-negative variants of 38C-13 cells isolated after immunoselection with anti-idiotypic Abs had undergone secondary gene rearrangements (22,23,26). Therefore, we analyzed the rearrangement status of 38C-13 and its spontaneously arising Id-negative variants obtained in vitro.…”

“…After restriction cleavage, 25 g of DNA were fractionated by electrophoresis on 0.7% agarose gel, transferred to Qiabrane Nylon Plus membrane (Appligene, Strasbourg, France) in 0.4 M NaOH, and hybridized with radiolabeled probes. The probes used are as follows: 3ЈJ 5 is a 620-bp HindIII-NcoI genomic fragment from the murine J -C intron (32); RS, a 800-bp Sau3A fragment (33); 3ЈJ 2, a 279-bp fragment obtained by PCR amplification using the forward primer 5Ј-CTTGTTCACTAAGTCTAAC CTTG-3Ј and reverse primer 5Ј-TTTCCAGTCTGGTCCCATCAC-3Ј; 3ЈJ 4, a 244-bp fragment generated using the forward PCR primer 5Ј-GGGTAACTTGTGTGAATTTGTG-3Ј and reverse primer 5Ј-GACTAT GACATGCCCCTCTC-3Ј; and V 38C, a 213-bp fragment generated using the forward PCR primer 5Ј-AAGGCAAGCCAAGACATTAAC-3Ј and reverse primer 5Ј-CAGATTATCATACTGTAGAC-3Ј (22).…”

“…The 38C-13 cells are surface IgM ϩ , IgD Ϫ and produce both and surrogate light chains (24). During studies on Id-specific immunotherapy of 38C-13 tumors, it has been observed that Id-negative variant tumors develop in surviving mice (22,25). The loss of idiotypic specificity was a result of secondary gene rearrangements leading to the synthesis of chains different from that of the parental cell line or of the lack of light chain production (22,23,26).…”

“…During studies on Id-specific immunotherapy of 38C-13 tumors, it has been observed that Id-negative variant tumors develop in surviving mice (22,25). The loss of idiotypic specificity was a result of secondary gene rearrangements leading to the synthesis of chains different from that of the parental cell line or of the lack of light chain production (22,23,26). In this study we show that 38C-13 cells undergo spontaneous V-J and RS rearrangement in culture, with recombination occurring on both the productive and the nonproductive alleles.…”

“…Given the controversy surrounding the nature of the B cells undergoing secondary V(D)J recombination and its regulation, we have analyzed the murine 38C-13 B cell line (21), which has previously been shown to undergo light chain gene replacement (22,23). The 38C-13 cells are surface IgM ϩ , IgD Ϫ and produce both and surrogate light chains (24).…”