Restricted immunoglobulin variable region (Ig V) gene expression accompanies secondary rearrangements of light chain Ig V genes in mouse plasmacytomas

Abstract

The many binding studies of monoclonal immunoglobulin (Ig) produced by plasmacytomas have found no universally common binding properties, but instead, groups of plasmacytomas with specific antigen-binding activities to haptens such as phosphorylcholine, dextrans, fructofuranans, or dinitrophenyl. Subsequently, it was found that plasmacytomas with similar binding chain specificities not only expressed the same idiotype, but rearranged the same light (V(L)) and heavy (V(H)) variable region genes to express a characteristic monoclonal antibody. In this study, we have examined by enzyme-linked immunosorbent assay five antibodies secreted by silicone-induced mouse plasmacytomas using a broader panel of antigens including actin, myosin, tubulin, single-stranded DNA, and double-stranded DNA. We have determined the Ig heavy and light chain V gene usage in these same plasmacytomas at the DNA and RNA level. Our studies reveal: (a) antibodies secreted by plasmacytomas bind to different antigens in a manner similar to that observed for natural autoantibodies; (b) the expressed Ig heavy genes are restricted in V gene usage to the V(H)-J558 family; and (c) secondary rearrangements occur at the light chain level with at least three plasmacytomas expressing both kappa and lambda light chain genes. These results suggest that plasmacytomas use a restricted population of B cells that may still be undergoing rearrangement, thereby bypassing the allelic exclusion normally associated with expression of antibody genes.

Figure 1

SKY™ images of SIPC tumors with variant T(6;15) chromosomal translocations, (A) SIPC3308 and (B) SIPC3282. The arrows mark chromosomal translocations that have occurred between CHR6 (yellow) and CHR15 (blue). There are two copies of each T(6;15) and the reciprocal T(15;6).

Figure 3

J_H rearrangements in SIPC tumors. Genomic DNAs from SIPC tumors and BALB/c liver were digested with EcoR1 and hybridized with the J_H probe: lane 1, SIPC3385; lane 2, SIPC3308; lane 3, SIPC3301; lane 4, SIPC3336; lane 5, SIPC3282; and lane 6, BALB/c. An arrow (left) depicts the germline (nonrearranged) J_H fragment, whereas the 3.5-kb EcoR1 rearrangement seen in several tumors is also highlighted.

Figure 2

Southern hybridization assay for c-Myc and Pvt 1 rearrangements in SIPC tumors. Shown top (c-Myc, EcoR1) and bottom (Pvt 1, BamH1) are the following tumors: lane 1, SIPC3385; lane 2, SIPC3308; lane 3, SIPC3301; lane 4, SIPC3336; lane 5, SIPC3282; and lane 6, BALB/c. Arrows depict the germline (nonrearranged fragments). Two samples (SIPC3385 and SIPC3301) contain rearranged bands with c-Myc, whereas no rearrangements are found with Pvt 1.

Figure 4

Nucleotide sequences of V_H genes from SIPC tumors. The sequences obtained with the five SIPC tumors have been compared with the most homologous germline genes which are members of the V_H-J558 family. Top: SIPC3336, SIPC3308, and SIPC3282 express V_H13-3; middle: SIPC3301 expresses 26.4.1-α; bottom: SIPC3385 expresses 4ma. Dashes indicate sequence identities. Germline amino acids in bold indicate that they are replaced in SIPCs. These sequence data are available from EMBL/GenBank/DDBJ under accession nos. AF154880–AF154882, AF154909, and AF154910.

Figure 5

Ig_L rearrangements of SIPC tumors. Genomic DNAs from SIPC tumors and BALB/c liver were digested with EcoR1 and hybridized with the IVS probe: lane 1, SIPC3385; lane 2, SIPC3308; lane 3, SIPC3301; lane 4, SIPC3336; lane 5, SIPC3282; and lane 6, BALB/c. An arrow (left) depicts the germline (nonrearranged) Ig_κ fragment, whereas the 18 and 9.5-kb EcoR1 rearrangements seen in SIPC3308, SIPC3336, and SIPC3282 are also highlighted (right).

Figure 6

Nucleotide sequence of the V_κ24C-J_κ4 gene from the SIPC tumors. Sequences obtained from SIPC3282, SIPC3336, and SIPC3308 (bottom line) have been compared to that of the germline V_κ24C (reference 15). Dashes indicate sequence identities. Substitutions at the amino acid level are indicated in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession no. AF154911.

Figure 7

Nucleotide sequences of V_κ1 genes expressed in SIPC tumors. Sequences were obtained from individual clones (designated A–F) of SIPC3282, SIPC3301, SIPC3336, SIPC3308, and SIPC3385. Although multiple subclones with identical sequences were obtained, only representative sequences that differ are shown. The sequences are compared with the most homologous germline gene K5.1 (V_κ1A, top) or K1A5 (V_κ1C, bottom). Dashes indicate sequence identities, and amino acid substitutions are indicated in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession nos. AF154883–AF154898.

Figure 8

Nucleotide sequences of V_λ1 genes expressed in SIPC tumors. Nucleotide sequences of subclones (designated A–F) of V_λ genes from SIPC3282, SIPC3336, and SIPC3301 are compared with the most homologous germline gene. Dashes indicate sequence identities, and amino acid substitutions are indicated in bold. These sequence data are available from EMBL/GenBank/DDBJ under accession nos. AF154899–AF154908.

Figure 9

Transcript levels of Ig_κ, Ig_λ, and RAG-2 in SIPC tumors. (Top) Equal amounts of RNA from mouse liver (lane 1), spleen (lane 2), SIPC3301 (lane 3), SIPC3308 (lane 4), SIPC3282 (lane 5), SIPC3336 (lane 6), and SIPC3385 (lane 7) were loaded into slots and hybridized to nick-translated probes from V_κ24 (row 1), V_κ1 (row 2), C_κ (row 3), or V_λ (row 4). (Bottom) RNA from mouse spleen (lane 1), thymus (lane 2), SIPC3282 (lane 3), SIPC3301 (lane 4), SIPC3308 (lane 5), SIPC3336 (lane 6), SIPC3385 (lane 7), ABPC18 (lane 8), and MOPC104E (lane 9) was reverse transcribed (RT) and PCR amplified using primers specific for RAG-2.