Ongoing somatic hypermutation of the rearranged VH but not of the V-lambda gene in EBV-transformed rheumatoid factor-producing lymphoblastoid cell line.
Epstein-Barr virus (EBV) transforms human peripheral B cells into lymphoblastoid cell lines (LCLs) that secrete specific antibodies. In contrast to peripheral blood B cells, LCLs express the activation-induced cytidine deaminase (AID) gene, a key enzyme in the generation of somatic hypermutation (SHM) in immunoglobulin variable genes. We have previously studied an LCL that secretes a rheumatoid factor (RF: an IgM(lambda) anti-IgG antibody) and identified the accumulation of SHM at a frequency of 1.5 x 10(-3)mut/bp in the rearranged variable region heavy chain gene (VH) of its RF sub-culture (i.e., RF-2004). The aim of the present work was to find out whether SHM was initiated as an early event following EBV transformation. Our results show that already the earliest RF-culture (RF-1983) mutates its VH at a somewhat higher frequency of 1.9 x 10(-3). Overall, we detected 17 point mutations in the RF-2004 culture and in 26 cellular clones derived from the RF-1983 and RF-2004 cultures. Most of the mutations were due to C to T or G to A transitions, with preferential targeting to WRCH/DGYW hotspot motifs, indicating that they were due to the initial phase of AID-directed mutations. A genealogical tree demonstrates that mutations were accumulated in a stepwise manner with 1-2 mutations per cell division. However, no mutations were found in the rearranged V-lambda (Vlambda) gene in the same RF-cultures and their subclones (i.e., <1.2 x 10(-4)mut/bp). To our knowledge this is the first reported clonal cell line that generates SHM in the VH, but not in the Vlambda. It may be due to abrogation of a cis-regulatory element(s) in the Vlambda or to a lack of a specific trans-acting factor which differentially direct the SHM machinery to this gene. Out of the 17 point mutations detected in both cell lines there were, 1 stop codon, 3 mutations which obliterated the binding of the RF antibody to its IgG antigen and 1 or 2 mutations which enhanced antigen-binding affinity. These results show that the evolutionary developed germline encoded antibody combining site is highly sensitive to amino acid replacements. Our combined findings that the RF cells accumulate in a stepwise manner up to 1-2 point mutations/sequence per cell division and the generation of high percentage of functionally deleterious mutations, are in accord with the 'multiphase-recycling model' of SHM, which states that B cells in the germinal center are subjected to multiple rounds of somatic mutations interchanged with periods of antigenic selection.
Chezar
Ilan
I
Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Lobel-Lavi
Liat
L
Steinitz
Michael
M
Laskov
Reuven
R
D000328:Adult; D000595:Amino Acid Sequence; D000941:Antigens / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000201:enzymology / Q000276:immunology*; D001483:Base Sequence; D002472:Cell Transformation, Viral; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D004854:Herpesvirus 4, Human / Q000472:pathogenicity*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D007146:Immunoglobulin lambda-Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D051718:MutS Homolog 2 Protein / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D051560:Proto-Oncogene Proteins c-bcl-6; D012217:Rheumatoid Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D027041:Somatic Hypermutation, Immunoglobulin; D051981:Uracil-DNA Glycosidase / Q000378:metabolism
D000941:Antigens; C084103:BCL6 protein, human; D004268:DNA-Binding Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains; D051560:Proto-Oncogene Proteins c-bcl-6; D012217:Rheumatoid Factor; D004259:DNA-Directed DNA Polymerase; D051981:Uracil-DNA Glycosidase; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein