Preferential utilization of specific immunoglobulin heavy chain diversity and joining segments in adult human peripheral blood B lymphocytes.

Abstract

SummaryWe have examined at the molecular level the CDR3 and adjacent regions in peripheral blood B lymphocytes of normal individuals. A total of 111 sequences (12-28 sequences from six individuals) were obtained after cloning of the polymerase chain reaction-amplified segments into plasmids or phage . The average length of the VDJ joining was 109 nucleotides, with a range from 79 to 151 . Approximately 75% of the sequences were in frame when translated into amino acids . Among the JH segments, J H4 was found most frequently (in 52 .5% of the sequences), and JH 1 and JH 2 segments the least frequently (-I% of the clones) . A polymorphic JH6 gene with a one-codnn deletion accompanied by a base change was present in two of six patients. Preferential breakpoints were found for JH 2, J.3, J 4, and Jx5, although the breakpoints of JH6 were distributed more heterogenously.In -90% of the cases, significant homology of the D regions with published D sequences was found . Preferential usage of a particular coding frame was observed in in-frame sequences utilizing DA, D21/9, and DMl segments. However, in general, all coding frames of germline D genes were used to generate CDR3s . Eight sequences that have a DN1-like D sequence with two base changes at the same positions were identified, suggesting the likely existence of a new germ line D gene belonging to the DN families. Using probes specific for a particular CDR3, the frequency of a specific B cell clone in the peripheral blood of normal individuals was estimated to be at most as high as 1/20,000 .T he most variable region of the immunoglobulin heavy chain is the third complementarity determining region (CDR3) (1, 2) . This region spans the junction between the variable (V ) diversity (D), and junctional 0,,) segments in the rearranged IgH genes (1, 2) . The hypervariability of this region is due to the combinatorial assortment of the many V , D, and JH segments that are utilized to generate a particular CDR3, to the imprecise joining mechanisms that include deletion of bases from the potential coding regions of each segment to be joined (3), and the addition of new bases that can be enzymatically added at the point of joining (N regions) (4) . Finally, somatic mutations of the rearranged region can contribute to the production of higher affinity antibodies (5) .The nucleotide sequences of all the human D genes, estimated to be -30 in number (6), have not been fully defined, and questions remain about the relative usage of different D genes used in VDJjoinings during development and in adult individuals . Furthermore, the characteristics of the extent of base excision and addition, including the identification of preferred sequence boundaries for the V., D, and J regions, have not been well delineated. The relative frequency of inframe translation products reflecting productive rearrangements, and the possibility of specific translation frames being preferred for particular D gene families have not been determined on a large sample size.To address these questions, we ha...

Evidence for a selected humoral immune response encoded by VH4 family genes in the synovial membrane of a patient with rheumatoid arthritis (RA)

“…In contrast, an extraordinary diversity in length and composition of the CDR3 was described for a set of RA-derived polyreactive antibodies and RF [30] and rearranged V H genes from PBL of a healthy donor [17]. For the J H segments we can confirm a predominant use of J H 4 [17,19].…”

Characterization of the immunoglobulin light chain variable region gene expressed in multiple myeloma

“…In the rearrangement of the IgH gene, the usage of J H segments revealed a developmental stage-specific trend. [7][8][9][10][11][12][13] At the earliest stage of B cell development, the initial D-J H rearrangements predominantly occur between D H Q52 and J H -1 segments which are located close to each other. 53 During B cell development, a second or a third D-J H joining might occur, then the more 5ЈD segments and more 3ЈJ H segments would join at a later stage.…”

“…6 Studies on IgH variable region gene sequences at various stages of development have revealed stage-specific trends in the usage of V H , D, and J H segments, the degree of N nucleotide addition, and the rate of somatic mutations. [7][8][9][10][11][12][13] However, little is known of the organization and diversification of the IgL variable region gene on -chain. On the other hand, multiple myeloma (MM) has a variety of clinical features relating to clinical stage, prognosis and complication.…”

Oligoclonal dominance of immunglobulin VH3 rearrangements following allogeneic bone marrow transplantation

“…After alkaline denaturation and UV cross-linking of the DNA, the filters were hybridized with the CDR3-specific oligonucleotide probes and a JHC probe (Figure 1). 25 Pre-hybridization was performed in 6 × SSC (20 × SSC: 3 mol/l NaCl, 0.3 mol/l Na citrate, pH 7.0), 10 × Denhardt's (0.2% w/v Ficoll, 0.2% w/v polyvinyl pyrrolidone, 0.2% w/v BSA), 1% SDS and 100 g/ l boiled herring sperm DNA at 55°C. After prehybridization the denatured 32 P-labeled olignucleotide probes were added and the filters were hybridized at 55°C overnight.…”

“…The JHC probe consists of a mixture of four different oligonucleotides derived from the sequences of JH2, JH3, JH4 and JH6 genes. 25 To ensure that the JH4 olignucleotide also hybridized to JH1 and JH5 gene sequences, the hybridization was performed at 35°C and the filters were washed at lower stringency, ie 1 × SSC, 1% SDS at RT.…”