No abstract

Expression of N-terminal-truncated Ig heavy chains without normal light chain expression has been shown to occur in human B cell tumor lines, and to be due to diverse types of structural alteration within the expressed Ig heavy and light chain genes. Due to the tumor cell origin of these lines, generation of aberrant Ig-encoding genes may only occur after malignant transformation, reflecting the release of the tumor B cell from the need to express functional Ig for continued clonal proliferation. The genetic basis for expression of VH-truncated mu chains without light chains in several Epstein-Barr virus (EBV)-transformed human B cell lines was investigated with the aim that this information would lead to detection of similarly aberrant Ig genes in normal human B lymphocytes. Analysis of the productive mu genes in three truncated mu-only human B cell lines showed a consistent structural change where a secondary VH-VHDJH gene rearrangement had occurred. The site of VH-VH joining was suggestive of a V(D)J recombinase-mediated event. A consistent pattern of mutation was also observed in the normal-sized, but non-functional kappa light chain transcripts, making them incapable of coding for a functional kappa chain. Using genomic DNA from peripheral blood lymphocytes of a donor whose B cells were originally used to make one of the truncated mu-only EBV B cell lines, a similarly mutated V kappa gene was detected and a similar composite VH-VH gene was cloned. The lack of such aberrant VH and V kappa genes in non-lymphoid cells of this individual showed that the structural abnormalities in the expressed Ig genes arose somatically during development of that B cell clone. The presence of these altered Ig heavy and light chain genes in the genomic DNA of untransformed lymphocytes also shows that generation of such variant B cell clones can be a pre-neoplastic event and occurs by genetic mechanisms active during normal B cell development.

The use of different viral promoters for the expression of the EBNA1 gene product appears to be a critical step in the regulation of Epstein-Barr virus latent gene expression and may reflect the extent of differentiation of B-cell hosts. Low-passage Burkitt lymphoma cell lines resemble immature B cells in that they express CD10 (CALLA) and do not express B-cell activation antigens. In these cells, transcription from a promoter located in the BamHI F fragment of the viral genome results in the exclusive expression of EBNA1, referred to as the latency I pattern of viral gene expression. In contrast, high-passage Burkitt lymphoma cells and lymphoblastoid cell lines resemble activated B cells in that they do not express CD10 but do express activation antigens such as CD23. In these cells, the use of two promoters located in the BamHI W and C fragments of the viral genome leads to the expression of all six EBNA gene products (latency III). We have found that four human B-cell lines, DB, .LBW2, LBW14, and Josh 7, stably express a pattern of B-cell differentiation antigens intermediate between those found in latency I and latency Ill cell lines and characterized by the coexpression of CD10 and CD23. The pattern of EBNA1 promoter usage in these cell lines was examined to determine whether their intermediate cellular phenotype was reflected in their patterns of viral gene expression. DB, LBW2, and LBW14 utilize both the BamHI F promoter region and BamHI W promoter region to transcribe the EBNA1 gene..This stable pattern of mixed promoter usage for the expression of the EBNA gene products in B cells has not previously been described. In addition, these three B-cell lines expressed lower levels of the viral latent gene product EBNA2 than those typically observed in latency III cells. The lower levels of activation of viral and cellular promoters known to be regulated by EBNA2 also correlated with the reduced levels of EBNA2 expression in these cells. These included the viral LMP1 and LMP2A promoters and the cellular CD23B promoter. Thefourth B-cell line, Josh 7, expressed EBNA1 mRNAs derived from both the BamHI W promoter and BamHI C promoter, similar to latency III cells. The intermediate cellular phenotype in Josh 7 cells appeared to be due, in part, to a deficiency in the expression of viral LMP1. These results show that the B-cell lines express unique intermediate patterns of expression of viral and cellular gene products and suggest that a continuum of latency states may exist for Epstein-Barr virus-infected B lymphocytes.