EBV Transformation of Tonsil Germinal Centre B Cells Carrying Functionally Inactivated IgV Gene.

Somatic hypermutation of immunoglobulin (Ig) gene sequences in the germinal centres of lymphoid tissues is necessary for affinity maturation of B cell responses to antigen challenge. This process generates a few clones with improved affinity that are selected into B cell memory and many clones with other non favourable Ig mutations, including some cells with functionally inactivated Ig gene that normally die by apoptosis. It is postulated that infection with Epstein-Barr virus (EBV), a B lymphotropic agent linked to several types of B cell lymphoma, can rescue germinal centre cells with unfavourable mutations. This creates a pool of infected cells at greater risk of developing into lymphomas. In the present work, CD38⁺ germinal centre B cells were separated from tonsil by negative selection for IgD and CD39. Peripheral blood naïve and memory B cell subpopulations were FACS sorted as IgD⁺, CD27⁻ and IgD⁻, CD27⁺ fractions respectively. These cells were infected with EBV (B95.8 strain) in vitro and seeded at limiting dilutions onto fibroblast feeders. EBV transformed lymphoblastoid cell lines (LCLs) from such cultures were analysed for surface Ig phenotype. Naïve B cell transformants were consistently IgM⁺, IgD⁺. Memory B cell transformants were IgM⁺ in some cases but more frequently IgG⁺ or IgA⁺. Germinal centre transformants showed the same spectrum of surface Ig phenotypes as memory cell transformants but in addition we identified six germinal centre derived LCLs which were consistently surface Ig negative. Sequencing from these lines confirmed that in at least three cases EBV had rescued cells with functionally inactivated Ig heavy chain gene.