IntroductionEpstein-Barr virus (EBV) has B-cell growth-transforming ability and is linked to a range of B-cell malignancies. 1,2 Of these, posttransplantation lymphoproliferative disease (PTLD) is pathogenetically the least complex. Thus, PTLD tumors arising early after transplantation, when immunosuppression is greatest, resemble EBV-positive in vitro-transformed lymphoblastoid cell lines (LCLs) in cellular phenotype and in expressing the full range of EBV-latent proteins. 3,4 Here, viral transformation appears to be sufficient for tumor growth, whereas in certain late-onset PTLD lesions, viral gene expression is more restricted and tumor evolution has involved additional cellular genetic changes. [5][6][7] Recent studies have found that most PTLD tumors carry hypermutated immunoglobulin sequences, many of which are atypical of conventional antigenselected memory cells. [8][9][10] Indeed, some tumors lacked surface immunoglobulin and had functionally inactivated immunoglobulin sequences, 8 highlighting parallels with another EBV-associated B-cell malignancy, Hodgkin lymphoma (HL), in which the tumor cells are again surface immunoglobulin negative and often carry similarly "crippled" immunoglobulin genes. 11 These findings suggest that EBV infection can promote the survival of atypical post-germinal center (GC) B cells carrying unfavorable or even inactivating immunoglobulin gene mutations. Such cells, arising as failed products of the somatic hypermutation process that occurs during GC transit, normally die by apoptosis 12 ; however, if rescued by EBV, they might form a pool of cells particularly prone to tumor development. Here we ask whether EBV infection of GC B cells in vitro leads to the outgrowth of LCLs with crippled immunoglobulin genes. Study design Target cell transformationFresh tonsils were obtained with informed consent from 3 adult patients after tonsillectomy. Mononuclear cells were isolated by Ficoll-Isopaque centrifugation and then T-depleted using CD3 Dynabeads (Dynal Biotech, Bromborough, United Kingdom). The resultant B-cell population was stained for the CD10 marker of GC origin 13 using phycoerythrin (PE)-Cy5-labeled anti-CD10 (BD Pharmingen, Oxford, United Kingdom) monoclonal antibody (mAb) and CD10 ϩ cells collected by fluorescence-activated cell sorter (FACS) on a Mo-Flo sorter (Dako Cytomation, Ely, United Kingdom). In parallel experiments involving different adult donors, naive, and memory B cells were isolated from peripheral blood B cells by FACS sorting of immunoglobulin D ϩ (IgD ϩ )CD27 Ϫ and IgD Ϫ CD27 ϩ subsets, respectively, after staining with FITC-labeled anti-IgD (Caltag, Buckingham, United Kingdom) and RPE (R-phycoerythrin)-labeled anti-CD27 (BD Pharmingen) mAbs. Target B cells were exposed to B95.8 EBV for 1 hour at 37°C and then were seeded at limiting dilutions onto wells containing a human fibroblast feeder layer. Cells were maintained in RPMI 1640 medium supplemented with 2 mM glutamine and 10% vol/vol fetal calf serum (FCS) for 3 to 4 weeks, at which time isolated wel...

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.

Following primary infection, Epstein-Barr virus (EBV) establishes life long persistence in the host IgD− CD27+ memory B cell compartment rather than the IgD+ CD27+ marginal zone (MZ)-like or the IgD+ CD27− naïve B cell compartments. One possible explanation for such exclusive persistence in memory B cells is that EBV preferentially infects memory B cells. Alternatively, the virus may infect all B cell subsets but then drive MZ and naïve B cells to acquire the Ig isotype-switched phenotype and hypermutated Ig genotype of memory cells. Here we ask whether there is any evidence for one or other hypothesis from in vitro experiments. B cells from healthy donor blood samples were FACS sorted on the basis of IgD/CD27 expression into naïve, MZ, and memory B cell subsets with purities of >99%, >97% and >98% respectively. Analysis of the IgVH sequence further confirmed purity of the FACS sorted B cell subsets. Accordingly, 102 of 105 IgVH sequences amplified from purified naïve B cells were germ-line where as the vast majority of sequences amplified from MZ and memory B cells were mutated. All three B cell subsets expressed equal amounts of CD21 (EBV receptor on B cells), bound similar amounts of virus, and transformed with equal efficiency to establish B lymphoblastoid cell lines (LCLs) in vitro. Naïve B cell transformants upregulated CD27 expression but retained the IgM+, IgD+ phenotype as determined by FACS analysis and RT-PCR; MZ-B derived LCLs likewise were IgM+, IgD+, CD27+; and memory-B derived LCLs were consistently CD27+, IgD− and expressed either IgG, IgA or in some cases IgM. Therefore, EBV infection per se did not induce class switching. However, both naïve and MZ-B derived LCLs could still be induced to switch to IgG in the presence of CD40 ligand and IL-4; signals that are normally provided by T cells in vivo. To assess if EBV infection might drive Ig hypermutation, we carried out IgVH sequence analysis on the naïve-B derived LCL clones. Interestingly, 42 of 114 clonal IgVH sequences amplified from naïve-B derived LCLs had 3 or more mutations and the patterns of mutation seen were consistent with that produced by somatic hypermutation (SHM). Furthermore, within some naïve-B cell derived LCL clones, there were both germ-line and mutated sequences all sharing the same VDJ rearrangement (CDR3 sequence), again implying sequence diversification following EBV transformation of a single naïve B cell. Some intraclonal variation of the already hypermutated IgVH sequence was also noted in memory and MZ-B derived LCLs further suggesting ongoing mutational activity. Consistent with this, activation-induced cytidine deaminase (AID) expression was upregulated in transformants as assessed by real time RT-PCR. Our in vitro data is therefore compatible with a model of EBV persistence where the virus infects all mature B cell subsets but then drives infected naïve B cells to acquire a memory genotype by inducing SHM. In addition, EBV infected naïve and MZ-B cells may undergo Ig class switching to acquire the IgD− CD27+ memory phenotype in the presence of T cell help in vivo. EBV’s ability to induce SHM may also contribute to the lymphomagenic potential of the virus in addition to its B cell transforming and growth promoting properties.