Andrew Bell mutated Ig Genotypes PDF

Epstein-Barr Virus Infection of Naı̈ve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology Emily Heath1., Noelia Begue-Pastor1., Sridhar Chaganti1., Debbie Croom-Carter1, Claire Shannon- Lowe1, Dieter Kube2, Regina Feederle1,3, Henri-Jacques Delecluse1,3, Alan B. Rickinson1, Andrew I. Bell1* 1 School of Cancer Sciences, College of Medicine and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom, 2 Department of Haematology and Oncology, Georg August University Göttingen, Göttingen, Germany, 3 Department of Virus-Associated Tumours, DKFZ, Heidelberg, Germany Abstract Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth- transforming infections yet establishes persistence only in IgD+ CD27+ non-switched memory (NSM) and IgD2 CD27+ switched memory (SM) B cells, not in IgD+ CD272 naı̈ve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV- driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively- harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naı̈ve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCL’s evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes. Citation: Heath E, Begue-Pastor N, Chaganti S, Croom-Carter D, Shannon-Lowe C, et al. (2012) Epstein-Barr Virus Infection of Naı̈ve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology. PLoS Pathog 8(5): e1002697. doi:10.1371/journal.ppat.1002697 Editor: Bill Sugden, University of Wisconsin-Madison, United States of America Received August 22, 2011; Accepted March 27, 2012; Published May 10, 2012 Copyright: ß 2012 Heath et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: DCC, CSL, HJD, ABR and AIB are funded by Cancer Research UK, grant number C910/A8829 (www.cancerresearchuk.org). EH was funded by a studentship from Cancer Research UK, grant number C8466/A6725. SC was funded by a LLR Clinical Research Fellowship, grant number 0338 (http:// leukaemialymphomaresearch.org.uk). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected]. These authors contributed equally to this work. Introduction evade detection by the host T cell response. A key finding was that the cells constituting this reservoir, whether in the blood of Epstein-Barr virus (EBV), an orally transmitted herpesvirus convalescent IM patients or of long-term EBV carriers, lie within widespread in human populations, first replicates in a permissive the IgD2 CD27+ memory B cell subset and not in IgD+ CD272 cell type in the oropharynx and then colonises the B cell system naı̈ve cells [7–9]. Furthermore, in IM cases where infected cell through a growth-transforming infection that drives the clonal numbers were sufficient to allow single cell analysis, these cells expansion of latently-infected cells [1–3]. This growth transfor- carried somatically-mutated immunoglobulin (Ig) gene sequences mation can be studied in vitro where infection of resting B cells typical of antigen-experienced memory cells , as do many of the occurs via CD21 receptor-mediated virus entry and leads to the EBV-driven lymphoproliferative disease lesions that arise in outgrowth of permanent lymphoblastoid cell lines (LCLs) immunocompromised patients where T cell control is relaxed expressing all eight EBV latent cycle proteins (six nuclear antigens [10–14]. EBNAs 1, 2, 3A, 3B, 3C and –LP, and two latent membrane The physiologic process of memory selection involves IgM+ proteins LMPs 1 and 2). Cells displaying these same markers of IgD+ CD272 naı̈ve B cells encountering cognate antigen in viral transformation are present in the tonsillar lymphoid tissues of lymphoid tissues and, with antigen-specific T cell help, prolifer- infectious mononucleosis (IM) patients undergoing primary EBV ating to form germinal centres (GCs). Here Ig variable gene infection [4,5]. Already, however, there is heterogeneity within sequences are subject to successive rounds of somatic hypermuta- these expanding B cell clones in IM tonsils [5,6], with some cells tion (SHM) to generate intra-clonal diversity before being re- apparently down-regulating viral antigen expression and switching expressed, usually in isotype-switched forms. Both SHM and out of cell cycle, thereby establishing a latent reservoir that can isotype-switching are critically dependent upon activation-induced PLoS Pathogens | www.plospathogens.org 1 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Author Summary blocked and there are no conventional switched memory B cells, EBV is sequestered in the tiny population of Ig gene-mutated, Epstein-Barr virus (EBV), a growth-transforming virus linked non-switched memory cells that exists in such patients rather than to several B cell lymphomas in man, is usually carried as an in the numerically dominant naive population [29,30]. The asymptomatic latent infection in B lymphocytes. Such virus chances of incoming virus selectively targeting such a small carriage selectively involves memory, but not naive, B cells. population by direct infection seems remote, again raising the How this selectivity is achieved is poorly understood since possibility that, in naive B cells, EBV infection per se may be able to we find that naive and memory cell types are equally impose a memory genotype and/or phenotype on these cells susceptible to infection and growth transformation to without recourse to GC signals. lymphoblastoid cell lines in vitro. Here we ask if EBV- Given these uncertainties, it is surprising that little attention has transformation of purified naı̈ve B cells can induce key been given to studying naı̈ve, non-switched-memory and switched- features of memory cells, namely immunoglobulin (Ig) memory (here designated N, NSM and SM respectively) B cell class switching and Ig gene mutation. We find that EBV does not induce Ig class switching (though the infected subsets as targets of EBV infection in vitro. Specifically, can naı̈ve cells remain responsive to exogenous switch signals) but cells acquire aspects of the memory cell Ig phenotype or genotype can induce Ig gene mutation. Thus, within 4 weeks of as a result of virus transformation? Here we show that EBV infecting naive B cell preparations, one can often detect infection per se does not alter the Ig phenotype of naive cells, cells carrying Ig mutations which appear to have arisen by although such infected cells remain susceptible to Ig class switch somatic hypermutation in vitro. Furthermore, in many induction by surrogate T cell signals. However, we did find cases such cells become dominant during clonal evolution evidence for Ig gene mutation among naive B cell transformants; of the emergent EBV-transformed cell line. Overall these thus, both under limiting dilution and bulk culture conditions, findings suggest a possible explanation as to why EBV is selection for successful LCL outgrowth from naı̈ve B cell infections selectively found in memory B cell populations in vivo and frequently involved clones with mutated Ig genotypes that appear why EBV-positive lymphoproliferative lesions/lymphomas to have arisen through virus-induced SHM in vitro. so frequently involve clones with mutated Ig genotypes. Results cytidine deaminase, AID [16,17], but are nevertheless distinct Characteristics of purified B cell subsets reactions that can take place independently of one another Circulating B cells, typically representing 4–8% of adult PBMC [18,19]. The small fraction of GC progeny cells with improved populations, were isolated by CD19 bead selection to purities affinity for antigen are then specifically selected by T cell-derived consistently.98%, as shown by staining for the pan-B cell marker survival signals, emerging as IgD2 CD27+ memory B cells; the CD20 (Figure 1A). Such preparations were co-stained with great majority of these are also IgM2 and have switched isotype to fluorochrome-labelled Abs to IgD and CD27 in order to identify IgG or IgA, (‘‘switched memory’’ cells). Given this the N (IgD+ CD272), NSM (IgD+CD27+) and SM (IgD2 CD27+) understanding of the physiology of memory cell selection, different B cell subsets. Naı̈ve B cells always represented the major subset, views have emerged as to how EBV might selectively colonise the accounting for 60–70% of total B cell numbers, with the other two IgD2 CD27+ memory cell pool. One view is that the virus first subsets each constituting between 8–25%. All three subsets were infects naı̈ve cells in vivo and, through mimicking the activation isolated to high purity by FACS sorting. Figure 1B illustrates the signals normally induced by cognate antigen, drives these cells to sort gates used and Figure 1C shows the results of re-analysing IgD initiate a GC reaction; the virus-infected clonal descendents of that and CD27 staining on the sorted populations. By these criteria, reaction thus acquire both the genotype and phenotype of purity was always.99% for N,.96% for NSM and.98% for memory cells via the natural process of GC transit, albeit with SM cell preparations. IgH sequence analysis further confirmed the virus-coded LMPs 1 and 2 substituting for affinity-based survival purity of these sorts. Thus, combining results from naive cell sorts signals. A second view, based mainly on the analysis of EBV- from 12 different individuals, 188 of 195 IgH sequences amplified infected B cell clones within IM tonsillar tissues, is that memory B from the N cell population were deemed to be non-mutated (i.e. cells are preferentially infected, or possibly have a proliferative/ less than 2 nucleotide changes from germline). In six of the above survival advantage during the phase of virus-driven B cell experiments, IgH sequencing was also extended to include the expansion, and that their progeny subsequently re-assume other subsets; as expected, the great majority of sequences memory characteristics with no requirement for GC transit. amplified from the NSM and SM cell populations (33/37 and An added complication to this debate is the more recent finding 64/66 respectively) were clearly mutated (Figure 1D). that EBV is harboured not just in the conventional IgD2 CD27+ memory pool of healthy virus carriers but also at lower Infectability of purified B cell subsets levels in a second distinct memory population comprising IgD+ Expression of the CD21 receptor and levels of virus binding CD27+ ‘‘non-switched memory’’ cells [20,22], that had hitherto were determined for the above B cell subsets from 4 successive been largely ignored in EBV studies. Although such cells do carry PBMC samples. Cell surface staining with a mAb to the EBV somatically mutated Ig genes, opinion is divided as to whether the receptor CD21 reproducibly showed that N, NSM and SM cells IgD+ CD27+ subset arises ontogenetically and is entirely express CD21 at similar levels (Figure 2A). Likewise these cells all independent of GC activity (as their presence in certain GC-null bound virus to similar amounts, as measured by exposure to a immune-deficiency states would imply [23,24]) or is populated at standard virus dose at 4uC to prevent internalisation of the virus, least in part by the products of abortive/incomplete GC reactions followed by extensive washing and quantitation of EBV genome involving Ig gene mutation without isotype switching [20,25–28]. copies bound per cell by quantitative PCR (data not shown). We EBV’s ability to colonise this subset in healthy individuals can then assayed matched N, NSM and SM preparations for therefore be explained in different ways depending upon one’s transformability using two different experimental designs. In one view of non-switched memory B cell origins. Interestingly set of experiments, N, NSM and SM cells were exposed to a however, in T cell-deficiency states where GC development is standard virus dose (50moi) before being seeded at a range of cell PLoS Pathogens | www.plospathogens.org 2 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Figure 2. Transformation efficiency of B cell subsets. (A) FACS analysis to measure surface CD21 expression on N (dotted line), NSM (thin line) and SM (thick line) B cell subsets. Staining with isotype control reagent is shown by shading. (B) Comparison of transformation efficiencies of N, NSM and SM B cell preparations in limiting dilution Figure 1. Sorting and purity of B cell subsets. (A) Isolation of cultures. Cells were exposed to EBV at 50 moi (EBV genomes per cell) for CD20+ B cells from peripheral blood. (B) Two colour FACS analysis of B 3 h, washed and then plated at 500 cells per well into 96 replicate wells cells stained with CD27 and IgD mAbs to identify IgD+ CD272 naı̈ve (N), of a 96 well plate containing irradiated fibroblast feeder cells. The IgD+ CD27+ non-switched memory (NSM) and IgD2 CD27+ switched numbers of wells containing growth transformed foci were scored after memory (SM) B cell populations. (C) Reanalysis to show purity of 6 weeks. Results are shown from two representative donors. (C) isolated N, NSM and SM B cell preparations. The figures in each panel Comparison of transformation efficiencies of N and NSM B cell indicate the purity of each isolated population D. Validation of cell preparations at a range of virus concentrations. Cells were exposed to purity by IgH analysis of sorted B cell subsets. 188 of 195 amplified IgH EBV at the indicated moi for 3 h, washed and plated at 1000 cells per sequences obtained from 12 independent naive B cell preparations well into replicate wells of a 96 well plate. The numbers of wells were classified as germline (0–2 mutations, open bars); by contrast, 33 showing transformation were scored after 6 weeks. Results are shown of 37 IgH sequences from 3 independent NSM B cell preparations, and from two representative donors. 64 of 66 IgH sequences from 6 SM B cell preparations were scored as doi:10.1371/journal.ppat.1002697.g002 mutated (shaded bars). doi:10.1371/journal.ppat.1002697.g001 comparing cultures derived from N (34%), NSM (30%) and SM densities in replicate wells in 96-well plates; in other experiments, (24%) cell infections. We therefore conclude that the N, NSM and N and NSM cells were exposed to a range of virus dilutions before SM subsets from peripheral blood are equally susceptible to EBV seeding into a 96-well plate at a standard cell number. In both infection and transformation in vitro. cases, end points in the transformation assay were scored after 6 weeks by microscopic inspection of cultures for characteristic foci Ig phenotype of EBV-transformed B cells of N-, NSM- and of EBV-transformed cells. Typical results from such experiments SM origin are shown in Figures 2B and 2C, and are expressed as the In several transformation experiments, we also set up LCLs percentage of replicate wells scoring positive at the limiting cell under non-limiting conditions by infecting 26106 N, NSM and seeding or limiting virus dose. Whilst absolute values for SM B cell preparations with EBV at 50moi and then culturing transformation efficiency varied between experiments, for any these cells in bulk, with subsequent expansion to bulk LCLs. To one individual donor the three B cell subsets always gave very investigate whether EBV induced transformation altered Ig isotype similar yields of transformed cultures. Thereafter, cultures scoring expression in the resultant lines, we first performed RT-PCR positive on plates set up under limiting conditions were first split analysis of IgH transcripts using isotype-specific primer combina- into duplicate wells and, where possible, further expanded over the tions. Figure 3A shows typical results obtained, using reference B following 3–6 weeks to yield a limiting dilution (LD)-LCL for cell lines of known isotype restriction and the Ig-negative Jurkat T analysis. Note that only a proportion of the wells transformed cell line as internal controls. Bulk lines derived from the N and under limiting conditions could be successfully expanded in this NSM cell subsets were consistently positive for IgM transcripts and way; overall, however, that proportion was not greatly different weakly positive for IgD but negative for the other isotypes. By PLoS Pathogens | www.plospathogens.org 3 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro IgA2) either untreated or exposed to CD40L/IL-4/IL21 for 7 days. Isotype control staining is shown in black lines; IgD, IgG and IgA staining shown in pink, green and red lines, respectively. doi:10.1371/journal.ppat.1002697.g003 contrast, bulk lines of SM cell origin expressed IgG and IgA transcripts, often accompanied by a weak signal for IgM but never for IgD; note that the weak IgM signal accords with the fact a very small proportion of cells within sorted IgD2 CD27+ memory populations are so-called ‘‘IgM-only’’ cells with an IgM+ IgD2 CD27+ phenotype. The clear inference from these transcriptional data, that viral transformation had not induced detectable class switching, was strongly supported by the results of staining with mAbs specific for IgM, IgD, IgG and IgA heavy chains. Figure 3B shows typical results where both the N-derived and NSM-derived bulk LCLs retained an IgM+ IgD+ phenotype, whereas the SM-derived LCL was dominated by IgG+ and IgA+ cells. Note also that, as others have reported , EBV transformation induces N cells to express CD27 such that all three groups of LCLs shared a CD27+ phenotype (data not shown). Table 1 summarises the Ig isotype data both from bulk LCLs of N, NSM or SM origin generated as above, and from the LD-LCLs expanded from cultures of all 3 B cell subsets as described earlier. Again, all N- and NSM-derived cell lines from bulk and LD cultures were IgM+ IgD+; by contrast the SM-derived bulk lines were predominantly mixtures of IgG+ and IgA+ cells, sometimes with a small IgM+ component, whereas SM-derived LD cultures were either only IgG+, only IgA+ or mixtures of both. Responsiveness of EBV-transformed B cells to isotype- switch signals We then asked whether EBV-transformed cells, though not induced to undergo isotype switching by viral infection, were still capable of switching given signals that mimic physiologic T cell help. CD40L and IL-4 stimulation is known to induce isotype switching to IgG in freshly isolated N cell preparations in vitro [32– 34], with switching also to IgA in the presence of IL21. Early passage bulk cultures (2–3 weeks post-infection) of N- and NSM-B cell origin were exposed to these inducing signals for 10 days; parallel cultures being maintained under normal conditions as a control. Figure 3C shows the results from an N-derived culture, typical of that seen generally with early passage cultures of N or NSM origin. Untreated cultures were uniformly IgD+ and lacked Table 1. Ig phenotype of N-, NSM- and SM-derived LCLs. No. IgM+ IgM+ Culture tested IgD+ IgG+ IgA+ IgD2 Mixed N LCL, bulk 13 13 0 0 0 0 N LCL, LD{ clone 82 82 0 0 0 0 Figure 3. Immunophenotype of LCLs derived from different B NSM LCL, bulk 4 4 0 0 0 0 cell subsets. (A) RT-PCR analysis of IgH gene expression. RNA from two naive (N)-LCLs, two non-switched memory (NSM)-LCLs and one NSM LCL, LD{ clone 8 8 0 0 0 0 switched memory (SM)-LCL was subjected to RT-PCR using a common SM LCL, bulk 6 0 0 0 0 6* primer in IgHV and a reverse primer specific for IgM, IgD, IgG or IgA SM LCL, LD{ clone 45 0 16 23 0 6{ transcripts. PCR products were analysed by agarose gel electrophoresis and visualised by ethidium bromide staining. Also shown are results { LD indicates cultures established under conditions of limiting cell numbers. from control IgG+, IgA+ and IgM+ IgD+ LCLs, while RNA from Jurkat cells *indicates bulk LCL cultures derived from infection of switched memory (SM) B was included as a negative control. (B) Two colour FACS analysis of bulk cells and containing predominant populations of IgG+ and IgA+ cells with small N, NSM and SM B cell-derived LCLs after dual staining cells with either numbers of IgM+ IgD2 cells. RPE-labelled anti-IgD and FITC-labelled anti-IgG Abs or RPE-labelled { indicates six LD LCLs derived from infection of SM B cell and containing both anti-IgM and FITC-labelled anti-IgA Abs. (C) FACS staining of surface IgG+ and IgA+ cells. IgD, IgG and IgA in a representative N-derived LCL (initially IgD+ IgG2 doi:10.1371/journal.ppat.1002697.t001 PLoS Pathogens | www.plospathogens.org 4 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro IgG and IgA, whereas significant fractions of cells in cultures In this experiment, of 25 cultures analysed, there were 13 exposed to CD40L+IL4+IL21switched to IgG or IgA, with monoclonal cultures (LCL6-1 to 6-13) and 2 biclonal cultures concomitant loss of IgD. (LCL6-14, 6-15) with non-mutated genotypes. Surprisingly, however, there were also 7 monoclonal cultures with mutated Ig genotype of EBV-transformed limiting dilution cultures genotypes (LCL6-16 to 6-22), plus 3 biclonal cultures (LCL6-23 to of N, NSM and SM origin 6-25) with both mutated and non-mutated genotypes. Figure S1 We now examined the Ig genotype of all LD-LCLs established presents the actual sequences for three of the above cultures; from the N, NSM and SM cell preparations whose pre-infection Ig LCL6-2, LCL6-18 and LCL6-20 with 0, 5 and 7 mutations, genotypes had been analysed in Figure 1D. For each LD-LCL, we respectively. sequenced several cloned IgH PCR products, identified the The above pattern of results, with a number of N-derived LD- constituent IgH V, D and J alleles and then assigned the sequences LCLs showing significant levels of IgH mutation, was observed in to individual CDR3 clones. By these criteria, the majority of LD- 8 successive experiments, each involving a different naive B cell LCLs (whether derived from N, NSM or SM cells) were preparation. Combining data from all 8 experiments, we analysed dominated by a single cellular clone; in addition, some were 594 IgH sequences from 140 LD cultures of EBV-infected N cell oligoclonal with 2 or 3 different clonotypes. Individual sequences preparations, and within these identified 198 distinct clones. The within each identified clone were assigned as germline or mutated majority of these cultures (89/140) yielded only germline as described above. sequences, usually with a single CDR3 clonotype or in some cases Table 2 shows results from one such experiment, here focusing with two co-resident clonotypes. However, the other 51/140 N- only on infections of the N cell subset. The individual clonotypes derived cultures yielded mutated sequences, in most cases in the detected within each LD culture are identified through their IgH absence of any detectable germline sequence. Overall, 72 of 198 V, D and J alleles and their signature CDR3 amino acid sequence. (36.3%) individual clonotypes identified within these cultures were Table 2. IgH genotype of LD LCL clones derived by naive B cell infection (donor 6). LD LCL IGHV IGHD IGHJ CDR3 translation Mutations 6-1 V3-11*01 D5-24 J6*02 CAREWGGYKPLTWVDYYCGMDVW 0 6-2 V3-23*01 D6-19 J4*02 CAKDRLAVAVFWDYW 0 6-3 V4-04*02 D3-3 J3*02 CARSITIFGVVSAELDAFDIW 0 6-4 V1-08*01 D4-17 J4*02 CARGGYYEGFDYW 0 6-5 V1-69*01 D6-13 J4*02 CASASRDSSSWYERPFDYW 0 6-6 V3-48*03 D3-22 J4*02 CARDQGGVTTGAEFDYW 0 6-7 V3-23*04 D3-10 J6*03 CAKEWVGSGSYYGKPTAGYYYYMDVW 0 6-8 V4-b*01 D6-19 J4*02 CARTLVVVDW 0 6-9 V3-74*02 D3-3 J4*02 CARTRITIFGVANLDYW 0 6-10 V3-48*03 D5-5 J4*02 CARDRSRDTAMVVFDYW 0 6-11 V3-11*01 D4-17 J6*02 CARDYGDYGAWDYYYYGMDVW 0 6-12 V5-51*01 D3-10 J4*02 CARQSFGAYYFDYW 1 6-13 V4-59*01 D3-10 J4*02 CARAPAITMVRGVIEYYFDYW 2 6-14 V3-15*01 D5-12 J4*02 CTTDQWLSWEAELCW 0 V1-69*06 D6-13 J4*02 CARDSSSSWYYFDYW 1 6-15 V4-31*01 D2-21 J6*02 CARVRLAGPLARNYYYGMDVW 1 V5-51*01 D6-19 J5*02 CARHEEAGEVSWFDPW 1 6-16 V3-30*04 D4-23 J4*02 CAREATEVPFDSW 3 6-17 V4-4*02 D2-2 J4*02 CARGRFEEDYW 4 6-18 V1-58*01 D3-10 J5*02 CAVELWFGDIRWFDPW 5 6-19 V3-49*04 D1-26 J6*02 CTNSGSYYFYGVDVW 6 6-20 V4-59*01 D6-19 J4*02 CASGSSGWLYYFDYW 7 6-21 V3-48*03 D3-10 J4*02 CAVLGSEYSDEPFDYW 9 6-22 V1-3*01 D4-11 J6*02 CARDPRTVTRHSYYYYMDVW 9 6-23 V3-21*01 D5-12 J4*02 CARGDRGDTLRVMDYW 0 V3-21*01 D5-12 J4*02 CARGDRGDTLRVMDYW 3 6-24 V3-21*01 D1-26 J4*02 CAREELRGYWG 0 V1-69*01 D3-22 J4*02 CARNQDTSGSLQFDYW 3 6-25 V3-21*01 D4-17 J4*02 CARMTTVTRLVDYW 0 V3-21*01 D3-3 J6*02 CVRAPWSGDYFYYYGLDVW 5 doi:10.1371/journal.ppat.1002697.t002 PLoS Pathogens | www.plospathogens.org 5 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro represented by mutated sequences. As a comparator, in 6 of the the derived LD-LCL cultures (shaded bars). The histograms show above experiments we also analysed 38 NSM-derived and 55 SM- the total number of unique clonotypes observed in relation to their derived LD cultures from infections of the memory cell subsets. degree of divergence from germline, sequences with 0, 1 or 2 Within these we identified 54 and 96 resident clones, respectively, changes deemed non-mutated. Note that the degree of IgH most cultures being dominated by a single or by two co-resident sequence divergence among NSM-derived and SM-derived LD- clonotypes. Not surprisingly, given the predominance of mutated LCLs is not markedly different from that within the matched pre- IgH sequences present in these B cell subsets pre-infection, all of infection populations. More importantly, however, mutated IgH the NSM-derived and all but two of the SM-derived cultures sequences are much more common in LD-LCLs derived the N cell carried mutated IgH sequences. subset than in their pre-infection counterparts. Figure 4 summarises the overall IgH genotype data from the N, NSM and SM cell preparations pre-infection (open bars) and from Characterising N-derived LD-LCLs with mutated and non- mutated Ig genotypes We were interested to know whether the frequent appearance of clones with mutated Ig genotypes in LD-LCLs derived from N cell infections reflected a growth rate advantage that these cells enjoyed, perhaps one that might be linked to differences in the degree to which cells were leaving the EBV-transformed latent state and entering lytic cycle. Early passage freezings of the emergent N-derived LCLs from individual experiments were therefore taken from cryostorage and compared in proliferation assays. Figure 5A shows typical results from one such experiment comparing 5 mutated and 4 germline clones from the same donor. The emergent LD-LCLs varied considerably in their growth rates, but this was not related to their Ig genotype status; clones with mutated genotypes (closed symbols) and clones with germline genotypes (open symbols) showed a similar spread of growth rates. Furthermore, as is clear from the immunoblots in Figure 5B, neither Ig genotype status nor growth rate showed any obvious correlation with expression levels of EBV latent proteins (here illustrated using the key transformation-associated proteins EBNA1, EBNA2 and LMP1) or with the degree of lytic cycle entry as detected from levels of the immediate early lytic protein BZLF1. Thus, although cells with mutated Ig genotypes are well represented among LD-LCLs from naive cell infections, this is not because they have an inherently faster growth rate than cells carrying germline IgH sequences. Ig genotype of mitogen-activated versus EBV- transformed bulk cultures of N origin The above evidence for mutated Ig genotypes in N-derived LCLs came entirely from mono- or bi-clonal populations expanded from individual limiting dilution cultures, i.e. from wells scored positive at the end of the 6 week transformation assay. Because many such wells still contained just small foci of transformed cells at that time, limitations on cell numbers precluded any prospective analysis of these cultures during their early period of expansion; as a result, sampling of LD-LCLs for genotypic analysis was often delayed until 9–12 weeks post-infection. As a second approach therefore, we turned to the analysis of resident clonotypes within bulk N cell cultures, harvesting aliquots of the same culture at regular intervals up to 12 weeks post infection. This allowed us to compare the clonal composition within an expanding EBV-infected culture with that seen in a matched culture driven to expand by repeated exposure to a non-viral proliferative trigger, CD40L/IL4. In our hands, this Figure 4. IgH mutation frequency in uninfected B cell subsets latter protocol induces expansions within the first 9 weeks which are and derived LCLs. Histograms show the number of mutations among at least as the equal of those driven by EBV, after which IgH clonotypes amplified from naı̈ve, non-switched memory and proliferation slows to a halt. Note that such mitogen-driven switched memory B cell preparations before EBV infection (open bars) proliferation has never been reported to induce Ig gene mutation and among sequences amplified from the derived limiting dilution LCLs [36–38]. (shaded bars). Mutations were only scored between IgH codons 9–92 (i.e. excluding CDR3). Where LD LCL cultures yielded multiple sequences We first used the approach of IgH CDR3 spectratyping to gain with differing numbers of mutation within the same CDR3 clone, the an overall picture of CDR3 length distribution (i.e. clonality) in the sequence with the maximum number of mutations was scored. two types of culture. Data from one such experiment are shown in doi:10.1371/journal.ppat.1002697.g004 Figure 6A. Clearly the CD40L/IL4-activated culture remains PLoS Pathogens | www.plospathogens.org 6 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Figure 5. Characterisation of LCL cultures derived from naive B cells. (A). Growth rate analysis of representative LD-LCLs, all derived from the same naive B cell preparation, were measured over 7 days. LCLs with germline IgH alleles are shown by the open symbols and LCLs with mutated IgH alleles are shown by solid symbols. (B). Immunoblots showing expression of EBV antigens EBNA1, EBNA2, LMP1 and BZLF1 in the same LD-LCLs as shown in panel (A). Bjab was used as a EBV-negative control; X50/7 LCL was used as a positive control for EBNA1, EBNA2 and LMP1, while Akata-BL cells induced with anti-IgG into lytic cycle were used as the control for BZLF1 expression. Calregulin was used as a loading control. doi:10.1371/journal.ppat.1002697.g005 polyclonal despite 9 weeks of in vitro expansion, retaining a similar lengths at both 6 and 9 weeks post-infection. The same samples Gaussian distribution of CDR3 lengths to that seen in the original were analysed by IgH sequencing as before, and the corresponding starting population. In contrast, the parallel EBV-transformed data are shown in Table 3. Thus all 18 sequences amplified from culture is dominated by sub-populations with distinct CDR3 the initial N cell population ex vivo were unique and 17/18 of these Figure 6. CDR3 spectratype analysis of mitogen-activated B blasts and LCL cultures derived from naive B cells. (A) Results of capillary gel electrophoresis of IgH PCR amplification products from uninfected naı̈ve B cells, B CD40L/IL4 stimulated naive B blasts and naive B cell-derived LCLs established from donor 11 (tested at 6 and 9 weeks). A bell-shaped curve of PCR product peak sizes seen in the uninfected control culture and in the naı̈ve B blast cultures indicates a polyclonal B cell population, while the small number of distinct peaks visible in the LCL cultures indicate that the population is dominated by a small number of clones. (B) Results of capillary gel electrophoresis of IgH PCR amplification products from naive B cell- derived LCLs established from donor 8. While a broad distribution of CDR3 lengths is observed in the uninfected control culture and 4 week LCL, only a small number of distinct peaks are visible in the LCL cultures at later time points indicating that the population is evolving towards monoclonality. doi:10.1371/journal.ppat.1002697.g006 PLoS Pathogens | www.plospathogens.org 7 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Table 3. IgH genotype of uninfected naive B cells, mitogen stimulated naive B blasts and a bulk LCL culture derived by naive B cell infection (donor 11). IgH sequence IGHV IGHD IGHJ CDR3 translation Mutations Naive B cells 1 V3-48*01 D3-10 J6*02 CARARHRWFGENYYYYGMDVW 0 2 V5-a*01 D6-19 J4*02 CARPDPGAAASGWYNW 0 3 V3-21*01 D3-10 J6*02 CARDPTVWFGEFEQHHPYYYYGMDVW 0 4 V3-23*01 D2-15 J3*02 CAKDRGYCSGGSCRDAFDIW 0 5 V4-59*01 D3-9 J4*02 CARGGAGYFDWDGDYW 0 6 V5-51*01 D1-1 J6*02 CARHFTTGYYYGMDVW 0 7 V1-24*01 D3-16 J6*02 CATPWGWGIFGTYYGMDVW 0 8 V5-51*01 D6-6 J4*02 CARHASIVDYYFDYW 0 9 V5-51*01 D2-21 J4*02 CARAGRDDFLDYW 0 10 V3-23*04 D3-3 J4*02 CANLGAYDFWSGYYDLSTDW 0 11 V4-59*01 D3-22 J5*01 CARASSGYYRRFDYW 0 12 V4-39*01 D3-10 J4*02 CARHQDYYVCPPDYW 0 13 V3-48*02 D2-2 J6*02 CASKVVPAAIGYYGMDVW 0 14 V4-31*03 D5-12 J6*02 CARGLWWLQYYGMDVW 0 15 V3-07*03 D4-4 J6*02 CARGMTTVTGYYYYGMDVW 0 16 V3-07*03 D2-15 J6*02 CARDDAGMGCSGGSCYSVSYYYYSMDVW 0 17 V4-31*03 D4-17 J5*02 CASSSTVTMGWFDPW 1 18 V3-11*03 D6-19 J6*02 CARDLRVDSSGWKGDYYYGMDVW 4 Naive B blasts 1 V3-15*07 D2-15 J6*02 CTTEGKPVAATQWGYYYYDMDVW 0 2 V1-46*01 D1-26 J6*02 CAGSTLVGAPWDYYYGMDVW 0 3 V4-31*03 D2-21 J5*02 CARGYCGGDCYMGGPW 0 4 V3-23*01 D4-23 J4*02 CAKEEWTTVVPGVFDYW 0 5 V5-a*01 D6-19 J4*02 CARPPKNSIAVAGARDYW 0 6 V3-21*01 D5-5 J6*02 CARDRAIDTAMWDYGMDVW 0 6 V4-31*03 D1-26 J2*01 CARDGNQWETIRW 0 7 V3-53*01 D6-19 J6*02 CARAGVGHYYYYGMDVW 0 8 V1-2*02 D1-26 J5*02 CARDLEWEPRRGNWFDPW 0 9 V3-21*01 D4-17 J4*02 CARDQGDYVVWHYFDYW 0 10 V3-21*01 D3-22 J4*02 CARDPGQPKNYYDSSGASGGYW 0 11 V6-1*01 D3-3 J4*02 CASSFGVGLDYW 0 12 V4-61*01 D4-23 J5*02 CASLSITMIVAW 1 13 V3-23*01 D4-23 J4*02 CAKVGGKRITMIVVGSW 1 14 V3-15*07 D6-6 J6*02 CTRPPSGMDVW 1 15 V4-4*07 D2-15 J6*02 CVGEVVVVAATPSYYYGMDVW 1 16 V4-31*03 D5-24 J5*02 CARDGVEMATRRAPNWFDPW 1 17 V1-69*09 D6-13 J6*02 CARPPGGGARTGYYGMDVW 1 Bulk LCL derived from naive B cell infection 1 V4-31*03 D6-19 J5*02 CARVRPRIAVAGTGGWFDPW 0 2 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 3 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 4 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 5 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 6 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 7 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 8 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 9 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 PLoS Pathogens | www.plospathogens.org 8 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Table 3. Cont. IgH sequence IGHV IGHD IGHJ CDR3 translation Mutations 9 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 10 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 11 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 12 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 13 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 14 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 15 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 16 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 17 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 18 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 19 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 20 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 21 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 22 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 23 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 24 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 0 25 V1-58*01 D3-16 J6*02 CAADFTFGGVIASSSYYYYGMDVW 1 26 V3-7*03 D6-13 J4*02 CALRIAAAGTRALPFDYW 2 27 V3-7*03 D6-13 J4*02 CALRIAAAGTRALPFDYW 3 28 V3-7*03 D6-13 J4*02 CALRIAAAGTRALPFDYW 3 29 V3-7*03 D6-13 J4*02 CALRIAAAGTRALPFDYW 3 30 V4-4*07 D3-22 J4*02 CARASQGYDSSGYYSYFFDYW 3 31 V4-4*07 D3-22 J4*02 CARASQGYDSSGYYSYFFDYW 3 32 V4-4*07 D3-22 J4*02 CARASQGYDSSGYYSYFFDYW 3 33 V4-4*07 D3-22 J4*02 CARASQGYDSSGYYSYFFDYW 4 34 V3-30*03 D5-5 J4*02 CAKTPRVATIMYYFDYW 5 doi:10.1371/journal.ppat.1002697.t003 were germline; likewise all 17 sequences amplified from the bulk Such EBV-infected bulk cultures were generated from six culture after 9 weeks expansion by CD40L/IL4 were unique and independent donors, in three cases with a matched CD40L/IL4- non-mutated. However, the parallel EBV-transformed culture stimulated B blast culture. Figure 7 presents a summary of the IgH analysed at 9 weeks contained only 5 distinct clonotypes, three of genotype data from the two types of cultures expressed as which appeared from their frequent detection to represent histograms; the height of the open bar indicates the percentage of numerically dominant cell clones. Of these three dominant clones, sequences with a particular level of mutation frequency that were one (in this case, the most frequent) had a germline IgH sequence amplified from the bulk cultures at different times, while the while the other two had 3–4 mutations relative to the nearest shaded area of the bar reflects the proportion of those amplified germline sequence. sequences that were members of a clonal family i.e. were detected In some experiments, greater cell yields in the N-subset sort twice or more. Thus, in starting polyclonal populations, all IgH allowed more frequent sampling of the EBV-infected bulk cultures. sequences are unrelated to one another and the great majority are The CDR3 spectratyping data from one such experiment are non-mutated. This remains the pattern seen at all times in the shown in Figure 6B, clearly showing that the population remains CD40L/IL4-expanded B blasts, whereas in EBV-infected cultures, broadly distributed in terms of CDR3 size up to 4 weeks post- the situation changes with time post-infection. Early on these infection but becomes much more focused by 6 weeks and further cultures are also polyclonal and non-mutated but, at around 4 focused by 9 and 12 weeks. The corresponding IgH sequence data weeks post-infection, mutated IgH sequences appear in significant from this experiment are presented in Table 4. They show that the numbers. From 4 to 12 weeks post-infection, the cultures then EBV-infected N cell culture is composed of multiple unique clones become increasingly dominated by a small number of individual both at 2 weeks, when all amplified sequences were germline, and clones; in some cases (e.g. Table 3) the most abundant clonotype is also at 4 weeks, at which point the first mutated sequences appear non-mutated, while in other cases (e.g. Table 4) it is mutated, as minor components. By 6 weeks, three clonotypes (one of which much as seen earlier in the limiting dilution culture experiments. was previously seen at week 4) together account for about half the amplified sequences and by 9–12 weeks the culture is dominated Mutations within the Ig gene sequences reflect SHM by just one of these clonotypes, with another remaining as a minor targeting component. In this case, the dominant IgH clonotype in the N- To ask whether the above Ig sequence changes seen in vitro derived bulk LCL is clearly mutated. might be products of SHM, we first screened N, NSM and SM PLoS Pathogens | www.plospathogens.org 9 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Table 4. Sequential analysis of the IgH genotype of a bulk LCL culture derived by naive B cell infection (donor 8). IgH sequence IGHV IGHD IGHJ CDR3 translation Mutations 2 week LCL 1 V4-59*01 D3-10 J6*02 CARHSGITMVRPLDYYYYYGMDVW 0 2 V1-46*01 D3-16 J4*02 CARDGGFVGATDFDYW 0 3 V1-18*01 D3-9 J4*02 CARSTGGYDILTGYFPFDYW 0 4 V1-58*01 D2-2 J6*02 CAAQNDIVVVPAAMGIGYGMDVW 0 5 V4-61*01 D3-22 J5*02 CARYEYYYDSSGFYWFDPW 0 6 V4-59*01 D1-26 J1*01 CARETIVGATTAYFQHW 0 7 V1-69*01 D3-22 J2*01 CASSSGYPDWYFDLW 0 8 V5-a*01 D6-19 J4*02 CARLSGWYSESHYW 0 9 V3-15*07 D6-19 J4*02 CTTGIRIAVAGPSFDYW 0 10 V4-31*03 D5-5 J4*02 CARENNEGSPFDYW 0 11 V4-31*01 D4-17 J4*02 CARSPRLRGPYYFDYW 0 12 V1-18*01 D3-3 J6*02 CARGSYDFWSGYYDYGMDVW 0 13 V1-46*01 D3-22 J5*02 CVTDSSGFHWFDPW 0 14 V5-51*01 D5-5 J4*02 CARHVTAMADYW 0 15 V3-48*03 D2-2 J6*02 CARDGVPAAMGYYYYYGMDVW 0 16 V3-30*01 D3-10 J6*02 CARDPGVTYYYGMDVW 0 18 V4-31*03 D2-2 J4*02 CARQVAAAVDYW 0 19 V1-3*01 D1-1 J4*02 CARGPTDYW 0 20 V3-74*01 D2-15 J6*02 CARRGGSDIYYYYGMDVW 0 22 V3-48*03 D4-4 J6*02 CARDYSNYDYYYYYGMDVW 0 23 V3-23*01 D3-22 J4*02 CAKSSNYYDSSGYYSAW 0 25 V5-51*01 D3-10 J6*02 CARLYYGSGPWGMDVW 0 26 V3-30*03 D5-12 J6*02 CARAGTRYYYYGMDVW 0 29 V1-69*06 D3-10 J6*02 CASSNTVLGGDYYYGMDVW 0 21 V1-2*04 D3-3 J3*02 CARDGNLGRGDAFDIW 1 24 V4-59*01 D3-3 J4*02 CARSPPGLNNYFDYW 1 30 V5-51*01 D3-16 J6*02 CARLGAAMVFYYGMDVW 1 31 V4-39*01 D5-5 J5*02 CARPRHDITMIVSW 1 32 V1-24*01 D2-15 J6*02 CACCSRSEDYYYGMDVW 1 4 week LCL 1 V4-61*01 D6-6 J4*02 CARDSSAARPDYW 0 2 V4-39*01 D2-2 J3*02 CARAGGKLVAAFDIW 0 3 V4-59*01 D3-16 J5*02 CARSGQVRFGFDPW 0 4 V1-69*01 D2-21 J6*02 CARAGGRDPPYYYYYYGMDVW 0 5 V4-59*01 D3-22 J4*02 CARAVGGPPFFNSSGYPIFDYW 0 6 V5-51*01 D6-19 J4*02 CARLQDSPPDYW 0 7 V1-46*01 D5-5 J4*02 CAREAVRYSYGHDYW 0 8 V1-f*01 D3-22 J4*02 CATARSTGYLAYW 0 9 V5-51*01 D4-4 J6*02 CARYGSYPYYYYGMDVW 0 10 V4-31*03 D5-5 J4*02 CARDGYGLDYW 1 11 V3-7*03 D1-1 J3*02 CARDTNWNGAPSAFDIW 1 12 V3-21*01 D6-13 J6*02 CARDVGSSSWYNYYGLDVW 1 13 V4-39*03 D1-26 J4*02 CARAASGSLDYW 1 14 V3-30*07 D3-22 J4*02 CAKSRYYYDSSFDYW 2 15 V3-23*04 D6-19 J4*02 CARGSSGWW 2 16 V4-59*01 D3-3 J4*02 CARVRLSGWYYFDYW 3 17 V4-4*02 D1-26 J4*02 CARDQGSYYGRWIDYW 5 18 V4-b*01 D1-7 J4*02 CARVSLEAVXFDYW 5 PLoS Pathogens | www.plospathogens.org 10 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Table 4. Cont. IgH sequence IGHV IGHD IGHJ CDR3 translation Mutations 19 V1-46*01 D6-19 J6*02 CARDSSGWYSTGYGMDVW 6 6 week LCL 1 V1-69*01 D4-23 J6*02 CARESTGDYYYYYGMDVW 0 2 V4-31*03 D5-24 J6*02 CARELKRWLQSGGGMDVW 0 3 V4-b*02 D3-22 J6*02 CARDTYYDSNGMDVW 0 4 V4-59*01 D1-26 J5*02 CARDLGSQWELGPW 0 5 V4-59*01 D1-26 J5*02 CARDLGSQWELGPW 4 6 V5-51*01 D4-23 J6*02 CARHGGGNSRYYGMDVW 0 7 V4-39*01 D4-23 J3*02 CLGGNDAFDIW 1 8 V5-51*01 D3-22 J4*02 CARQTDDSSGYYDYW 1 9 V4-59*01 D6-19 J6*02 CASMPSIAVAGDYYYYGMDVW 1 10 V3-64*05 D6-19 J6*02 CVKEGSSYYYYYYGMDVW 2 11 V3-23*04 D6-19 J4*02 CARGSSGWW 2 12 V3-23*04 D6-19 J4*02 CARGSSGWW 5 13 V3-48*02 D3-10 J6*02 CARVRGWTTYGMDVW 2 14 V3-48*02 D3-10 J6*02 CARVRGWTTYGMDVW 5 15 V3-48*02 D3-10 J6*02 CARVRGWTTYGMDVW 6 16 V3-48*02 D3-10 J6*02 CARVRGWTTYGMDVW 6 17 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 2 18 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 19 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 20 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 21 V3-23*04 D2-2 J6*02 CAKDLGYCSSTSCYADRYGMDVW 4 9/12 week LCL 1 V3-23*04 D6-19 J4*02 CARGSSGWW 2 2 V3-23*04 D6-19 J4*02 CARGSSGWW 2 3 V3-23*04 D6-19 J4*02 CARGSSGWW 3 4 V3-23*04 D3-3 J4*02 CAKGCGGVKTIGCDCW 5 5 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 4 6 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 7 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 8 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 9 V3-23*04 D3-10 J6*02 CAKGRGFGELHGMDVW 7 10 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 11 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 12 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 13 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 14 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 15 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 16 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 17 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 18 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 19 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 20 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 21 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 22 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 23 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 24 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 25 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 7 PLoS Pathogens | www.plospathogens.org 11 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Table 4. Cont. IgH sequence IGHV IGHD IGHJ CDR3 translation Mutations 26 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 8 27 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 8 28 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 8 29 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 8 30 V3-23*04 D3-10 J6*02 CAKDRGFGELHGMDVW 8 31 V3-23*04 D2-2 J6*02 CAKDLGYCSSTSCYADRYGMDVW 7 doi:10.1371/journal.ppat.1002697.t004 subsets pre- and post-infection for expression of AID, an enzyme IgD+ phenotype of the mutated LCLs discounted their being which is essential (but itself not sufficient) for SHM to occur. In derived from isotype-switched SM cells. However we could not each case, AID transcription was undetectable before infection and formally discount a contribution from NSM contaminants from was activated by EBV. Figure S2A shows the relevant data from N the Ig phenotype since both N-derived and NSM-derived cell preparations. Interestingly, even though all cells in the culture transformants would give the same IgM+ IgD+ LCL signature. were actively infected and proliferating by day 7 post-infection, AID The possibility of resolving this issue by genotype was raised by a levels rose only slowly, not reaching their steady state level until day recent report that a proportion of NSM cells carry mutations 35, kinetics that are at least compatible with the delayed appearance in the Bcl6 intronic major mutation cluster (MMC), mutations that of mutated IgH sequences in such cultures. However, we found no (as in conventional memory cells) are thought to arise through correlation between AID expression and Ig gene mutation status. SHM mis-targeting during a cell’s residence in GCs where Bcl6 is Thus AID transcript levels were similar in N-derived LCLs with and highly expressed [44,45]. If such mis-targeting is indeed dependent without mutation as well as in NSM- and SM-derived lines; upon active bcl6 transcription, then this would not be expected to interestingly these AID levels were not only lower than seen in occur during EBV-induced B cell transformation in vitro because, freshly-isolated GC B cells and in 4 Burkitt lymphoma (BL)-derived as already reported and as we confirmed in the present work cell lines, included as SHM-positive controls, but also much lower (Figure S3), Bcl6 expression is suppressed by growth-transforming than those in CD40L/IL4-stimulated B blasts which lack detectable EBV infection. We therefore selected 18 LD-LCLs that had a SHM (Figure S2B,C). Moreover N-derived LCLs with and without mutated Ig genotype and were derived from N cell preparations, IgH mutations gave similar results in quantitative RT-PCR assays amplified a 718bp region of the Bcl6 MMC, then cloned and specific for alternatively-spliced AID mRNAs and for the Polg and sequenced multiple independent amplification products. As UNG co-factors involved in the SHM process [40,41] (Figure S2C). internal controls, we included parallel amplifications from sorted As a second approach, we asked whether the IgH gene naive and memory B cell populations ex vivo. As shown in Table mutations seen in N-derived LCLs had the hallmark of SHM S1, 17/18 Bcl6 MMC sequences amplified from naive B cells were targeting by mapping the location of all IgHV sequence changes germline, whereas 10/24 sequences from memory cells were seen in pre-infection N, NSM and SM populations and in their mutated; such findings are in close accord with an earlier report derived LCLs. To avoid distortion of the LCL data by numerically. Turning to the Ig-mutated LD-LCLs from N cell infections, dominant clones, here any one mutated clonotypic sequence we detected both germline and mutated allelic sequences in just 2 identified within an LCL only contributes once to the cumulative lines; the great majority of lines (16/18) only ever yielded germline data. The overall findings are summarised in Figure 8, where the sequences. Overall, therefore, the bcl6 data are again consistent height of the bars indicates the number of times a change in a with the majority of these Ig-mutated LD-LCLs being truly particular IgHV codon was identified. Sites in the IgHV sequence derived from naive cells. known to be favoured by the SHM machinery (hot-spots) [42,43] are identified by filled bars. Focusing first on the data from pre- infection B cell subsets, as already described we found very few Intra-clonal diversification of Ig sequences in EBV- mutations in the N-subset, while the NSM and SM subsets showed transformed bulk cultures of N subset-origin the expected distribution of mutated sites, with frequent To pursue the question another way, we reasoned that if the involvement of known SHM hotspots and avoidance of coldspots. mutated clonotypes arising in EBV-infected N cell cultures were Not surprisingly, the NSM- and SM-derived LCLs showed a truly being generated from naive precursors in vitro, then we should similar distribution of mutations as in their pre-infection be able to detect evidence of intra-clonal sequence diversification populations. However, it was striking that the same pattern of within such cultures. In this regard, 28 of the 72 LD-LCLs in which distribution was also seen among the mutated IgH clonotypes the dominant clonotypic sequence was mutated also contained found in N-derived LCLs, at least consistent with these changes variants (typically with 1 to 3 nucleotide changes) of that sequence at being a product of SHM. the single point of harvesting. More informative, however, are the Such findings nevertheless leave open the possibility that the data from bulk N-cell infections sampled over time. Summing data mutated clonotypes frequently detected in N-derived LCLs have from all 6 experiments, each involving a different N-cell donor, we not arisen from authentic naive cells induced into SHM in vitro identified a total of 17 mutated clonotypes that were present on two but from memory cells already carrying IgH mutations that were or more occasions in the same culture between 4 and 12 weeks post- present as minor contaminants of the original N cell preparations. infection; of these, 14/17 clonotypes showed evidence of intra- Note that the transformation assays on sorted B cell subsets clonal sequence variation, with a total range of 1–12 sequence (Figure 2) implied that memory cell contaminants would enjoy no changes per clonotype. Two such examples are presented in competitive advantage in such a situation. Furthermore, the IgM+ Figure 9, where in each case the variants can be linked into family PLoS Pathogens | www.plospathogens.org 12 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro this was GC-dependent, involving preferential infection of naı̈ve cells that the virus then induced into memory via GC transit , or GC-independent, involving preferential infection/expansion of pre-existing memory cells. Neither view can fully accommo- date the more recent finding that EBV also colonises the IgD+ CD27+ NSM subset, not just in healthy carriers but also in patients congenitally devoid of GCs and therefore of SM cells; in such patients the NSM subset is hugely outnumbered by naı̈ve B cells (and is therefore very unlikely to be a preferential target of primary infection) yet it harbours essentially all the latent virus. Given the difficulties posed by such in vivo findings, the present work took the reductionist approach of focusing on experimental infection of N, NSM and SM cells in vitro. This showed that, while EBV itself does not induce Ig isotype switching, N-derived LCLs remain susceptible to switching induced by surrogate T cell signals. More importantly, in at least a proportion of N cells, EBV infection induces IgH sequence changes which bear the hallmarks of SHM; furthermore, B cell clones with such changes frequently become numerically dominant as the emerging LCL evolves towards monoclonality, typically beginning between 4–6 weeks post-infection. These findings not only suggest alternative routes whereby EBV might become embedded in B cell memory in vivo but also strengthen the argument that EBV- induced SHM could contribute to clonal evolution in EBV- associated lymphoproliferative lesions/lymphoma. It was first necessary to check the susceptibility of the different B cell subsets to EBV infection/transformation. This has been a surprisingly neglected issue after an early study, examining B cell subsets within EBV-infected tonsillar B cell cultures up to 48 hr post-infection, found no differences in the infectability of cells with different surface Ig isotypes. More recently, Dorner et al. also reported that naı̈ve (CD272) and total memory (CD27+) B cells from tonsils were equally infectable in short-term assays, although subsequently naı̈ve cells grew slightly quicker and gave slightly better LCL yields from limiting dilution seedings. While naı̈ve (CD272) cells from peripheral blood resembled their tonsillar counterparts, they were more infectable than peripheral blood memory (CD27+) preparations; this could not be explained at the level of receptor (CD21)/co-receptor (HLAII) expression but was ascribed to heterogeneity among circulating memory cells in expression of another putative co-receptor, a5b1 integrin. Comparisons with the present work are difficult because Dorner et Figure 7. Summary of IgH mutation frequency in mitogen- al. employed an unusual recombinant EBV strain (lacking one of activated B blasts and LCLs derived from naı̈ve B cells. the latent proteins, LMP2A) whose low titre preparations Histograms show the number of mutations in IgH sequences amplified from CD40L/IL4-stimulated B blasts and EBV-infected LCLs derived from necessitated the use of spinoculation to achieve measurable rates naı̈ve B cell preparations at 0, 4, 6 and 12 week time points. The height of infection. The present work, using wild-type EBV and of the open bars indicate the percentage of total IgH sequences conventional infection protocols, did not detect any significant analysed with a particular frequency of mutations, while the shaded difference in virus binding, infectability or transformability under bars indicate the proportion of these IgH sequences detected more limiting conditions between peripheral blood N, NSM and SM than once in each culture i.e. belonging to a CDR3-related clonal family. preparations (Figure 2). Likewise, another recent report comparing nt: not tested. doi:10.1371/journal.ppat.1002697.g007 naı̈ve (CD272) and total memory (CD27+) B cells from blood also found no significant difference in transformability. Interest- trees with different branches arising from a co-resident germline ingly we noticed that, in contrast to the ease with which LCLs parental sequence. In one case (Figure 9A), we found the parental grow out from positive wells at the high end of transformation IgH sequence and 9 clonally-related variants with up to 8 sequence assays, only a subset of positive wells arising under limiting changes; in another case (Figure 9B and Table 4), we found the conditions could be expanded to establish LCLs. This likely parental IgH sequence and a further 5 related variants with up to 6 reflects the fact, recently noted by others [51,52] and also apparent sequence changes. The detailed sequences used to construct these from our CDR3 spectratyping of EBV-infected bulk cultures trees are shown in Figures S4A and S4B. (Figure 6), that the process of LCL establishment is associated with significant clonal selection, a hurdle which cultures with small seed Discussion populations may fail to overcome. The basis of this selection remains to be determined. However, from the point of view of the As originally stated, the different views as to how EBV present work, we can conclude that N, NSM and SM populations selectively colonises the SM B cell subset in vivo hinged on whether show similar levels of attrition at this stage. PLoS Pathogens | www.plospathogens.org 13 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Figure 8. Distribution of IgVH mutations in uninfected B cell subsets and derived LCLs. Histograms show the cumulative pattern of IgVH somatic mutations in naive (N), non-switched memory (NSM) and switched memory (SM) B cell subsets pre-infection and in their derived LCLs. Mutations were scored between codons 9–92 (according to the Kabat numbering system) with codons known to be SHM hotspots shown as filled bars Note that each mutated clonotypic sequence identified within an LCL only contributes once to the overall data. doi:10.1371/journal.ppat.1002697.g008 With respect to virus-induced changes in cellular phenotype, as by transcript-specific RT/PCR assay or by protein expression many have observed [52–56], EBV-transformed LCLs converge (Figure 3A, B and Table 1). This accords with early work on the on a similar ‘‘lymphoblastoid’’ phenotype, irrespective of the IgM+ IgD+ cell fraction from peripheral blood where EBV precise differentiation stage of the target B cell; importantly, that infection induced IgM but not IgG or IgA production. phenotype includes the memory marker CD27, which is induced However it apparently contradicts another study in which on N-derived LCLs to levels similar to those retained on memory switch circles, considered an early marker of class switching, and LCLs. By contrast, EBV transformation does not lead to some switch transcripts were detected in naı̈ve B cells after EBV convergence of Ig isotype expression. Thus we detected no Ig infection in vitro. In that same study, however, evidence of isotype isotype switching in N- or NSM-derived LCLs, whether analysed switching at the protein level was only given for clones of the EBV- PLoS Pathogens | www.plospathogens.org 14 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro Figure 9. Presumed genealogical trees showing examples of sequence divergence within CDR3-related clones. Each tree is based on clonally-related IgH sequences amplified at different time points from an independent bulk LCL culture derived from EBV-infected naı̈ve B cells. In each case, functional IgH sequences were first aligned with the nearest germline IGH V, D and J alleles to determine the number and location of mutations. Each clonally-related IgH sequence is represented as a box, with the number of incremental mutations shown on each branch; the sequence name and the position of the codon changes (according to the Kabat numbering system) are indicated inside the box while newly acquired mutations are underlined. (A) Prospective analysis of LCL5 identified a sequence (clone w4s6) with zero mutations relative to the nearest germline allele IGHV4-59*01 and a further 9 clonally-related variants (with up to 8 additional changes) which segregated into two major branches. Note that the same mutation at codon 70 was seen in both branch 1 and branch 2 and was therefore assumed to have occurred twice independently. (B) Serial analysis of LCL8 identified a sequence (w6s17) with two mutations relative to the nearest germline allele IGHV3-23*04 and a further 5 clonally-related variants with up to 6 additional sequence changes (note the change in codon 31 involved two substitutions). In this case the nearest germline IGVH allele was a presumed hypothetical intermediate and is therefore represented as a dotted box. doi:10.1371/journal.ppat.1002697.g009 negative BL line, Ramos, that had been transfected to express higher than the background that would expected from PCR LMP1 , the EBV latent cycle protein that mimics many of the amplification error and in stark contrast to the mean values of 0.4 effects of CD40 ligation [59,60]. Likewise another study substitutions per sequence seen in the original N cell-sorts and of linking EBV with the induction of switch recombinase activity was 0.5 substitutions per sequence seen in CD40L/IL4-expanded N based on viral infection of a B lymphoma line BJAB or on EBV- cell cultures. Interestingly the 9.2 value is below the mean number positive BL cells. In neither study, therefore, was there definitive of mutations (15.2) we observed in SM cell preparations but evidence of EBV-induced isotype switching in the setting of similar to the mean (7.2) seen in NSM cells and their derived normal B cells; indeed others have used EBV-infected B cells as LCLs. This similarity, and the fact that all N-derived transfor- isotype-stable substrates in order to study switching induced by mants were IgM+ IgD+, meant that if such mutated clonotypes exposure to cytokines and/or CD40 ligation [62,63]. We indeed were arising from pre-existing memory cells in the original N cell confirmed that EBV transformation still leaves early passage N- sorts, then the source of contamination must be NSM cells. and NSM-derived LCLs responsive to exogenous signals, CD40L/ However, such a possibility seems at odds both with the high IL4/IL21, that mimic T cell-derived switch signals in vivo purity of N cell preparations, shown to be.99% by IgD/CD27 (Figure 3C). Thus EBV infection itself can induce naı̈ve B cells staining and 97% by Ig gene sequencing (Figure 1), and with the to acquire an NSM-like surface phenotype (IgD+ CD27+) and, fact that NSM cells were not more transformable than N cells with appropriate T cell help, a SM-like phenotype (IgD2 CD27+). when compared in parallel assays (Figure 2) nor more likely to Key findings arose when the study turned to virus-induced survive the clonal selection that then occurs with transition from changes Ig genotype. In limiting dilution seeding experiments, transformed cell focus to established LCL. Having said that, we designed to generate transformed populations of limited clonality, cannot entirely discount the possibility that some of these mutated we were surprised to find that.30% N-derived LD-LCLs clonotypes derive from NSM cells contaminating the N cell sorts, contained mutated IgH sequences, a result seen consistently particularly in the rare cases of clones that also carry bcl6 across experiments on 8 different B cell donors (Figure 4). mutations. Were NSM cells to be the source of even a small Combining data from all these mutated clonotypes gives a mean of fraction of the Ig-mutated LCLs detected in the present work, this 9.2 nucleotide substitutions per IgH sequence, a value substantially would be worthy of further attention since it implies that NSM- PLoS Pathogens | www.plospathogens.org 15 May 2012 | Volume 8 | Issue 5 | e1002697 EBV Infection of Naive B Cells In Vitro derived transformants enjoy an advantage over EBV-infected the possibility that EBV infection per se could induce an NSM Ig naive cells that is only apparent when the two are competing in genotype/phenotype without germinal centre transit, while T cell mixed culture. signals, perhaps in the extra-follicular e

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