1 Introduction. Andreas Bräuninger 1, Tilmann Spieker 1, Anja Mottok 1,AudreySylviaBaur 2,Ralf Küppers 3 and Martin-Leo Hansmann 1

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1 Eur. J. Immunol : Somatic hypermutation in EBV + lymphoproliferations 1593 Epstein-Barr virus (EBV)-positive lymphoproliferations in post-transplant patients show immunoglobulin V gene mutation patterns suggesting interference of EBV with normal B cell differentiation processes Andreas Bräuninger 1, Tilmann Spieker 1, Anja Mottok 1,AudreySylviaBaur 2,Ralf Küppers 3 and Martin-Leo Hansmann 1 1 Department of Pathology, University of Frankfurt, Frankfurt, Germany 2 Department of Pathology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland 3 Institute for Genetics and Department of Internal Medicine I, University of Cologne, Cologne, Germany In a model for persistent infection, Epstein-Barr virus (EBV) uses the germinal center (GC) reaction to establish persistence in memory B cells. To study whether EBV adopts to normal B cell differentiation processes also in EBV-associated lymphoproliferative diseases, we micromanipulated EBV + cells from biopsies of five patients with post-transplantation lymphoproliferative disease (PTLD) and one unusual Hodgkin lymphoma with many small EBV + cells, and analyzed rearranged V genes of single cells. In all cases clonal expansions of EBV + B cells were identified. The vast majority of these clones carried mutated V gene rearrangements and a fraction of clones showed ongoing hypermutation. Hence, PTLD likely derive from GC and/or post-gc B cells. In two clones hypermutation occurred in the absence of follicular dendritic and CD4 + T cells, important interaction partners of normal GC B cells. Furthermore, in one case sustained somatic hypermutation occurred without expression of a functional antigen receptor. Hence, EBV + B cells in PTLD can retain or acquire features of GC B cells in an unphysiological setting and may continue to undergo somatic hypermutation uncoupled from normal selection processes, suggesting that EBV interferes with normal B cell differentiation and selection processes in PTLD. Key words: Epstein-Barr virus / Somatic hypermutation / Germinal center reaction / Hodgkin lymphoma / Post-transplantation lymphoproliferative disease Received 12/12/02 Revised 17/3/03 Accepted 8/4/03 1 Introduction [DOI /eji ] Abbreviations: AID: Activation-induced cytidine deaminase AILD: Angioimmunoblastic T cell lymphoma BCR: B cellreceptor BL: Burkitt lymphoma EBER: EBV-encoded RNA EBNA: EBV nuclear antigen FDC: Follicular dendritic cell GC: Germinal center HL: Hodgkin lymphoma HRS: Hodgkin/Reed-Sternberg LMP: Latent membrane protein PTLD: Post-transplantation lymphoproliferative disease Epstein-Barr virus (EBV) infects about 90% of human individuals and usually persists asymptomatically in lifelong carriers due to a balance between its capability to transform B cells into proliferating lymphoblasts and a tight control of EBV + B cells mainly by CD8 + T cells [1]. Immunosuppression in transplantation patients unmasks the transforming potential of EBV, and expansions of EBV + B cells with development of EBV + B cell lymphomas are frequently observed [2]. Furthermore, in 80% of endemic Burkitt lymphoma (BL) and 40% of Hodgkin lymphoma (HL) cases the tumor cells are EBV + [1]. The EBV-associated lymphomas are characterized by expression of distinct sets of EBV-encoded genes, defining latency programs I III. In post-transplantation lymphoproliferative disease (PTLD), for example, all nine latent genes are usually expressed (latency III), while in HL, only the EBV nuclear antigen (EBNA)1 and the latent membrane proteins (LMP)1 and LMP2a are expressed (latency II) [1]. Also in latently infected healthy individuals distinct EBV genes are expressed in the different EBVinfected B cell populations [3]. Based on these observations, a model has been developed in which the virus uses normal B cell differentiation processes to establish a persistent infection [3, 4]. An important aspect of this model is the germinal center (GC) reaction, in which B cells that encountered a specific antigen proliferate 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim /03/ $ /0

2 1594 A. Bräuninger et al. Eur. J. Immunol : vigorously and modify their rearranged V genes by somatic hypermutation [5]. Only B cells carrying antigen receptors with improved affinity survive and are selected into the pool of memory B cells [6]. In the model of persistent infection, EBV primarily infects naive B cells and induces their proliferation [4]. When such cells subsequently undergo a GC reaction and differentiate into memory B cells, the virus gains access to the memory B cell pool, the site of viral persistence. While this model suggests that EBV uses normal B cell differentiation processes to establish a lifelong persistent infection in healthy carriers, in other situations EBV + B cells show features not compatible with this model. Thus, in infectious mononucleosis, the acute primary infection by EBV, no GC B cell clones with intraclonal sequence diversity were observed among expanding EBV + clones, suggesting that in this situation either EBV does not reside in GC B cells, or EBV-infected GC B cells loose key features of such B cells [7]. On the other hand, ongoing somatic hypermutation during clonal expansion is typical for BL and EBV + Bcells in angioimmunoblastic T cell lymphoma (AILD), showing that EBV + B cells may show this hallmark of GC B cells [8 10]. Notably, in AILD, sustained somatic hypermutation appears to occur uncoupled from selection for a functional B cell receptor (BCR) [10]. On the basis of these indications that EBV may either adopt to normal B cell developmental processes to establish a persistent infection or dramatically alter the differentiation processes of infected B cells in particular diseases, we wanted to characterize the impact of EBV on infected B cells in another EBV-associated disease, namely PTLD, with a cellular micromilieu different from that of the follicular dendritic cell (FDC)- and T cell-rich AILD. V gene rearrangements of single EBV + Bcells of five patients affected by PTLD and one patient with an EBV + lymphoproliferation beside an HL were analyzed. This approach is well suited to identify clonal expansions, and analysis of somatic mutation patterns in rearranged V genes can be instrumental not only to assign B cells to the naive, GC or memory compartments, but also to analyze whether normal selection processes took place in GC reactions [11, 12]. 2 Results 2.1 Immunophenotypic characterization In two polymorphic PTLD (cases 1 and 2) no morphologically or immunophenotypically conspicuous tumor cells were observed. Frequencies of EBV-infected B cells, identified as cells expressing the EBV-encoded RNA (EBER) transcripts, among all cells were more than 10% and less than 1% in cases 1 and 2, respectively (Table 1). The EBER + cells of both cases expressed EBNA2, which is indicative for latency type III. In case 3, a monomorphic centroblastic lymphoma in a post-transplantation patient with EBER + tumor cells, EBNA2 LMP1 X Table 1. In-situ hybridization and immunohistochemistry for expression of EBV-specific genes in EBV + lymphoproliferations a) Case Diagnosis Cells EBER + (%) a) 1 Polymorphic PTLD All /+ 2 Polymorphic PTLD All 1 + +/ 3 Centroblastic lymphoma, PTLD All /+ +/ 4 Hodgkin lymphoma, HRS 100 /+ Post transplant Other 1 10 /+ 5 Hodgkin lymphoma, HRS 100 +/ Post transplant Other 1 10 /+ 6 Hodgkin lymphoma, HRS 100 +/ Others 1 10 a) b) ne: not evaluable; +, +/ and /+: fraction of EBNA2 + or LMP1 + cells in the same range as EBER + cells(+),onlyafraction(+/ ) or a small fraction compared to EBER + cells ( /+). For EBNA2, sections of tonsils from patients affected by infectious mononucleosis were stained in parallel as positive controls. Percent of all cells.

3 Eur. J. Immunol : Somatic hypermutation in EBV + lymphoproliferations 1595 more than 10% of the lymphoma cells expressed LMP1 and less than 10% EBNA2. A minor fraction of tumor cells ( X 10%) expressed the GC marker BCL6 [13], and only single scattered CD21 + or KiMP4 + FDC and CD4 + T cells, both of which are important constituents of normal GC [6], were observed. Cases 4 6 were HL with large amounts of small- to medium-sized EBER + cells (between 1% and 10% of all cells) beside the EBER + Hodgkin/Reed-Sternberg (HRS) tumor cells. The CD30 + HRS cells and also the small EBV + cells were EBNA2. In cases 4 and 5, both of which are HL occurring after transplantation, and in case 6, various fractions of the HRS cells expressed LMP1, indicative of latency type II usually found in HRS cells [1]. LMP1 expression was also observed for a fraction of small EBV + cells in cases 4 and 5. In case 5, BCL6 + cells and CD4 + T cells and FDC were detected only outside the tumor-infiltrated region from which the EBV + cells were micromanipulated (Fig. 1). In case 6, remnants of GC and large numbers of FDC and CD4 + T cells and also BCL6-expressing cells were found. 2.2 Analysis of rearranged immunoglobulin V genes of single EBV + cells From cases 1 3, between 50 and 70 EBER + cells, and from the three HL, EBER + HRS cells (between 30 and 60) and small EBER + non-hrs cells (between 41 and 105) were micromanipulated as single cells from frozen tissue sections and subjected to semi-nested PCR for V gene rearrangements at IgH, Ig and Ig Q loci. V gene rearrangements were directly sequenced. To verify the micromanipulation of EBV-infected cells, also a seminested PCR for the EBNA1 gene was performed and only EBNA1 PCR-positive cells were included in the analysis (Table 2). At least one Ig gene rearrangement in addition to the EBNA1 fragment was amplified from 48% to 69% of cells. 2.3 Analysis of clonal expansions among EBV + B cells In both polymorphic PTLD, several EBV + B cell clones ranging from small to larger expansions (2 to 28 cells) were identified (Table 2). While the three clones in case 2 carried mutated V gene rearrangements, in case 1 three of four clones carried unmutated rearrangements. In both cases the clones lacked intraclonal sequence diversity. Table 2. PCR results and sequence analysis a) Case Cells analyzed Cells with mutated rearrangements/ cells informative (%) %ofcells assigned to clones Number of clones Cells per clone Clones with mutated rearrangements Clones with intraclonal diversity 1 EBER + 14/30 (47) / EBER + 34/34 (100) /3/ EBER + 39/39 (100) EBER + HRS 19/19 (100) small EBER + 18/19 (95) 0 5 EBER + HRS 35/35 (100) small EBER + 44/45 (98) EBER + HRS 19/19 (100) small EBER + 60/60 (100) /4/3 3/ a) Only cells positive for EBNA1 and an Ig rearrangement are considered. Cells from which only unmutated V rearrangements were amplified were not considered informative regarding somatic hypermutation, as (nonfunctional) V J joints are often inactivated by rearrangement of the kappa-deleting element and hence exempted from somatic hypermutation [39 41]. Aliquots of buffer covering the sections during micromanipulation served as negative controls. For each case, buffer controls were analyzed in parallel with the cells. Overall, from 7 of 170 buffer controls one or more V gene rearrangements were amplified, and from 7 of 182 buffer controls an EBNA1 product was obtained (plus very weak EBNA1 products from all buffers of case 5). Hence, there was a low level of contamination in the experiments (3.4% for both, V gene rearrangements and EBNA1), which is, however, much lower than the frequency ofamplificatesobtainedfrom the microdissected cells.

4 1596 A. Bräuninger et al. Eur. J. Immunol : Fig. 1. EBER in-situ hybridization and immunohistochemical stainings of consecutive sections of case 5. (A C) EBER in-situ hybridization of case 5 in a 40, 100 and 400 magnification. The follicle remnants on the left side of all pictures contain only few EBER + cells compared to the tumor-infiltrated region on the right side of all pictures. An EBER + HRS cell is marked by an arrowhead and two small EBER + cellsbyarrows(c).(d,e)overview(40 )andhigher power magnification (100 ) of a CD21 immunostaining for detection of FDC. FDC were only found in the follicle remnants and not in the tumor-infiltrated region from which the EBER + cells were micromanipulated. (F, G) CD4 staining (40 and 100 magnification) showing that most CD4 + T cells are found in the follicle remnants. (H, I) Overview (40 ) and higher power magnification (100 ) of a BCL6 immunostaining showing that stained cells were only found in the follicle remnants and not in the tumor-infiltrated region.

5 Eur. J. Immunol : Somatic hypermutation in EBV + lymphoproliferations 1597 Table 3. Clonal rearrangements of EBV + lymphoproliferations a) a) b) c) d) pf: potentially functional; nf: non functional. Only rearrangements amplified at least twice are shown. For rearrangements with intraclonal diversity, mutation frequencies for sequences with greatest homologies to germ-line sequences are given. All sequences were deposited in the EMBL database under accession nos. AJ and AJ From six cells in addition to the rearrangements shown, PCR products likely due to cellular contamination were amplified. A 128-bp fragment of the V H 4 4 gene segment from the V H L primer binding site to amino acid 20 of the FRI was followed by a 215-bp fragment without significant homologies, a 486-bp fragment from the J H 2/3 intron extending into the J H 3/4 intron (the J H 3 primer binding sites were heavily mutated) and a 198-bp fragment beginning in the J H 5/6 intron and ending at the J H 6 primer binding site. Nonfunctional due to two stop mutations and a 41-bp deletion. A sequence for codons 1 77 of V H 4 34 (codons are missing) was followed by 43 bp without any homology (CDR3?). As there were no sequence homologies to any of the six J H genes 5 to the J H primer, the J H segment could not be identified. The rearrangement is out-of-frame relative to the reading frame in the J H primer.

6 1598 A. Bräuninger et al. Eur. J. Immunol : As expected, in the four cases diagnosed as lymphomas (cases 3 6), monoclonal populations of EBV + tumor cells were identified. In the centroblastic lymphoma, the EBV + cells belonged to a single tumor clone with mutated V gene rearrangements and extensive intraclonal sequence diversity (Table 2, 3; Fig. 2). The HRS cells of the three HL were in each case a single clone and carried mutated V gene rearrangements without intraclonal diversity. In one case, crippling mutations (two nonsense mutations and a 41-bp deletion) rendering the originally functional V gene rearrangement nonfunctional were observed (Table 3), in accordance with previous findings [14]. In two of the HL, clonal expansions unrelated to the HRS cells were also identified among the small EBV + cells. In case 5, a single clonal expansion with extensive intraclonal sequence diversity encompassed 78% of small EBV + cells (Table 2, Fig. 2). Whether case 5 represents a PTLD in which two malignant B cell clones (the HRS cell clone and the clone of small EBV + B cells) developed concurrently, or a HL in which an EBV-infected B cell underwent a large clonal expansion, is unclear. In case 6, nine smaller expansions were observed, all with mutated V gene rearrangements and four of them with intraclonal sequence diversity. Taken together, besides the mutating tumor clone of the centroblastic lymphoma and the mutated HRS cell clones without intraclonal diversity, 17 EBV + B cell clones were identified, 14 with mutated V gene rearrangements, and 5 of these showed intraclonal sequence diversity. 2.4 Differences of EBV + B cell clones with intraclonal diversity to normal GC B cell clones Fig. 2. Genealogical trees for clones with intraclonal sequence diversity. Genealogical trees are based on sequence alignments obtained with the GeneWorks software (Intelligenetics, Oxford, GB). Presumed precursors are boxed and assumed intermediates are included and marked with X. Each circle represents, if not otherwise stated below the circle, a single cell. The numbers given beside the lines indicate the numbers of mutations defining a branch in the genealogical tree. The detection of intraclonal diversity in several EBV + B cell clones indicated that EBV + B cells had retained or acquired features of GC B cells. However, in several aspects these clones showed striking differences to normalgcbcells:(1)inthelargeebv + non-hrs clone of case 5, no BCL6 expression was detected despite extensive intraclonal sequence diversity (Fig. 1). (2) In the centroblastic lymphoma and in the large EBV + clone of non-hrs cells of case 5, somatic hypermutation occurred in the absence of FDC and CD4 + T cells in the tumor-infiltrated region from which the cells were obtained. As somatic hypermutation is strictly dependent on expression of activation-induced cytidine deaminase (AID) [15], a reverse transcription (RT)-PCR analysis of RNA isolated from tissue sections of the centroblastic lymphoma was performed. AID transcripts were indeed detected, supporting the idea that hypermutation is active in the centroblastic lymphoma (Fig. 3). (3) In the centroblastic lymphoma, sustained somatic hypermutation occurred without expression of a functional antigen receptor as a clonal D H J H rearrangement was amplified from one and a clonal V H D H J H rearrangement with a large crippling deletion from the other IgH allele (Table 3). In normal GC and also in other B cell subsets, cells without functional antigen receptors are usually quickly eliminated by apoptosis [6, 16]. Notably, also the V H rearrangement of the large clone of non-hrs cells in case 5 that showed ongoing hypermutation was non-

7 Eur. J. Immunol : Somatic hypermutation in EBV + lymphoproliferations Discussion Fig. 3. RT-PCR analysis of AID expression with normalized cdna from two different tonsils (lanes 1, 2), normalized cdna from the centroblastic lymphoma (lane 3), the double amount of normalized cdna from a second RNA preparation of the centroblastic lymphoma (lane 4), normalized cdna from an infectious mononucleosis (lane 5) and normalized cdna from placenta (lane 6). Shown are the 160-bp AID PCR products on agarose gels after 37, 39 and 41 PCR cycles (lanes 1 6: 37 cycles, lanes 7 12: 39 cycles with the same loading pattern as for lanes 1 6, lanes 13 18: 41 cycles with the same loading pattern as before, lanes 19 and 20 with RT controls with RNA from the centroblastic lymphoma and placenta after 41 cycles, and lane 21 with PCR negative control; length standard is a 100-bp ladder). Amplification of AID cdna was verified by direct sequencing of PCR products. productive. However, as attempts to characterize the second IgH allele failed (using also V H leader primers or primers for D H J H joints and germ-line configuration), it is not clear whether the amplified V H gene was the originally functional one. Because of the differences of mutating EBV-infected B cells to normal GC B cells, we also analyzed whether EBV might interfere with the somatic hypermutation process itself. Somatic hypermutation is characterized by two intrinsic features: a predominance of transitions and the targeting of the G in the RGYW hotspot motif more frequently than expected, assuming a random distribution of mutations [17, 18]. Taken all EBV-infected cells from this study together, we observed two times more transitions than expected and the G in the RGYW hotspot was mutated 3.6 times more frequently than expected. The predominance of transitions and the preferential targeting of the hotspot motifs was also observed when distinct groups of cells or rearrangements were analyzed (e.g. HRS cells, unique cells, clones with or without ongoing mutation, in-frame and out-of-frame rearrangements), indicating that EBV infection does not interfere with the mutation process itself. The histogenetic origin of EBV + B cell proliferations in PTLD has not yet been studied in detail. The present analysis of two polymorphic and one monomorphic PTLD and two HL developing in transplantation patients shows that the expanding EBV + B cells, including three frank lymphomas, are nearly exclusively derived from GC and/or post-gc B cells. Perhaps the derivation of the lymphomas from GC-derived B cells is related to the fact that GC B cells undergo DNA-modifying processes (somatic hypermutation and class-switch recombination) that have been suggested to pose a significant risk for the malignant transformation of B cells [19]. Besides EBV + tumor clones (the three HRS cell clones and the tumor clone of the centroblastic lymphoma) we identified clonal expansions among EBV + cells in four of six cases. In 5 of the 17 non-tumor clones and in the tumor clone of the centroblastic lymphoma, significant intraclonal sequence diversity was observed, demonstrating that ongoing somatic hypermutation in EBV + clones is not restricted to AILD and BL [8 10]. That somatic hypermutation was indeed active in EBV + B cell clones was supported for the centroblastic lymphoma by the detection of AID transcripts. In most instances where considerable ongoing hypermutation is observed outside classical GC, FDC and CD4 + T cells are nevertheless found. This holds true for follicular lymphoma, lymphocyte predominant HL, progressively transformed GC, and AILD [10, 20 26]. However, typical GC were not observed in any of the three cases with ongoing hypermutation analyzed here, and neither FDC nor CD4 + cells were detected in cases 3 and 5 in the regions from which cells were micromanipulated. Thus, in contrast to other settings, somatic hypermutation occurred in these two EBV + clones [the centroblastic lymphoma and the large (pre-tumor?) clone besides the HRS cells in case 5] in the absence of FDC and CD4 + cells, indicating that somatic hypermutation has become independent from a GC-like environment. This may be due to a specific combination of transforming event(s) and expression of EBV genes. Expression of the transcriptional repressor BCL6, a hallmark of GC B cells, is usually also retained in lymphomas thought to be derived from GC B cells and showing intraclonal sequence diversity like follicular lymphoma, BL, GC-derived diffuse large B cell lymphoma and lymphocyte predominant HL [12, 13, 27 30]. However, in case 5 no BCL6 expression could be detected in the region from which cells were micromanipulated. Thus, the features of the clone of small EBV-infected B cells in case 5 indicate that somatic hypermutation activity can

8 1600 A. Bräuninger et al. Eur. J. Immunol : in particular instances occur independent of BCL6 expression. For at least one of the six clones with ongoing mutation (the centroblastic lymphoma) it is likely that the tumor cells survived and even showed sustained somatic hypermutation without expression of a functional BCR. B cells of all maturation stages are usually stringently selected for expression of a functional BCR [16], and even most B cell lymphomas seem to depend on expression of a BCR [31]. So far, the survival of receptor-less B cells in vivo has been described in two instances: In about 25% of cases of classical HL, the HRS cells carry obviously crippling mutations in originally potentially functional Ig gene rearrangements. In 40% of cases of classical HL, the HRS cells are EBV-infected [14]. In AILD the survival of oligo- or monoclonal populations of receptor-less EBV + B cells was observed, and these cells even showed sustained hypermutation activity after loss of a functional BCR [10]. In EBV + classical HL and AILD, LMP1 and LMP2a are expressed and may deliver the survival signals normally generated by CD40 and BCR [32, 33], respectively, and thus allow the survival of receptor-less B cells. LMP1 expression was also detected in some small EBV-infected B cells in the centroblastic lymphoma described here, pointing to a role of LMP1 expression in the survival of EBV + receptordeficient B cells. In conclusion, we observed ongoing somatic hypermutation in a considerable fraction of EBV + expansions in patients with EBV + lymphoproliferations, demonstrating that EBV + GC-like B cell clones are not restricted to AILD and BL. Ongoing somatic hypermutation without detectable BCL6 expression in the absence of FDC and CD4 + T cells or without expression of a functional antigen receptor indicates that EBV can substantially interfere with normal B cell differentiation processes. 4 Materials and methods 4.1 Tissues and clinical data All biopsies were taken for diagnostic purposes. Biopsies for cases 1, 2 and 4 6 were from lymph nodes, while in case 3 a subcutaneous infiltrate was obtained. Cases 1 and 2 were both polymorphic PTLD, case 3 a monomorphic centroblastic lymphoma and cases 4 6 HL. Cases 1 5 were patients after (5 and 2 months, 9, 7 and 7 years, respectively) organ transplantation (kidney, bone marrow, kidney, kidney and heart, respectively). The biopsy of case 6 was diagnosed as HL, but the only clinical symptoms were recurrent lymph node swellings. 4.2 Immunohistology and EBER in-situ hybridization Sections (5? m) of formalin-fixed paraffin-embedded tissues were used for immunostainings with antibodies against EBNA2, LMP1 (CS1 and CS2) and CD21 (all from Dako, Hamburg, Germany). Deparaffinated and rehydrated sections were subjected to 2 min of high-pressure cooking in citrate buffer, ph 6, for antigen retrieval. Frozen sections were used for BCL6 (Novocastra, Newcastle upon Tyne, GB) immunostainings. For CD4 (Dako) and KiM4P (a kind gift of M. Parwaresch, University of Kiel, Kiel, Germany) immunostainings, sections of formalin-fixed and frozen tissues were used. Bound primary antibodies were visualized with biotinylated secondary antibodies, avidin-coupled alkaline phosphatase and Fast Red as substrate (all from Dako). In-situ hybridization was performed on frozen sections with EBER1 and EBER2 probes as described [34]. 4.3 RT-PCR for AID RNA was extracted from 2-? m sections of formalin-fixed tissues using trizol (Invitrogen, Carlsbad, CA) and resuspended in 50? lh 2 O. Eight microliters RNA were used for cdna synthesis with the first-strand AMV cdna synthesis kit (Roche, Mannheim, Germany), and 1 5? lofcdnawereusedas templatesinpcr.cdnawerenormalizedbypcrforthe housekeeping gene HPRT (HPRTE 7US2: 5 -CCCAC- GAAGTGTTGGATATAAGC-3 ; HPRTE 9LS1: 5 -GCTTTT- CCAGTTTCACTAATGACAC-3 ) and subsequent dilution. Equal amounts of cdna were subsequently used for AID expression analysis (AIDE 2US2: 5 -GTAGTGAAGAGGC- GTGACAGTGC-3 ; AIDE 3LS2: 5 -ACCAGGTGACGCGG- TAGCAGC-3 ). Primer pairs for HPRT and AID are intronspanning. 4.4 Micromanipulation and single-cell PCR Using a hydraulic micromanipulator, single EBER + cells were micromanipulated from 5-? m frozen tissue sections covered with buffer and transferred to PCR tubes containing 20? lof 1 Expand PCR buffer (Roche) as described [35]. Aliquots of buffer covering the sections were taken as negative controls, usually four buffer controls for each ten micromanipulated cells. After 2 h proteinase K digestion semi-nested PCR for amplification of rearranged IgH, Ig and Ig Q V genes and a fragment of the EBNA1 gene were performed as described [36 38]. In brief, in the first round of PCR, V gene family-specific primers from the framework region I of all three Ig loci were used together with sets of primers for the corresponding J segments. For the IgH locus, in several experiments a set of family-specificleader primers was used instead of the framework region I primers. For cases 3 and 5, in some experiments a set of family-specific D H segment primers for amplification of D H J H rearrangements and a fragment specific for germ-line configuration of the IgH locuswereusedtogetherwiththev H /J H and V /J primer

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