Immunohistology, EBER In Situ Hybridization, RT-PCR, and TCR-γ Fragment Length Analysis.
All selected cases displayed the typical histological and immunohistological picture of AILD, i.e., proliferation of (partly atypical) CD3+
T cells, admixed with scattered CD20+
B immunoblasts and hyperplastic FDC networks. By EBER in situ hybridization, which detects all virus-harboring B cells, moderate to high amounts of EBV-positive cells were observed in six cases from which EBV-infected cells were micromanipulated (), while in three other cases no or few EBV-infected cells were detected (). Immunohistology for BZLF1 (whose expression indicates that cells switched from latent to lytic infection 3031
) and EBNA2 (typical for cells expressing all latent EBV proteins) was negative in all cases analyzed. Scattered LMP1+
cells were observed in cases 2 and 4. In case 5, LMP1-positive cells accounted for about one- third of the EBER-positive cells, partly displayed the morphology of large blasts and were predominantly arranged in a nodular fashion throughout the lymph node. As LMP2a immunostainings could not be evaluated because of unspecific staining, RT-PCR of RNA isolated from whole tissue sections of cases 1–6 was used to detect LMP2a expression. Cases 4, 5, and 6 tested positive ().
In a fraction of AILD cases, another human herpes virus, HHV8, associated with B cell malignancies and with transforming potential in animal models, has been detected 323334
. In a sensitive seminested PCR with DNA from cases 1–6, HHV8 DNA was detected only in case 5 (data not shown). However, when single EBV-infected cells from this same case were analyzed, no coinfection of EBV-positive cells with HHV8 was observed (see Materials and Methods). Therefore, the features of the EBV-infected B cells analyzed in this study and described below are not influenced by a coinfection of the cells with HHV8.
To analyze the cases for the presence of clonal T cell populations, a PCR amplification of TCR-γ gene rearrangements from DNA extracted from whole tissue sections and subsequent determination of length and intensity distributions of the fluorescent-labeled PCR products was performed 2335
. This approach is suitable to detect clonal expansions among TCR-γδ– as well as TCR-αβ–expressing T cells, since also the latter usually carry TCR-γ gene rearrangements. In all four of the six EBV-rich cases informative for TCR-γ rearrangements (cases 1, 2, 4, and 5; in cases 3 and 6 signals were too low for evaluation; ), the results were indicative of a single T cell expansion in each patient, thus supporting the diagnosis of T cell lymphoma at the molecular level. Among the three cases used for analysis of EBV-negative B cells only in case 9 a dominant T cell clone was detected.
Clonal Expansion among EBER+ and CD20+ Cells.
Between 50 and 115 single EBER+ cells were micromanipulated from frozen tissue sections of six EBV-rich cases (). All cells were subjected to seminested PCR for rearrangements at the IgH, Igκ, and Igλ loci and in most instances also tested for EBV-infection in an EBNA1-specific PCR. From 31 to 58% of the micromanipulated cells at least one Ig gene rearrangement was obtained, and between 24 and 125 Ig rearrangements were amplified per case. Comparison of the Ig gene sequences identified clonal expansions among the EBV+ cells in all six cases (). However, the number of clones and the extent of clonal expansion differed markedly between cases. In patients 1–3, 60–90% of the EBER+ cells could be assigned to several clones of varying sizes (case 1: 10 clones with 2–7 members; case 2: seven clones with 2–18 members; and case 3: six clones composed of 2–4 cells). In case 4, a dominant clone encompassing nearly half of the EBER+ cells was identified. In cases 5 and 6, all EBER+ cells belonged to a single clone.
PCR and Sequence Analysis of Rearranged Ig Genes of Single EBER+ and CD20+ Cells of AILD
To analyze whether the large clonal expansions of cases 5 and 6 were restricted to EBV-positive cells and/or accounted for most B cells in the tissue, for both cases also CD20+ cells were micromanipulated and tested for infection with EBV in an EBNA1-specific PCR (). In case 5, 14 of 17 CD20+ cells were positive for EBNA1. Thus, in this case ~80% of the CD20+ B cells are EBV-infected. Furthermore, nearly all EBV-positive cells of this case (33 of 35 cells taking EBER+ and CD20+/EBNA1+ cells together) belonged to one clone. The three EBNA1 PCR-negative cells carried unique rearrangements. In contrast to case 5, in case 6 only a small fraction of the CD20+ cells was EBV-infected (4 of 21 Ig-PCR positive cells were also positive in the EBNA1-PCR). The four EBV-positive cells belonged to the same clone identified among the EBER+ cells. Surprisingly, also 14 of 17 CD20+ and EBNA1 PCR-negative cells could be assigned to this clone. Since the EBNA1-specific PCR works very efficiently and since 9 of the EBNA1-PCR-negative cells define a distinct subclone in the genealogical tree of this clone (), we conclude that in this case, the B cell population consisted largely of a single dominant clone and only about a quarter of the cells in this clone are EBV-infected, perhaps due to a single infectious event in the history of the clone.
Figure 2 Genealogical trees for examples of clonal expansions of EBV-infected B cells and an EBV-negative clone (case 7) with ongoing somatic hypermutation from six cases of AILD. Genealogical trees are based on alignments obtained with the GeneWorks software (more ...)
Some CD20+/EBER− B cells were also micromanipulated from case 2 to analyze the clonal composition and differentiation status of EBV-negative B cells. Among the 42 rearrangements amplified from 24 EBER− and EBNA1 PCR-negative samples (two to three cells were analyzed together in each PCR) none was clonally related to those amplified from EBER+ cells. Only two pairs of clonally related cells were identified.
In addition, for a comprehensive comparison of EBV+
B cells in AILD, from three further cases with no or few EBV+
cells, single proliferating (Ki67+
) cells, B cells (CD20+
) and proliferating B cells (Ki67+
) were micromanipulated (Ki67 staining was chosen because the analysis of these cases was originally focused on proliferating B cells in EBV−
cases of AILD). In case 8, no clonally related B cells were identified and in case 9, only 2 of 71 informative cells were clonally related. A single EBV−
B cell clone encompassing nearly 2/3 of the proliferating B cells was identified in case 7. As in this case no T cell clone was detected, this B cell clone may represent a B cell tumor that developed in the background of AILD as described previously 91011.
Taken together, in each of the six EBV-rich cases clonal B cell expansions were detected among the EBV-infected cells, ranging from polyclonal populations with several small clones to monoclonal expansions dominating the B cell population in the tissue. One case with a large monoclonal expansion included EBV+ as well as EBV− B cells. With the exception of one large clone in an EBV− case, EBV− B cells showed little tendency for clonal expansion.
Somatic Mutation in Rearranged Ig Genes of EBV-infected B Cells.
Rearranged Ig genes of EBV-infected B cells were evaluated for the presence of somatic mutations, considering members of clones and EBV-harboring cells not assigned to clones (unique cells) separately. Analysis of the sequences amplified from unique EBV-infected cells revealed that 55 of 58 informative cells from cases 1–5 carried mutated V genes. The average mutation frequency of 5.4% for VH
= 29; range 0.4–11.9%) is within the range typical for memory B cells 36
Among the EBV-infected cells belonging to expanded clones, three types of clonal expansions were identified based on the presence and pattern of somatic mutation (two clones are not considered, see legend to ): (i) only three of the 26 clones carried unmutated V region genes. These three clones were all identified in case 3 and consisted of only 2–4 members. (ii) Nine clones identified in cases 1, 2, and 4 had mutated V genes with an average mutation frequency of 7% for VH rearrangements (n = 8, range 3.5–11.6%) but did not show intraclonal diversity. Most of these nine clones were small and defined by only two members. (iii) In 14 clones with mutated V genes intraclonal diversity was observed ( and ). Such clones were identified in all six cases. The extent of intraclonal diversity varied considerably between the clones, ranging from three sequence variants among 17 sequences (clone 1 of case 4) to 12 variants among 15 sequences (clone 2 of case 2). Also the numbers of shared mutations differed markedly between clones. For the dominant clones of cases 4–6 several shared mutations were identified, whereas for clones 1 and 2 of case 2 no mutations common to all clone members were observed (). The average mutation frequency of VH rearrangements for clones with ongoing mutation from cases 1–4 was 2.8% (identical mutated rearrangements were counted only once; n = 35, range 0–7.2%), and for the two clones of cases 5 and 6 the least mutated members showed 10 and 17% mutation frequency, respectively.
The clone detected among EBV− cells in case 7 showed also intraclonal diversity besides 20 and 33 shared mutations in its VH and Vλ rearrangements, respectively ().
Taken together, the vast majority of EBV-infected B cells carried somatically mutated V region genes, and many members of EBV+ B cell clones showed ongoing hypermutation, indicating that the virus preferentially resides in GC and perhaps also memory B cells.
Frequent Crippling Somatic Mutations in Clonally Expanding EBV-infected B Cells.
Surprisingly, the analysis of the mutation pattern disclosed a large number of crippling mutations in originally potentially functional V gene rearrangements. These destructive mutations were either nonsense mutations or duplications or deletions resulting in loss of the correct reading frame. Most destructive mutations were found in clones showing ongoing somatic mutation (). For each of the large clones of cases 4–6, inactivating mutations were found in the in-frame V gene rearrangements (). In cases 5 and 6, amplification of DHJH rearrangements from the second IgH alleles confirmed that the inactivated VHDHJH rearrangements were indeed the originally functional ones (see legend to ). The 11 cells belonging to the EBV− clone of case 7 carried various different mutations rendering either the originally potentially functional VH or the functional Vλ rearrangement nonfunctional, while none of the shared mutations was crippling ().
Somatic Mutation Pattern of Ig Gene Rearrangements from EBER+ Cells of AILD
Nonsense mutations were found in originally potentially functional rearrangements of clones with ongoing mutation with an average frequency of 7.7% of all mutations (or 6.5% if, to estimate the frequency of destructive
nonsense mutations, in V genes with several nonsense mutations only one is counted). This contrasts with the 0% (0 in 143 mutations) and 0.2% (1 in 515 mutations) nonsense mutations found in clones without ongoing mutation and unique cells, respectively (). For mutations that a priori could not be subject to selection (i.e., mutations in out-of-frame rearrangements and mutations that occurred in already clonally inactivated rearrangements), a frequency of 5.2% nonsense mutations was observed, which is close to the calculated theoretical value of 4.7% assuming random mutagenesis without selection 37
. Thus, the originally functional V genes of clones with ongoing mutations accumulated nonsense mutations with a frequency somewhat higher than in the case of nonproductive, nonselected rearrangements.
In normal GC B cells, deletions or duplications account for ~6% of somatic mutation events, as has been calculated from their frequency in mutated out-of-frame rearrangements 38
. In this study, the frequency of deletions/duplications in nonproductive rearrangements was somewhat lower (3.5%). In functional V region genes most deletions/duplications result in loss of the correct reading frame or cripple a V gene rearrangement because a large part of the rearrangement is lost or duplicated. Such deletions/duplications are therefore stringently counterselected, so that expressed V genes of post GC B cells only rarely carry such mutations (usually 3- or 6-bp deletions/duplications in the complementary determining regions). In line with this, deletions/duplications were only rarely observed in the present study in in-frame rearrangements of clones without ongoing mutation and in unique rearrangements, with frequencies of 0.7 and 0.6%, respectively (). However, in originally functional rearrangements of clones with ongoing mutation, deletions and duplications were found with a frequency of 6.4%, and many of those were destructive, further indicating that these rearrangements acquired somatic mutations without selection for functionality.
In addition to the counterselection of destructive mutations, also the ratio of replacement (R) to silent (S) mutations in FRs of functional Ig gene rearrangements is indicative for selective pressure on a cell to express a functional antigen receptor. In cells selected for expression of a functional B cell receptor, R mutations in FRs are usually counterselected. Thus, for selected memory B cells a R/S ratio of 1.0 to 1.6 is usually observed, contrasting with the ratio of ~ 3.0 for V gene rearrangements that acquire somatic mutations without selection, like out-of-frame rearrangements (). The out-of-frame rearrangements analyzed in this study showed an average R/S value of 2.5. For the in-frame rearrangements of clones without ongoing mutation and the unique EBER+ cells, we observed R/S ratios of 1.1 and 1.3, respectively, indicative of stringent selection for expression of a functional antigen receptor, like in normal B cells. In contrast, the R/S ratio for clones with intraclonal diversity is 2.7, and thus similar to that of rearrangements that acquire mutations without selection for functionality.
Taken together, each of the three features of somatic hypermutation analyzed (the frequency of nonsense mutations, the frequency of deletions/duplications, and the R/S ratios) indicate that expanding EBV-infected B cells in AILD acquire somatic mutations without selection for expression of a functional antigen receptor.
Considering the obvious crippling Ig mutations, the frequencies of Ig-less B cells among all B cells can be calculated. In case 2, ~1% of all B cells lost the capacity to express a functional antigen receptor, whereas in cases 5, 6, and 7 the fraction of Ig-less B cells amounts to 71, 86, and 56% of all B cells, respectively. For cases 1, 2, and 4 fractions of 4, 0, and 43% of Ig-less B cells among the EBER+ B cells were calculated.