Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) gene. AID has been reported to be specifically expressed in the germinal center (GC). Follicular lymphoma (FL) cells are known to be exposed to GC reaction, as characterized by a high degree of SHM with some heterogeneity in terms of intraclonal microheterogeneity and antigen selection. The heterogeneity of SHM pattern in FL intrigued us to investigate the AID expression. AID expression was investigated in 19 FL materials consisting of 15 cases of FL fresh cells and four cell lines. In all, 10 fresh cells and three cell lines expressed AID, but the others did not. SHM was investigated in 12 fresh cells and four cell lines. The ongoing mutation was significantly different between AID-positive and AID-negative FL fresh cells (unpaired Student's t-test, P=0.047). Ongoing mutation was not seen in any of the cell lines. AID expression was associated with the ongoing mutation in FL fresh cells (two-tailed Pearson's coefficient correlation, r=0.899, P=0.01). The switch off of AID expression may start in the B-lineage differentiation stage counterpart of FL after optimizing SHM, indicated by the cessation of the ongoing mutation in AID-negative FL fresh cells.
The immunoglobulin (Ig) gene undergoes V(D)J recombination, class switch recombination (CSR) and somatic hypermutation (SHM) to encode a high-affinity antibody. The latter two processes mainly occur in the germinal center (GC) of secondary lymphoid organ, which is formed by the activated B-cell.1 The isolation of the activation-induced cytidine deaminase (AID) gene, whose product is an enzyme required for SHM and CSR, was a major achievement for clarifying the molecular mechanism of the SHM and CSR processes despite the fact that its precise actions remain elusive.2 An initial report showed that AID is specifically expressed in GC.3
Follicular lymphomas (FLs) are thought to originate from GC B cells, based on the accumulation of SHM in their Ig genes.4 The tumor growth of FLs may be driven by antigen selection,5,6 which involves an active SHM process reflected by the ongoing mutations that result in considerable intraclonal microheterogeneity.7 Ongoing mutation has also been reported to play a role in the resistance of some FL cases to a certain therapy.8 The mutational analysis of the Ig gene in FLs has revealed some heterogeneity in SHM characteristic in terms of intraclonal microheterogeneity and antigen selection.9,10
Based on the reported heterogeneity of the SHM pattern in FL and the possible role of AID involved, AID expression was investigated in the present study. AID mRNA was investigated in the fresh cells of 15 FL cases and four FL cell lines. AID mRNA was expressed in 10 cases and three cell lines. Furthermore, SHM in IgVH genes was analyzed in 12 FL cases and all cell lines. Independent of the expression or absence of AID transcripts, the IgVH genes in all FL samples were somatically mutated, indicating that AID can start to be switched off in FL. The switch off of AID expression in some FL cases may be related to the achievement of high-affinity antibody, as indicated by the cessation of the ongoing mutation in AID-negative FL fresh cells.
Materials and methods
Patients and cell lines
Our series consisted of 19 FL materials comprising 15 fresh cells of FL cases derived from lymph node tissues and four FL cell lines, namely FL18,11 FL 218,12 FL31812 and FL518. The diagnosis of the 15 cases and the donor patient of FL-518 were based on the pathological diagnosis, cell-surface antigen analysis of lymph-node cell suspension and chromosomal analysis of t(14;18)(q32;q21) (Table 1).
Reverse transcription polymerase chain reaction (RT-PCR) for AID
cDNA was synthesized using random primer (TaKaRa, Kyoto, Japan) and M-MLV reverse transcriptase (GibcoBRL, NY, USA) from 2 μg of sample derived RNA extracted by Trizol (Invitrogen, Carlsbad, CA, USA). cDNA (2 μl) was then amplified using the PCR reaction mixture from Takara Ex-Taq (Takara, Shiga, Japan) in a total volume of 50 μl, following the supplier’s instruction, using a specific AID primers covering the region from exons 1 to 5, yielding a product of 646 bp.13,14 Briefly, 5 pmol each of an upstream primer (5′-IndexTermGAG GCA AGA AGA CAC TCT GG-3′) and a downstream primer (5′-IndexTermGTG ACA TTC CTG GAA GTT GC-3′) were used. After 5 min of incubation at 94°C, 30 cycles of PCR were performed under the following conditions: a denaturation step at 94°C for 1 min, an annealing step at 56°C for 1 min and an extension step at 72°C for 2 min (10 min in the last cycle). PCR products (10 μl) were electrophoresed in a 3% low melting NuSieve GTG Agarose gel (BMA, Rockland, ME, USA) in 1 × TBE and visualized by staining with ethidium bromide. This primer pair can also detect splice variants consisting of a longer (939 bp) and a shorter (530 bp) product, which were reported to perturb the functionality of wild-type AID by preventing SHM or interrupting ongoing SHM in CLL cases.14
Densitometry of AID mRNA levels was carried out using Scion Image-Release Beta 4.0.2. for Windows. The expression levels were normalized to the expression level of β-actin mRNA15 of each sample.
SHM and ongoing mutation study
A seminested PCR was performed as described previously,16 with 500 ng of genomic DNA extracted from lymph node tissues for fresh cells and from cell pellets for cell lines, using a Genomix kit (Talent SRL, Trieste, Italy). In brief, DNA samples were first amplified using the PCR reaction mixture from Ex-Taq Takara in a total volume of 50 μl by using 5 pmol of an upstream primer (FR2A 5′-IndexTermTGG ATC CGC CAG GCT TCN GG-3′) and 5 pmol of a downstream primer (LJH 5′-IndexTermTGA GGA GAC GGT GAC C-3′). After 10 min at 95°C, 30 cycles of PCR were performed under the following conditions: a denaturation step at 96°C for 15 s, an annealing step at 60°C for 45 s and extension step at 72°C for 45 s (7 min for the last cycle). For the reamplification, the downstream primer was replaced by nested consensus primer (VLJH 5′-IndexTermGGT GAC CAG GGT CCC TTG GCC CCA G 3′), and 1% of the amplified product (0.5 μl) was used as template. PCR was performed according to the same protocol described above for a total of 24 cycles. PCR products (10 μl) were electrophoresed in a 3% low melting agarose gel in 1 × TBE buffer and visualized by staining with ethidium bromide.
PCR product (100μl) was electrophoresed in a 3% low melting agarose gel in 1 × TAE buffer and recovered from the gel, purified using a MinElute Gel Extraction Kit (QIAGEN, MD, USA) and ligated into the pCR 2.1 vector to transfect TOP10 competent cells (TA cloning kit; Invitrogen, Carlsbad, CA, USA). Following an overnight culture, 15 colonies were picked from a Luria Bertani (LB) agar plate based on X-Gal screening (Nacalai Tesque.Inc, Kyoto, Japan). After the insert was checked by PCR, five to 10 positive colonies were subcultured overnight in 5 ml of LB medium. Minipreps of plasmid were prepared from the cultures by alkaline lysis and purified on DNA affinity column by QIAprep Miniprep kit (QIAGENGmbH, Germany). Sequencing was carried out on an ABI Prism 310 sequencer (Applied Biosystems, Fostercity, CA, USA) using the dye terminator cycle sequencing method (BigDye version 3, Applied Biosystems).
Final sequences were compared to those of the published germline genes with the highest homology in the V-Base (http://www.mrc-cpe.cam.ac.uk/). The number of somatic mutations within the complementarity determining region (CDR)2 and framework (FR)3 in the consensus sequence of each sample were determined. As mutation patterns within CDRs are difficult to interpret, only the R (replacement) to S (silent) mutation ratio in the FR3 was considered as an indication for antigen selection.17 A sequence was regarded as being antigen selected when the R to S ratio in FR was less than 1.6.18
Ongoing mutation analysis
Sequences from the same DNA insert were compared within clones and aligned with the consensus one. The consensus sequence was derived from the most dominant sequences among clones.19 Ongoing mutation was determined by dividing the cumulative number of partially shared mutations (mutations shared by some clones but not by all the IgVH gene clones) and unique mutations (mutations unique to a distinct IgVH gene clone) with the expected number of mutations calculated based on the error rate of PCR19 (in our experimental conditions, it was 4.5 × 10−4 change/base/PCR cycle).
Statistical analysis of the difference of the ongoing mutation between AID-positive and AID-negative FL fresh cell categories was performed by the unpaired Student's t-test, and P<0.05 was considered as statistically significant. The correlation between AID expression and ongoing mutation was analyzed by determining the two-tailed Pearson's correlation coefficient, with r>0.5 considered as statistically significant. Both tests were performed using SPSS version 10 for Windows.
AID mRNA expression
The AID transcript by was detected RT-PCR in 13 out of 19 FL materials, representing 10 out of 15 fresh cells of FL cases (66.6%) and in three out of four cell lines (75%) (Table 2). The expression of the two splice variant products was always detected in cases expressing wild-type AID, but not in the cases in which the wild-type AID was not expressed (data not shown).
IgVH gene usage and mutational analysis
The SHM analysis of IgVH gene was carried out for 16 materials, consisting of 12 out of 15 fresh cells of FL cases and four cell lines. The SHM of IgVH gene was not analyzed in three FL cases due to the lack of clonality as revealed by the blurred band of IgH DNA PCR product visualized on the agarose gel (data not shown). This may have resulted from the limited amount of tumor cells in the tissue or the lack of primer site in the IgVH gene, which may have been caused by frequent mutations in the IgVH gene.20 The AID/β-actin ratios determined by densitometry analysis in 12 fresh cells of FL cases analyzed for SHM are shown in Table 3.
The IgVH sequences of the 16 FL materials (12 fresh cells and four cell lines) analyzed in our study were deposited in the GenBank under accession numbers AY245254-480, AY450647-662 and AY450667-9. The VH genes were derived from the VH3 in 10 materials, VH4 in five materials and VH1 in one material. Such usage without apparent bias from that in normal peripheral blood lymphocytes has also been reported (Table 2).21
The distribution of mutations in CDR2 and FR3, the mutation rate and the antigen selection calculation are also shown in Table 2. Irrespective of the AID expression, all materials displayed SHM in their IgVH genes, with mutation rates ranging from 4.16 to 15.6%, with an average of 10.07%. Similar frequencies have been reported by others.21,22 The possible role of a specific antigen in the clonal growth of the tumor cells was indicated by the evidence of antigenic selection in eight of 16 materials (50%) as shown by an R to S ratio in FR3 of less than 1.6.18
Ongoing mutation analysis
As shown in Table 3, the ongoing mutation in seven AID-positive FL fresh cells was generally high, except in case 7, as indicated by an average of 17.93-fold higher than the expected number of additional mutations due to PCR error. Besides expressing the wild-type AID, all AID-positive FL fresh cells also expressed splice variants of AID mRNA. The products of these splice variants in the FL cases did not seem to inhibit the function of the wild-type AID mRNA products, in contrast to those in CLL cases.14 For these seven cases, the AID/β-actin ratio was plotted in Figure 1.
In case 7, there were two additional partially shared mutations of the transition type (codon 78: C to T), which was only 4.4-fold higher than the number of mutations expected due to PCR error (0.45). In clone 5 of case 10, there was one additional base substitution (codon 83: A to T) that caused a stop codon. Besides the additional partially shared and unique mutations, there was one base deletion in codon 91(A) of clone 5 of case 1, which might have resulted from the ongoing mutation activity.23
Among the five AID-negative FL fresh cells, the ongoing mutation was generally low, yielding an average of 2.96-fold higher than the expected number of additional mutations due to PCR error. Two AID-negative FL cases did not have any additional mutation (cases 6 and 11). In three cases (case 4, 5 and 12), some additional mutations were observed in low frequency. Cases 4 and 5 showed only one and two additional unique nucleotide changes, respectively, which was 2.6-fold higher (case 4) and 4.3-fold higher (case 5) than the number of expected mutations due to PCR error (0.38 and 0.46, respectively). Moreover, the one unique nucleotide change in case 4 was of transition type (codon 80: T to C), and so were the two unique nucleotide changes in case 5 (codon 54: T to C and codon 94: A to G). Thus, one or two additional unique mutations in these cases is hardly an evidence for the ongoing mutation. In case 12, there were two additional partially shared mutations of transition type (codon 79: T to C) and one additional unique mutation of transversion type (codon 63: C to G), which was 7.9-fold higher than the number of expected additional mutations due to PCR error (0.38).
The difference in the ongoing mutation between the AID-positive and AID-negative FL fresh cells was significant (P=0.047). AID expression and ongoing mutation were also well correlated in the FL fresh cells (r=0.899, P=0.01).
None of the four cell lines showed considerable intraclonal microheterogeneity indicating the ongoing mutation. FL18 and FL318, both of which are AID positive, did not have any additional mutation. Each of FL218 (AID positive) and FL518 (AID negative) had only one additional unique nucleotide change, which is only 3.1-fold (FL218) and 1.5-fold (FL518) higher than the expected number of mutations due to PCR error (0.32 and 0.66, respectively). The one additional unique mutation in FL218 was of transition type (codon 93: G to A) and so was the one in FL518 (codon 64: A to G).
Switch-off point of AID expression in B-lineage differentiation
While an initial report showed that AID is specifically expressed in the GC,3 the exact point in B-lineage differentiation where AID expression is switched off remains to be identified. It was suggested that the expression of AID is probably restricted only to a short period of time during normal B-cell maturation, while the constitutive expression of AID could be observed in the neoplastic condition.24 However, broad studies on the SHM pattern, which is known to be useful for tracing the progenitors of human B-cell lymphomas,18 suggested the concordance of the expression of AID in both the normal and neoplastic condition.
In our previous study,25 the AID expression was found in 13 of 15 Burkitt lymphoma (BL) cell lines and five of five cases of fresh BL cells. SHM was found in four of four analyzed AID-positive BL lines, whereas no SHM was found in the two rare AID-negative BL lines. One of the two AID-negative BL lines (Tree92) expressed terminal deoxynucleotidyl transferase and recombination activation gene 1 and 2.26 Thus, the absence of AID in the two rare BL lines was of primary-negative type (negative before the primary expression). This primary-negative AID expression in the two rare BL cell lines is in contrast to the absence of AID expression in our FL materials, in which AID was switched off after the completion of SHM. In this situation, the absence of AID can be referred to as secondary negative.
It appeared from our data that the SHM rate was generally lower in AID-positive FL fresh cells than in AID-negative ones, except for two cases (cases 7 and 10) (Table 2). When the ratio of AID/β-actin of AID-positive FL fresh cells were plotted, there seemed to be a tendency for SHM rate to increase along with AID expression level (Figure 1). Considerable intraclonal microheterogeneity, indicating the ongoing mutation, was a common feature in those AID-positive FL fresh cells, except in 1 case (case 7). In contrast, in the five AID-negative FL fresh cells, the SHM rate was generally high (Table 2), accompanied by a low degree or lack of detectable ongoing mutation (Table 3). Case 7, which was AID positive but had a high SHM rate and low intraclonal microheterogeneity, is of importance, as it possibly represents a borderline stage between common AID-positive FL cases and AID-negative FL cases, before AID expression is switched off. Some mechanisms to switch off the expression of AID in FL fresh cells might be affected by the level of SHM and ongoing mutation activity.
The possible switch-off point of AID expression in the normal B-cell counterpart of FL in our results accords with the result of Greeve et al,24 where AID was expressed in normal centroblasts but not in centrocytes, both of which are thought to be possible progenitors of FL. However, our results contradicted another part of their results, in which they found AID being expressed in five of five FL cases. This difference may be resolved by analyzing more samples, since there have been several reports showing heterogeneity in the SHM pattern in FL, suggesting the possible heterogeneous expression of AID in this entity.10,22 Another report by Smit et al27 showed the expression of AID mRNA above the limit of detection only in nine out of 36 cases (25%). The quantitative RT-PCR method used in their study might have caused this lower incidence than ours.
Association between AID expression and ongoing mutation in FL cells
Previous studies of Ig gene SHM in FL reported abundant heterogeneity of the SHM pattern. Evidence of possible antigenic selection was found in 30% and intraclonal variation in 78.2% of FL cases,10 while another study observed antigenic selection in 84% and intraclonal variation in 71% of cases.22 Based on the reported heterogeneity in FL,10,22 the difference in the AID expression found among the FL materials in our series and others,24,27 and based on the basic proposed function of AID in activating the SHM machinery,2 it can be presumed that some difference in the SHM characteristic might exist between the AID-positive and AID-negative FL.
Our primary finding was that a significant difference in intraclonal microheterogeneity, indicating the ongoing mutation, was found between AID-positive FL fresh cells and AID-negative ones (P=0.047). In the fresh cells of FL cases, the AID expression was associated with ongoing mutation (r=0.899, P=0.01). This could explain the reported heterogeneity found in fresh FL cells.10,22
None of the FL cell lines (three AID-positive and one AID-negative) showed ongoing mutation. A given subclone might have prevailed due to some growth advantage during maintenance, resulting in the disappearance of subclones that might have exhibited the ongoing mutation. The absence of B-cell receptor crosslinking and intimate T–B cell contacts through various surface molecules in the in vitro culture leads to the cessation of the operation of the SHM machinery, as concluded based on one study using the BL2 cell line.28 Interaction through CD80-CD28 and CD40-CD40L was also reported to be necessary but not sufficient for inducing SHM.29 A very rare FL cell line exhibiting the ongoing mutation, HF-1.3.4, was derived from the parental HF-1 FL cell line after stimulation with B-cell-specific growth factor.30 So far, Ramos is the only cell line that naturally displays the ongoing mutation in vitro due to the clonal instability of its Ig variable region.31 AID expression was shown to be correlated with the mutation rate in Ramos subclones, and a long-term culture period was shown to favor the outgrowth of nonmutating cells expressing lower levels of AID.
Concerning our previously reported finding that BL generally expressed AID25 irrespective of the variety of the ongoing mutation,32,33,34 AID is necessary to initiate but may not be sufficient to maintain an active ongoing mutation process,35 as seen in some BL not displaying the ongoing mutation.32 An additional factor may be required to maintain the ongoing mutation process in some cases of BL.36 Alternatively, some impairment in AID function may cause the arrest of the ongoing mutation in some cases of BL, as suggested by an experiment where a nonfunctional AID mutant arrested the process of ongoing mutation in a hypermutating BL cell line.37 Impairment of AID function by the products of splice variant AID mRNAs was also reported in an AID-positive mutated CLL case (homology of 93%) that lacked intraclonal microheterogeneity.14 These reports14,37 and our results suggest a possible association between AID and ongoing mutation in FL. The association between AID and CSR has been observed in stage of CLL,38 and such point needs to be clarified as a next step in FL stage.
Since analyzing the ongoing mutation clarifies the properties of B-cell lymphoma in disease progression and the resistance of FL to certain therapy, such analyses should be beneficial for predicting the disease prognosis.8,39 Thus, the association between AID and ongoing mutation in FL deserves further investigation.
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MSH is a Graduate Student of Foreign Scholarship of Japan (MONBUSHO) under the superiority of Professor Masafumi Matsuo.
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