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From the Division of Pathology,*
City of Hope National
Medical Center, Duarte, California; the Department of
Pathology,
University of Southern California,
Los Angeles, California; the Department of
Pathology,
Okayama University Medical School,
Okayama, Japan; and the Department of
Pathology,§
State University of Sao Paulo,
Botucatu, Brazil
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An individual's human leukocyte antigen (HLA) Class I genotype strongly influences the EBV-specific CTL response; strong CTL responses are frequently associated with restriction through certain HLA Class I alleles. For example, CTLs restricted through the HLA-A11 allele are often dominant in polyclonal CTL cultures reactivated in vitro from HLA-A11-positive individuals. Investigators have shown this A11-restricted response to be directed largely to the transformation-associated viral antigen EBNA-4, which contains multiple HLA-A11-restricted epitopes with differing immunogenicities.6
Investigators have determined the relative immunogenicities of overlapping truncated EBNA-4 (also known as EBNA-3B) fragments (14-15 amino acids in length) by a cytotoxic assay.6 Among those fragments, HLA-A11-restricted CTLs have demonstrable reactivities against peptides of epitopes 399-408 and 416-424 of the EBNA-4 antigen, whereas reactivities against other fragments were either never detected or had a tendency to be lost on serial passage.6 Lymphoblastoid cell lines with mutant EBNA-4 antigen, particularly within epitopes 416-424, are capable of escaping from HLA-A11-restricted CTLs.7-9
It has recently been proposed that a high prevalence of EBNA-4 mutations serves as a mechanism of escaping the CTL response in certain HLA types. Previous studies of a high HLA-A11-prevalence population in Papua New Guinea found the same EBNA-4 point mutation at residue 424 in several Type A EBV isolates.7 Study of another high HLA-A11-prevalence population in southern China revealed only two point mutations in 23 different EBV isolates, at epitopes 417 or 424 of the EBNA-4 antigen.10 These findings suggested that immune pressure selected for resident EBV strains lacking immunodominant HLA-A11-restricted CTL epitopes. In contrast, two recent polymerase chain reaction (PCR) studies from two geographically distinct Papua New Guinea populations identified epitope-loss variants of EBV with the identical amino acid substitutions in all EBV isolates restricted through several class I HLA types.11 Furthermore, the substitutions did not correlate with the contemporary distribution of HLA types in the different Papua New Guinea populations, suggesting that immune pressure plays a minimal role in the long-term evolution of EBV.
EBNA-4 has not been consistently identified in EBV-associated tumors in immunocompetent patients. However, it is possible that EBNA-4 may be expressed in early tumorigenesis, perhaps prior to or concurrent with neoplastic transformation, at or around the time that immune selection occurs, since this protein has been shown to up-regulate and down-regulate a variety of host proteins, including the Burkitt's lymphoma-associated antigen CD77.12,13 EBNA-4 mutations have not been previously extensively investigated in tumor tissues. To test the hypothesis that EBNA-4 mutations are a mechanism of escaping the normal immune surveillance, we examined EBNA-4 mutations in several different EBV-associated malignancies, including Hodgkin's disease (HD) from the United States and Brazil, AIDs-related non-Hodgkin's lymphoma from the United States, and gastric carcinoma (GC) from the United States and Japan, as well as normal tissues from all three countries. We found that i) mutations in positions 1 and 2 of epitope 399-408 and positions 2 and 9 of epitope 416-424 are very common (67% of all mutations detected) in HD and GC; ii) the substituted amino acids in these positions are virtually identical to those seen in endemic EBV-associated nasopharyngeal carcinoma (southern China) and Burkitt's lymphoma (Papua New Guinea); iii) the reactive tissues show similar mutations to their neoplastic counterparts; iv) the mutation rate may vary in different geographic areas; and v) the mutations show no relationship to HLA-A11 positivity.
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Materials and Methods |
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We studied 37 cases of known EBV-associated GC (12 from the United States, 25 from Japan), 25 cases of known EBV-associated Hodgkin's disease (HD) (12 from the United States and 13 from Brazil), and 9 cases of acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (from the United States). We also studied 24 reactive lymphoid tissues (10 from the United States, 8 from Japan, and 6 from Brazil). The EBV status in GC, HD, AIDS lymphoma, and reactive lymphoid tissues have been previously reported.14-17 Briefly, all cases in this study were previously shown to be EBV-positive by in situ hybridization.14-17 As previously reported, all 37 GC cases, all 24 reactive lymphoid tissues, 8/9 AIDS lymphoma cases, and 23/25 HD cases had Type A EBV. Only two United States HD and one AIDS lymphoma case had type B EBV.14,17
PCR Studies for Epitopes 399-408 and 416-424 of EBNA-4
For each case, viral genomic DNA was extracted from 5-µm sections cut from formalin-fixed, paraffin-embedded tissue blocks, using 0.2 mg/ml protease K digestion buffer overnight, followed by denaturation by boiling. PCR studies were performed with 2 µl of extracted DNA in a 30-µl mixture containing 50 mmol/L KCl, 10 mmol/L Tris buffer (pH 8.3), 50 µm of each deoxynucleotide triphosphate, 2.5 mmol/L MgCl2, 1 unit of Taq polymerase (Perkin Elmer, Foster City, CA), and 20 pmol of each primer. Two sets of appropriate oligonucleotide primers were chosen, flanking the DNA region coding for epitopes 399-408 and 416-424 EBNA-4 of the prototype B95.8 EBV virus:18 EBNA-4+ primer 5'-GAGGAGGAAGACAAGAGTGG-3' and EBNA-4- primer 5'-GATTCAGGCGTGGCTCTTGG-3'; nested EBNA-4 PCR primer+ 5'-TACCGCAAACACTGCCGTAC-3' and nested EBNA-4 primer- 5'-CTGTTCTGGCTGCCTTCTTC-3'. After initial denaturation for 3 minutes at 95°C, 45 amplification cycles were performed as follows: denaturing at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 40 seconds. A final extension at 72°C for 3 minutes completed the PCR amplification. The PCR setup and the post-PCR work were performed in separate laboratories to minimize the possibility of contamination.
Bidirectional PCR Amplification of Specific Alleles (Bi-PASA) for the HLA-A11 Locus
PCR amplification of specific alleles (PASA) was originally developed for assaying a known mutation in one allele.19 Bi-PASA was designed for heterozygous gene analysis, such as HLA type determination.20 HLA-Class I genes are located in human chromosome 6 and are highly polymorphic, with each of the HLA genes having multiple alleles in the population.
Genomic DNA was extracted as above. A unique HLA-A11 sequence was
previously identified in HLA-A exon-3 from 524-560, which is specific
to HLA-A1101, A1102 and A1103.21
This fragment was first
amplified by PCR from 456-617 using HLA-A11 PCR primer+
5'-GGACCTGCGCTCTTGGAC-3' and HLA-A11 PCR primer-
5'-GTGCGCTGCAGCGTCTCC-3'. The HLA-A11-specific sequence was determined
by Bi-PASA method (Figure 1)
.19,20,22
Briefly, two
outer (P 464-481 and Q 585-602) and two inner (A 543-560 and B 559-574)
HLA-A-11-specific primers were made. The two outer primers were P
(primer+ 5'-ATGGCAGCTCAGATCACC-3') and Q
(primer- 5'-TCCTTCCCGTTCTCCAGG-3'). The two inner primers
were A (wild-type) (primer+
5'-ggggcgggcAGCCTACCTGGAGGGCAC-3') and B (mutant)
(primer- 5'-gcgggcgggGCCACTCCACGCACCG-3'). The
italic letters in primers A and B represent the noncomplementary 5'
tail sequences that switch from inefficient amplification of genomic
DNA to efficient amplification of previously amplified template by
PCR.18
The Bi-PASA products were run on an agarose gel. The
mutant bands were cut out for DNA sequencing (Figure 1)
.
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The sequencing primers were: for EBNA-4: EBNA-4+ 5'-ACTGCCGTACAATCCAACAG-3' and EBNA-4- 5'-CCTTCTTCTTTCTGTGTTCC-3', and for HLA-A11:HLA-A11 5'-ATGGCAGCTCAGATCAAC. Thirty microliters of the PCR products were run on an agarose gel and the product bands were cut out, purified using a Qiaex gel extraction kit (Qiagen, Hilden, Germany), and resuspended in 30 µl of water. The products were sequenced with an AmpliCycle sequencing kit (Perkin Elmer), using the manufacturer's recommended conditions. The products of the sequencing reaction were then separated by gel electrophoresis, dried, and exposed to film. The gel consisted of 7 mol/L urea and 8% polyacrylamide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Figures 2 and 3
)
and one case of United States AIDS lymphoma had seven mutations (Figure 4)
.
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, and
positions 2 and 9 of second epitope 416-424, which we have termed S2
and S9, respectively (Figures 2–4)
. Among the 45 detected mutations,
15 occurred in S9 (33%), six in S2 (13%), five in F1 (11%), and four
in F2 (9%). All except two of these mutations were identified in
type-A EBV-positive cases. The F1/F2 mutations occurred in 2/6 cases of
United States HD, 1/3 United States reactive tissues, 1/6 United States
GC, and 1/2 United States AIDS lymphoma cases. It was not observed in
any of the tissues from Brazil or Japan. The difference between the
incidence of F1/F2 mutations in tissues from the United States compared
to other countries was not statistically significant
(P = 0.06). The S2 mutations occurred in 1/3
United States reactive tissues, 1/6 United States GC cases, 1/2 United
States AIDS-lymphoma cases, and 3/7 Japanese GC. The difference between
the incidence of S2 mutations in tissues from the United States
compared to other countries was not statistically significant (all
tissues, P = 0.37; malignant tissues only,
P = 0.11). The S9 substitutions were observed in all
tissue types, from all countries, and included 3/6 United States HD,
2/3 United States reactive, 2/2 Brazilian HD, 5/6 United States GC, 1/2
United States AIDS-lymphomas, 1/7 Japanese GC, and 1/2 Japanese
reactive tissues.
In five of six cases involving an amino acid substitution at S2, Val
was replaced by Leu or Ile. The other was a silent mutation
involving no amino acid substitution. In 12 of 15 cases involving an
amino acid substitution at S9, Lys was replaced by Asn. F1 and F2
mutations from Ala-Val to Ser-Leu occurred together in four cases
involving those loci (Figures 2 and 3)
. In the case involving an F1
mutation without F2 mutation, the amino acid substitution was also
Ser-for-Ala. Substitution of position 5 (S5) with or without
substitution of position 7 (S7) of second epitope 416-424 was detected
in six cases. Four out of six S5-mutated cases were type A EBV-positive
and two were type B EBV-positive.
All 28 cases with EBNA-4 mutations had HLA-A locus Bi-PASA-amplifiable products. Only two of the 28 patients were shown to be HLA-A11-positive, including one case each of United States reactive and United States GC. Fourteen of the 28 (50%) cases had no AQ band, con-sistent with no HLA-A11 amplification. Fourteen of the 28 (50%) cases had AQ bands, which were sequenced. However, only two of 14 AQ bands were demonstrated to have the HLA-A11 sequence (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent epidemiological studies have shown that mutation of the antigenically determined epitope of EBNA-4 may play a key role in the development of EBV-associated malignancies. The Papua New Guinea and southern Chinese populations have an unusually high HLA-A11 prevalence, with a frequency of ~60%.7,10 HLA-A11 epitope-loss variants of EBV were first identified from six Papua New Guinea Type A EBV isolates by PCR studies.7 De Campos-Lima et al10 reported 23/23 southern Chinese NPC isolates to show mutations in EBNA-4 at epitope positions S2 and S9. These studies suggested that the viral strains lacking the major A11-restricted CTL epitope enjoyed a selective advantage in this particular population due to their failure to elicit a strong CTL response.
We examined EBNA-4 mutations in different EBV-associated malignancies, including Hodgkin's disease from the United States and Brazil and gastric carcinoma from the United States and Japan, as well as AIDS-related non-Hodgkin's lymphomas from the United States and normal tissues from all three countries. Many AIDS lymphomas are known to express a latency pattern consistent with EBNA-4 expression, whereas the other two neoplasms are thought to not express EBNA-4, at least at the time of biopsy. However, one could hypothesize that EBNA-4 may be expressed early in transformation, because it has been shown to up-regulate and down-regulate a variety of host proteins, including the Burkitt's lymphoma-associated antigen CD77.13 We found that approximately one-third of our malignant cases, regardless of tumor type or geographic origin, had EBNA-4 mutations. In addition, the reactive tissues showed a similar percentage of EBNA-4 mutations.
Two-third of mutations detected in United States and Brazilian HD, US
and Japanese GC and US AIDS-lymphoma occurred in the same EBNA-4
epitope positions as those reported in Papua New Guinea and Southern
China (Figures 2 and 3)
.7,10
We also found a limited number
of specific EBNA-4 mutations that accounted for a high percentage (67%
of all mutations detected) of mutations in cases of HD and GC from all
three countries. The amino acid substitutions at the four commonly
mutated epitopes in our cases were similar to those found in isolates
of nasopharyngeal carcinoma patients from southern China and Burkitt's
lymphoma patients from Papua New Guinea.7,10,29
In five of
our six cases involving an amino acid substitution at S2, Val was
substituted by Leu or Ile. In the southern Chinese isolates, one-half
of the cases had the Val at S2 substituted by Leu.7
In 12
of our 15 cases involving an amino acid substitution at S9, Lys was
substituted by Asn. All of the Papua New Guinea isolates showed the
single-point mutation Lys to Thr at S9.7
In the southern
Chinese cases, the Lys at S9 was substituted by Asn or more rarely by
Arg or Thr.10
F1 and F2 mutations from Ala-Val to Ser-Leu
occurred together in all four of our cases involving those loci
(Figures 2 and 3)
and the single case with F1 mutation without F2
mutation also had Ser replacement of Ala. The Papua New Guinea isolates
all showed Ser-Leu or Ser-Phe mutation in F1 and F2.10
In
addition, 13/23 Chinese isolates also showed mutations that altered F1
and F2 from Ala-Val to either Ser-Leu, Pro-Leu, or
Ser-Phe.10
Experiments have shown that A11-positive LCLs
carrying these mutations are not lysed by B95.8-induced CTL clones
because the substitutions of Val by Leu at S2 and Lys by Asn at
S9 are likely to abrogate binding of the peptide to the HLA-A11
groove.7,10
Epitope positions S2 and S9 of EBNA-4 are known
to be important determinants for HLA-A11 and peptide interaction
because both residues are positively charged and form a small groove
for HLA-A11-peptide binding.29
Thus, the CTLs of
susceptible individuals cannot eliminate mutant EBV strains before
viral persistence is established. Furthermore, the viral strains
lacking the target peptides may have a greater chance of successful
transmission to such individuals.
In our study, although the observed amino acid substitutions were nearly identical to those seen in isolates from EBV-associated nasopharyngeal carcinoma and Burkitt's lymphoma, the large majority of our cases were not in a high HLA-A11 prevalence population. The phenomenon that epitope loss variants of EBV have enjoyed a selective advantage in HLA-A11 high prevalence populations was not observed in our EBV-associated HD, GC, and AIDS-lymphoma cases. This is in contrast to earlier studies of a high HLA-A11-prevalence population in Papua New Guinea that found the same EBNA-4 point mutation at residue 424 in several Type A EBV isolates.7,10 Study of another high HLA-A11-prevalence population in Southern China revealed only two point mutations in 23 different EBV isolates, at epitopes 417 or 424 of the EBNA-4 antigen. These findings suggested that immune pressure selected for resident EBV strains lacking immunodominant HLA-A11-restricted CTL epitopes. In their original studies,7,10 de Compos-Lima et al predicted HLA-A11 prevalence based on population data, not on HLA-A11 phenotyping of individual cases as performed in the current study. Several recent studies on EBV isolates from coastal and highland Papua New Guinea showed that sequence analysis within the CTL epitope regions was identical in isolates from these two regions, whereas the prevalence of HLA-A11 is >50% in the coastal population and <5% in the highland population.11 The substituted amino acids were virtually identical to those seen in a recent PCR study from two geographically distinct Papua New Guinea populations that identified epitope-loss variants of EBV with the identical amino acid substitutions in all EBV isolates restricted through several class I HLA types.11 The substitutions did not correlate with the contemporary distribution of HLA types in the different Papua New Guinea populations, suggesting that immune pressure plays a minimal role in the long-term evolution of EBV. These studies strongly suggested that the mutations within CTL epitope sequences are not due to selective pressure exerted by the immune system, but as a result of local dissemination of a single origin. Although we did not observe identical mutations in GC and HD of different ethnic groups in the current study, the positions and substitutions of mutated amino acids in epitopes 399-408 and 416-424 of EBNA-4 are very similar among the cases from different ethnic groups and geographical locations with no correlation with the contemporary distribution of HLA-A11.11,30 Our results further support that amino acid changes within the CTL epitope regions of the EBV are fortuitous events of a random genetic drift and are not influenced by the host genetic background and immune system.30 Similar results were also documented in some cases of Hodgkin's disease, from which no relationship was found between LMP-2 mutation and HLA-A0201, though HLA-A0201 allele is known to present one epitope of LMP-2 protein and generate a CTL response.31 EBV is a virus that is highly stable through evolution. It is possible that EBV strains detected in EBV-associated malignancies or in EBV-associated reactive conditions are similar regardless of ethnic groups and geographical locations.
The incidence of EBV-positive HD in Brazil and elsewhere in South America has been shown to be much higher (70–90%) than that in the United States.32,33 It is very interesting to note that the Brazilian HDs have a borderline significantly lower EBNA-4 mutation rate than United States HD cases (P = 0.06). However, the HLA-A11 antigen frequency (AF) in South America is about the same as United States (AF < 10).10
Two cases showed substitution at epitope position S5, with Phe by Leu and at epitope position S7, with Phe by Ile. Both of these cases had type B EBV. Identical amino acid substitutions were previously detected in three type B EBV isolates.7 Type B EBV, whether wild-type or mutant at these epitopes, does not bind to HLA-A117; instead, type B EBV interacts with the T cell receptor.34 It remains to be determined whether the peptide actually represents a CTL epitope in individuals infected with type B EBV strains.7
Although the precise role of EBV in the etiology of many EBV-associated tumors remain unclear, the presence of this virus in malignant cells offers a potential target for a CTL-based therapy, because immunodominant epitopes are likely to elicit an effective response in vivo.35-38 Different EBV-associated malignancies express different set of EBV antigens.39 Although individuals show clear differences (HLA background) in target antigen choice, the most frequent target antigens are EBNA-3, -4 and -6. Other latency antigens, including EBNA-2, EBNA-LP, LMP-1, and LMP-2 are sometimes targeted.40,41 EBV-specific CTLs to LMPs have already been used to treat EBV-associated malignancies.37,42 Before initiating such studies, an immunodominant epitope must be demonstrated for a given intracellular pathogen. For EBV-associated neoplasms that express EBNA-4, epitope 412-424 of EBNA-4 could be a candidate for such purpose. However, it is important to first determine whether the gene is mutated such that it no longer constitutes a target for CTLs. For those patients with specific epitope mutations, both vaccination and immunotherapy using these epitopes are likely to fail.
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Footnotes |
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Accepted for publication May 18, 1999.
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