Immune Netw.  2011 Feb;11(1):50-58. 10.4110/in.2011.11.1.50.

Expression of Epstein-Barr Virus Gene and Clonality of Infiltrated T Lymphocytes in Epstein-Barr Virus-associated Gastric Carcinoma

• Lee JM
• Kim H
• Noh SH
• Lee WY
• Kim SJ
• Park JH

• KMID: 2150692
• DOI: http://doi.org/10.4110/in.2011.11.1.50

Abstract

BACKGROUND
Epstein-Barr virus associated gastric lymphoepithelioma-like carcinoma (LELC) is characterized by the intensive infiltration of lymphoid cells, the presence of EBV, and the better prognosis over typical adenocarcinoma. Thus, it was assumable that viral latent proteins may be responsible for the recruitment of a certain T cell repertoire to EBV-associated gastric carcinoma.
METHODS
To examine above possibility, EBV gene expression in gastric carcinoma tissues and usage of TCR among the tumor infiltrating lymphocytes were analyzed.
RESULTS
EBV specific DNA and EBERs RNA were detected in 4 out of 30 patients. RT-PCR analysis revealed that all 4 of EBV-positive tumor tissues expressed EBNA1 mRNA and BARTs and LMP2a was detected only one sample out of 4. However, the EBNA2 and LMP-1 transcripts were not detected in these tissues. CD8+ T cells were the predominant population of infiltrating lymphocytes in the EBV-positive gastric carcinoma. According to spectra type analysis of infiltrating T cells, 10 predominant bands were detected by TCR Vbeta CDR3 specific RT-PCR from 4 EBV-positive tumor tissues. Sequence analysis of these bands revealed oligoclonal expansion of T cells.
CONCLUSION
These findings suggest that clonally expanded T cells in vivo might be a population of cytotoxic T cells reactive to EBV-associated gastric carcinoma.

Keyword

Epstein-Barr virus-associated gastric carcinoma; EBNA1; BARTs; Cytotoxic T lymphocyte; CDR3

MeSH Terms

Adenocarcinoma
DNA
Gene Expression
Herpesvirus 4, Human
Humans
Lymphocytes
Lymphocytes, Tumor-Infiltrating
Prognosis
Proteins
RNA
RNA, Messenger
Sequence Analysis
T-Lymphocytes
DNA
Proteins
RNA
RNA, Messenger

Figure

• 

  Figure 1 Detection of EBV DNA in gastric carcinoma tissues using PCR. (A) DNA was extracted from 30 gastric carcinoma tissue samples, and PCR was performed using the EBV specific primers SL18 and SL19. PCR products were electrophoresed in a 2% agarose gel. The EBV genome was detected in 4 of the samples (#967, #974, #1010, and #1016). No EBV genome was detected in the gastric carcinoma cell line, MKN45 (negative control), and 26 gastric carcinoma tissue samples. Lane 1: DNA size marker, 1 kb DNA ladder; MKN45: gastric carcinoma cell line showing no EBV infection; 967, 974, 1010, 1016: Gastric carcinoma tissues samples with the EBV genome. (B) VNTR DNA of EBV LMP1 was amplified and electrophoresis was performed. M: DNA size marker, 100 bp DNA ladder; DW: distilled water; MKN45: gastric carcinoma cell line (negative control); LCL: B lymphoblastoid cell line (positive control) transformed with EBV produced with the B95.8 cell line; 967, 974, 1010, 1016: gastric carcinoma tissue samples with EBV genes detected.

• 

  Figure 2 Pathological findings of gastric carcinoma tissue samples and EBER in situ hybridization. (A) The pathological findings of gastric carcinoma tissue sample #967 containing the EBV genome. This carcinoma was an undifferentiated adenocarcinoma and shows severe infiltration of lymphocytes (H-E staining, ×400 magnification). (B) EBERs in situ hybridization. Nuclei of gastric carcinoma cells show a positive response, while lymphocytes infiltrating the gastric carcinoma tissues and the surrounding connective tissue show a negative response.

• 

  Figure 3 Determination of EBV type in gastric carcinoma tissues. PCR was performed using EBNA3C specific primers in order to determine the type of EBV in gastric carcinoma tissues. The resulting PCR product was electrophoresed. Lane 1: DNA size marker, 100 bp DNA ladder; MKN45: negative control; B95.8: positive control; 967, 974, 1010, and 1016: gastric carcinoma tissue samples with EBV genes detected.

• 

  Figure 4 Detection of EBV specific gene transcripts in gastric carcinoma tissue samples using RT-PCR. Total RNA was isolated from gastric carcinoma tissues and cDNA was prepared. RT-PCR was performed using the cDNA as template and specific primers for EBV genes, i.e., EBNA1, EBNA2, BARTs and LMP-1. The resulting products were electrophoresed in a 2% agarose gel. MKN45: negative control; LCL: B lymphoblastoid cell line (positive control) transformed with EBV produced with the B95.8 cell line; 967, 974, 1010, 1016: gastric carcinoma tissue samples with EBV gene detected.

• 

  Figure 5 Distribution of lymphocytes infiltrating in gastric carcinoma tissues. Immunohistochemical staining of gastric carcinoma tissue #967 using anti-CD8 (A), anti-CD4 (B), and anti-CD20 (C) antibodies (200×).

• 

  Figure 6 TCR Vβ chain usage of DR3 of CD8+ T cells infiltrating gastric carcinoma tissues. After performing RT-PCR using specific primers to each Vβ family, electrophoresis was done in a sequencing gel and CDR3 was divided according to sizes. The predominant band (arrow) could be observed in Vβ family 2, 6, and 11 of sample #967; Vβ family 12 and 14 of sample #974; Vβ family 3, 6, 8, and 15 of sample #1010;Vβ family 8 of sample #1016. 967, 974, 1010, and 1016: Gastric carcinoma tissue samples with EBV gene detected.

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