Biclonal expansion of T cells infected with monoclonal Epstein-Barr virus (EBV) in a patient with chronic, active EBV infection

Abstract

Recent studies have suggested that a high percentage of Epstein-Barr virus (EBV)-infected lymphocytes in peripheral blood of patients with chronic, active EBV infection (CAEBV) is of T cell origin. Although T cells are expanded oligoclonally in CAEBV, it is not clear whether the restricted diversity of T cells arise from immune reaction against EBV-related antigens or from proliferation of EBV-infected cells. We experienced a patient with CAEBV who had biclonal expansion of peripheral blood T cells. We identified clonotypes of these two T cell clones in detail and purified the T cell clones. EBV infected mainly the two T cell clones, whereas the viral loads in peripheral blood cells other than these T cell clones were low or undetectable. The EBV strains infecting the two T cells clones were indistinguishable from each other by a series of genotype analyses of the virus. These results suggest that the two T cell clones infected with the same monoclonal EBV proliferated in peripheral blood of the patient.

Fig. 1

Southern blot analysis of TCRβ gene rearrangement of the patient's PBMNC. The genomic DNA was extracted from PBMNC and digested with EcoRI (E), BamHI (B) and HindIII (H). The digested DNA was subjected to Southern blotting and hybridized with TCRJβ1 (Jβ1) and TCRJβ2 (Jβ2) probes. ‘G’ and arrows indicate the germline bands and clonally rearranged bands, respectively. The figure is a montage from two independent experiments.

Fig. 2

Infection of CD4⁺ TCRαβ⁺ T cells in peripheral blood of the patient with EBV. Indicated cell populations were purified from the patient's PBMNC using MACS. The purity of each cell subpopulation is 98·1% in CD3⁺, 97·2% in CD20⁺, 95·5% in CD56⁺, 97·8% in CD14⁺, 98·5% in TCRαβ⁺, 96·2% in TCRγδ⁺, 97·3% in CD8⁺, 98·7% in CD4⁺, 97·5% in TRBV7⁺, 98·2% in TRBV9⁺ and 95·2% in TRBV7^– TRBV9^– T cells, respectively. Each cell population was subjected to the ISH assay using EBER probe (a) and PCR analysis for detection of EBNA2 (b).

Fig. 3

Expansion of TRBV7⁺ T cells and TRBV9⁺ T cells in peripheral blood of the patient. PBMNC of the patient were stained with FITC- or with PE-conjugated anti-CD3, anti-TRBV7 and anti-TRBV9 MoAb and then analysed using FACScan.

Fig. 4

Analysis of EBV termini in TRBV7⁺ T cells and TRBV9⁺ T cells of the patient. TRBV7⁺ cells, TRBV9⁺ cells and TRBV7^– TRBV9^– cells were purified from the patient's PBMNC using MACS. Genomic DNA were extracted from the purified cells and Raji cell line. Southern blots of BamI-digested DNA (0·01 or 0·1 mg per lane) were hybridized to probe for terminal repeats.