[Transgenic expression of T cell receptors on HBV-specific cytotoxic T lymphocytes].

OBJECTIVE

To express T lymphocyte receptors (TCRs) on hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) mediated by retrovirus and investigate their binding affinity.

METHODS

Peripheral blood mononuclear cells were isolated from acute hepatitis B patients with HLA-A2⁺; phenotype, and after induced, HBV-specific CTLs were sorted out followed by cloning and proliferating. Cell RNAs were extracted. The sequences of TCR's α and β chains were obtained by means of RT-PCR, 5'RACE and OVER-LAP PCR, for constructing TCR retrovirus vectors. Through retrovirus-mediated transduction, HBV-specific TCRs were expressed on Jurkat cells and CD8⁺; T cells from HLA-A⁺; healthy subjects.

RESULTS

Two paired TCR Vα and Vβ, respectively named α21β13 and α15β13, were obtained from one patient with acute hepatitis B and HLA-A2⁺;. The titers of packaged recombinant retroviruses were 1.5×10;-5.0×10⁵; IU/mL. Immunofluorescence staining by anti-Vβ13 TCR-PE targeting the specific TCR and HLA-A2 restricted epitope-specific pentamer showed a positive expression of reconstructed TCR on T cells. The positive cells accounted for 1.06%-2.25% for Vβ13 on Jurkat cells, 1.03%-2.06% for Vβ13 chain and 1.05%-1.12% for the epitope-specific pentamer on T cells from healthy HLA-A2⁺; subjects respectively. By contrast, only less than 0.05% cells from healthy HLA-A2⁻ ;subjects were positive for either Vβ13 or the pentamer.

CONCLUSION

TCRs on HBV-specific CTLs could be expressed by TCR gene transfer mediated by retrovirus, and they were proved with binding affinity to HLA-A2-restricted epitope.