Detection of antigen-epitope-specific cytotoxic T lymphocytes in patients with hepatitis B virus infection by enzyme linked immunospot assay

Abstract

To investigate the ability of secreting interferon-gamma (IFN-gamma) of the peripheral blood monocular cells (PBMC) stimulated by hepatitis B virus (HBV)-specific cytotoxic T lymphocyte (CTL) epitopes peptides and to analyze the difference of CTL immune response in patients with HBV infection. Four HLA-A2-restricted HBV cytotoxic T lymphocyte epitopes [Tp: HBV polymerase 575-583 (FLLSLGIHL), Te1: envelope 28-39 (IPQSLDSWWTSL), Te2: envelope 183-191 (FLLTRILTI) and Tc: core 18-27 (FLPSDFFPSV)] were synthesized. Human leucocyte antigen (HLA)-A2 typing was detected by Flow cytometry. PBMCs which were isolated from patients with chronic hepatitis B(CHB), patients with chronic severe hepatitis B(CSH), subjects with past HBV infection(N1) and healthy blood donors (N2) were stimulated by the four HLA-A2-restricted HBV CTLs epitopes. Enzyme linked immunospot (ELISPOT) assay was used to detect the frequency of secreting IFN-gamma CTL in each group. (1) HLA-A2 typing: 20 of 44 patients with CHB (45.5%) were HLA-A2 positive, 10/18 (55.6%) in CSH and 6/10 (60%) in group N1 were HLA-A2 positive.10 healthy blood donors' HLA-typing was detected in the early study.(2) ELISPOT results: (1) The total responses to the four epitopes in CHB, CSH, N1 and N2 groups were 50% (10/20), 10% (1/10), 83.3% (5/6) and 10% (1/10), respectively. The response in N1 group was significantly higher than that in CSH group (chi(2) = 9.000, P = 0.008) and N2 group (chi(2) = 9.000, P = 0.008). (2) The CTL average magnitude response to Tp epitope, Te1 epitope, Te2 epitope and Tc epitope was also significantly higher in past HBV infection group (77 SFC/10(6) PBMC, 59 SFC/10(6) PBMC, 100 SFC/10(6) PBMC and 57 SFC/10(6) PBMC, respectively) than that of CSH group (10 SFC/10(6) PBMC, 0 SFC/10(6) PBMC, 0 SFC/10(6) PBMC and 20 SFC/10(6) PBMC respectively, all P < 0.01) and N2 group (15 SFC/10(6) PBMC, 0 SFC/10(6) PBMC, 22 SFC/10(6) PBMC and 30 SFC/10(6) PBMC respectively, all P < 0.01). This study indicates that the T cell immune response to HBV-specific epitopes might be detected either in patient with chronic HBV infection or with previous HBV infection. This response should be much higher in patients with past HBV infection, even the virus had been cleared for long time. These results demonstrate that HBV-specific CTL might play an important role in the clearance of the virus.