丙型肝炎病毒CTL表位的HLA-A2限制性及其免疫学效应研究

目的 研究前期鉴定的5条丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位的HLA-A2限制性及其免疫学效应.方法 基于T2胞株,采用MHC-肽复合物稳定性试验研究前期鉴定的HCV特异性CTL表位与HLA-A2分子的亲和力;采用细胞内细胞因子染色(intracellular cy-tokine staining,ICS)和ELISPOT研究七述HLA-A2限制性CTL表位体外刺激HLA-A2刚性外周血单个核细胞(PBMC)产生肽特异性CTL的效应;采用CTL杀伤试验研究上述肽特异性CTL杀伤靶细胞(负载相同肽的T2细胞)的效应.结果 前期研究鉴定的5条CTL表位肽中,NS4b_78(SMMAF-SAAL)和NS5a_367(TVSSALAEL)与HLA-A2分子有高亲和力(FI1);ELISPOT结果显示NS4b_78(SMMAFSAAL)和NS5a_367(TVSSALAEL)可诱导高水平的分泌IFN-γ的效应细胞[分别为(60±6)SFC/10~4PBMC vs(4±1)SFC/10~4PBMC,P<0.001;(10±3)SFC/10~4PBMC vs(2±1)SFC/10~4PBMC,P<0.001];ICS结果证实这两条肽刺激HLA-A2阳性PBMC后,在CD8~+T细胞中产生了高百分比的CD8~+IFN-γ~+T[分别为(2.33±0.22)%vs(0.05±0.01)%,P<0.001;(0.36±0.06)%vs(0.03±0.01)%,P<0.001];并且,肽特异性CTL可特异性杀伤负载相同肽的T2细胞.结论 NS4b_78(SMMAFSAAL)和NS5a_367(TVSSALAEL)受HIA-A2限制,并具有较好的免疫原性.

The HLA-A2 restriction and immunogenicity of hepatitis C virus-spedfic cytotoxic T lymphocyte epitopes

Objective To explore the HLA-A2 restriction and immunogenicity of 5 previously identified HCV-speeific CTL epitopes. Methods Based on T2 cell, to explore the HLA-A2 restriction of previously identified HCV-specific CTL epitopes by MHC-peptide complex stabilization assay;To detect pep-tide-specific CTL in HLA-A2~+ PBMC stimulated by HLA-A2-restricted peptides by intracellular cytokine staining(ICS) and ELISPOT; To explore the cytotoxicity of peptide-specific CTL to same peptide-loaded T2 cells (target cells) by CTL cytotoxicity test. Results Among 5 previously identified CTL epitopes NS4b_78 (SMMAFSAAL) and NS5a_367 (TVSSALAEL) have high-affinity for HLA-A2 molecules(FI 1) ;ELISPOT results shown that NS4b_78(SMMAFSAAL) and NSSa_367(TVSSALAEL) induced high levels of IFN-γ-se-creting cells [(60±6) SFC/10~4 PBMC vs (4±1 ) SFC/10~4 PBMC, P < 0.01 ; (10 ± 3 ) SFC/10~4 PBMC vs (2±1 ) SFC/10~4 PBMC, P <0.01, respectively] ;ICS results indicated that there were high percentages of CD8~+ IFN-γ~+ T cells in total CD8~+T cells stimulated by these peptides [(2.33 ±0.22 ) % vs (0.05±0.01)%, P <0.001 ; (0.36±0.06)% vs (0.03±0.01)%, P <0.001, respectively]. Furthermore,peptide-specific CTL could effectively kill same peptide-loadcd T2 cells. Conclusion NS4b_78 (SMMAF-SAAL) and NSSa_367 (TVSSALAEL) were identified as HLA-A2-restricted CTL epitopes which could in-duce immune response in vitro.