用酶联免疫斑点法检测肝癌患者抗原特异性免疫应答能力

Application of the IFN gamma ELISPOT assay for monitoring CD8^+ T cell response to specific antigen from hepatocellular carcinoma patients

目的 研究HLA A2阳性肝细胞癌 (HCC)患者及正常人外周血CD8+T细胞对流感病毒抗原肽 (Flup58 66)的免疫应答能力。方法 用免疫磁珠法分离出HCC患者与正常人外周血CD8+T细胞 ,以照射后自体CD8- 外周血单个核细胞 (PBMC)或树突状细胞 (DC)作为抗原提呈细胞 (APC) ,加载Flu抗原肽 ,经 7d培养诱导 ,用酶联免疫斑点 (ELISPOT)法检测诱导后产生γ干扰素的效应CD8+T细胞频数。结果 当效应细胞为 5× 1 0 4个 /孔时 ,CD8- PBMC作为APC ,Flu抗原肽特异的CD8+T细胞频数在HCC患者组 (n =8)为 2 2个± 9个 /孔 ,正常人组 (n =1 2 )为 59个± 2 7个 /孔 ,两组间差异有非常显著意义 (P <0 0 1 ) ,但均为阳性应答 ;在 5名正常人 ,Flu抗原特异的CD8+T细胞频数以DC为APC时高于以CD8- PBMC为APC时 (P <0 0 5)。结论 HCC患者对Flup58 66的免疫应答程度虽较正常人低 ,但大多数中晚期HCC患者仍然具有对抗原肽产生特异免疫应答的能力。

Objective To study the specific CD8 + T cell response to HLA A2 binding peptide Flu p58 66 from HLA A2 positive hepatocellular carcinoma patients and healthy donors. Methods The CD8 + T cells were separated with immunobeads from PBMC of HCC patients and healthy donors, respectively. The irradiated autologous CD8 - PBMC or isolated dendritic cells were loaded with influenza matrix peptide as APC. After 7 days′culture, the frequency of effector cells to secrete IFN gamma in response to Flu p58 66 was detected in ELISPOT assay. Results With CD8 - PBMC as APC, the frequency of effector cells to secrete IFN gamma in response to Flu peptide was 22±9/well in HCC patients ( n =8) and 59±27/well in healthy donors ( n =12) when the effector cells were 5×10 4/well( P <0.01) To compare the antigen presenting capacity of APC derived from 5 healthy donors, DC was better than CD8 - PBMC. Conclusion Although the frequency of specific effector CTL to secrete IFN gamma in response to Flu p58 66 was lower in HCC patients than in healthy individuals, the majority of HCC patients have the cellular immunity specific to antigen peptides.