巨细胞病毒特异性免疫应答体外检测系统的建立

Establishment of an in vitro system for testing cytomegalovirus-speciflc immune response

目的以巨细胞病毒（CMV）感染的成纤维母细胞为抗原提呈细胞，建立CMV特异性细胞免疫应答的体外检测模型。方法将CMV-AD169、RV-TB40E-4突变株分别感染人胚肺成纤维母细胞（HELF），流式细胞仪（FCM）检测感染细胞HLA-A＊0201表达强度；将感染的HELF、荷载外源性多肽的自身外周血单个核细胞（PBMC）以及未感染的HELF分别与PBMC共同孵育，FCM检测CD8^＋T细胞内IFN-γ的表达作为应答指标。结果CMV-AD169感染细胞HLA-A＊0201表达强度较未感染组降低78．24％±19．72％，而突变株病毒不影响其表达，而且对AD169感染的HELF无应答的PBMC却对突变株病毒感染的细胞显示出了强阳性应答。AD169感染并荷载外源性多肽的HELF产生的刺激应答率，是感染的HELF和荷载多肽的未感染HELF所诱导应答比率的总和。结论CMV-AD169下调MHC Ⅰ分子表达，但不减弱细胞提呈外源多肽的能力。RV-TB40E-4是US2-6／US11基因被删除的CMV突变株，不抑制MHC Ⅰ分子表达，从而代表了一种更适宜研究CMV特异性免疫应答的工具。

Objective To establish an in vitro test system for testing CMV specific immune respense. Methods Human embryonic lung fibroblasts （HELF） infected with CMV-AD169 and a mutant strain RV-TB40E-4, respectively, were stained with anti-HLA-A ＊ 0201-PE and then measured for the expression of HLA-A ＊ 0201. The infected HELF were incubated with individual pp65 peptide NLVPMVATV and cultured together with PBMC , then intracellular IFN -γwas tested by flow cytometry （ FCM ） . Results The expression of HLA-A ＊ 0201 was markedly suppressed on AD169-infected fibroblasts, while it was not affected on RV-TB40E-4 -infected cells. PBMC showing no response to AD169-infected cells responded significantly to RV-TB40E-4-infected cells. The HELF infected with AD169 and loaded with external peptides induced a significantly stronger response which was the sum of the induced response ratios of the infected HELF and the uninfected HELF loaded with peptides. Conclusion CMV-AD169 downregulates the expression of MHC Ⅰ , while it does not decrease the capacity of cells to present external peptides. RV-TB40E-4, a CMV mutant in which the gene of US2-6/US11 has been deleted, does not affect MHC Ⅰ expression, and therefore represents a more suitable tool to explore CMV-specific immune response.