Pozzi Eleana, Zanotto Carlo, Pacchioni Sole, De Giuli Morghen Carlo, Radaelli Antonia
Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy.
J Virol Methods. 2009 Jan;155(1):77-81. doi: 10.1016/j.jviromet.2008.09.029. Epub 2008 Nov 12.
Although several techniques are available to evaluate cell-mediated immunity, numerous difficulties have prevented their use in rabbits. Cytotoxic T-lymphocyte (CTL) assays have been used to determine the ex vivo cytolytic activity of CD8+ T-lymphocytes in immunization protocols. However, this assay cannot be performed with rabbit peripheral blood mononuclear cell (PBMC) targets because of their high spontaneous (51)Cr release. To overcome this intrinsic difficulty shown by rabbit cells, syngeneic normal and SV40-immortalized cells were prepared from skin biopsies. The results show that: (i) skin-derived rabbit fibroblasts can be used as target cells after infection with a fowlpox virus recombinant; (ii) SV40-immortalized skin fibroblasts appear to be more appropriate for repeated assays; (iii) antigen-expanded T-cells and fresh PBMCs can be used as effectors with a similar efficiency; and (iv) dissociation of adherent skin fibroblast target cells with EDTA is to be preferred over TrypLE enzymatic treatment.
尽管有多种技术可用于评估细胞介导的免疫，但众多困难阻碍了它们在兔子身上的应用。细胞毒性T淋巴细胞（CTL）检测已被用于在免疫方案中确定CD8 + T淋巴细胞的体外细胞溶解活性。然而，由于兔外周血单个核细胞（PBMC）靶细胞的高自发性（51）Cr释放，无法对其进行该检测。为克服兔细胞显示出的这一内在困难，从皮肤活检组织制备了同基因正常细胞和SV40永生化细胞。结果表明：（i）感染鸡痘病毒重组体后，源自皮肤的兔成纤维细胞可用作靶细胞；（ii）SV40永生化皮肤成纤维细胞似乎更适合重复检测；（iii）抗原扩增的T细胞和新鲜PBMC可作为效应细胞，效率相似；（iv）与TrypLE酶处理相比，用EDTA解离贴壁皮肤成纤维细胞靶细胞更可取。
