International AIDS Vaccine Initiative Human Immunology Laboratory (IAVI HIL), London, UK.
J Immunol Methods. 2011 Jul 29;370(1-2):43-54. doi: 10.1016/j.jim.2011.05.007. Epub 2011 Jun 12.
The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from local tissue before any conclusions regarding the induction of mucosal responses are made. Here we describe the optimisation and subsequent qualification of a qualitative and quantitative polychromatic flow cytometry assay to assess antigen specific CD8+ T cells isolated from the gut, using samples from HIV positive and negative volunteers. Internal quality controls monitored over time, combined with the use of quality gating and standard operating procedures were used to demonstrate the generation of robust and reliable data.
下一代候选 HIV 疫苗包括选择对黏膜部位具有嗜性的复制载体，需要有效的 T 细胞反应来限制 T 细胞复制和与 HIV 相关的 CD4 T 细胞丢失。为了充分评估此类候选疫苗的免疫原性，需要开发稳健的质量控制分析方法，以检测肠道中 HIV 特异性 CD8+T 细胞反应。尽管在获得足够量的组织方面存在明显的挑战，但黏膜免疫反应的高度分隔性质要求在得出关于黏膜反应诱导的任何结论之前，直接从局部组织中分离 CD8 T 细胞进行评估。在这里，我们描述了一种优化和随后定性和定量多色流式细胞术分析的方法，用于评估从肠道中分离的抗原特异性 CD8+T 细胞，使用来自 HIV 阳性和阴性志愿者的样本。随着时间的推移进行的内部质量控制，结合使用质量门控和标准操作程序，证明了生成稳健和可靠数据的能力。
