A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis
A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis
复制标题
DOI：
10.1172/jci58653
复制
发表时间：
2011-11-01
影响因子：
15.9
通讯作者：
Love, J. Christopher
中科院分区：
文献类型：
--
作者：
Varadarajan, Navin;Julg, Boris;Love, J. Christopher
CD8(+)T细胞是病毒感染的获得性免疫反应的重要组成部分。CD8(+)T细胞反应不足被认为是感染HIV后持续慢性感染的部分原因。因此，至关重要的是要找出各种方法来界定什么构成适当或不适当的反应。干扰素-γ的产生已被用来衡量T细胞的功能，但细胞因子的产生与细胞裂解病毒感染细胞的能力之间的关系尚不清楚。此外，使用新分离的血液样本以单细胞分辨率评估多个CD8(+)T细胞功能，并随后回收这些细胞进行进一步功能分析的能力尚未实现。正如这里所描述的，为了满足这一需求，我们开发了一种在125-p1微孔中高通量、自动化的检测方法，以同时评估来自HIV感染患者的数千个CD8(+)T细胞介导裂解和产生细胞因子的能力。这一并行的、直接的分析使我们能够调查即时细胞毒活性和短期细胞因子分泌之间的相关性。大多数体内启动的、循环中的HIV特异性CD8(+)T细胞在遇到特定数量的细胞上呈现的同源抗原时，在细胞溶解和细胞因子分泌方面不协调，尤其是干扰素-γ。我们的方法应该有助于确定单个效应者CD8(+)T细胞之间的功能差异特征，包括来自粘膜样本的特征和疫苗诱导的特征。
CD8(+) T cells are a key component of the adaptive immune response to viral infection. An inadequate CD8(+) T cell response is thought to be partly responsible for the persistent chronic infection that arises following infection with HIV. It is therefore critical to identify ways to define what constitutes an adequate or inadequate response. IFN-gamma production has been used as a measure of T cell function, but the relationship between cytokine production and the ability of a cell to lyse virus-infected cells is not clear. Moreover, the ability to assess multiple CD8(+) T cell functions with single-cell resolution using freshly isolated blood samples, and subsequently to recover these cells for further functional analyses, has not been achieved. As described here, to address this need, we have developed a high-throughput, automated assay in 125-p1 microwells to simultaneously evaluate the ability of thousands of individual CD8(+) T cells from HIV-infected patients to mediate lysis and to produce cytokines. This concurrent, direct analysis enabled us to investigate the correlation between immediate cytotoxic activity and short-term cytokine secretion. The majority of in vivo primed, circulating HIV-specific CD8(+) T cells were discordant for cytolysis and cytokine secretion, notably IFN-gamma, when encountering cognate antigen presented on defined numbers of cells. Our approach should facilitate determination of signatures of functional variance among individual effector CD8(+) T cells, including those from mucosal samples and those induced by vaccines.