Markey Kate A, Gartlan Kate H, Kuns Rachel D, MacDonald Kelli P A, Hill Geoffrey R
QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia; Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia.
QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia.
J Immunol Methods. 2015 Aug;423:40-4. doi: 10.1016/j.jim.2015.04.029. Epub 2015 May 9.
Immunological synapse formation between antigen-specific T cells and antigen presenting cells (APC) involves reorganization of the cellular cytoskeleton (polymerization of filamentous actin) and recruitment of adhesion molecules (e.g. LFA-1, ICAM-1). This engagement is critical for the generation of specific immune responses. Until recently, quantitative, high-throughput measurements of these interactions have not been possible. Instead, previous assessment was reliant on qualitative microscopy of live cells, where typically the APC is adhered to a surface and the suspended T cell is required to migrate to facilitate synapse formation. While this methodology can demonstrate the capacity for synapse formation, it cannot accommodate quantification of large numbers of interacting cell pairs, nor does it allow for statistically robust comparison between test conditions. We have developed a method for assessing immunological synapse formation between purified ex vivo dendritic cells (DCs) and responder antigen-specific CD4(+) T cells using imaging flow cytometry, allowing us to quantify LFA-1 and f-actin rearrangement at the interface between DC/T cell pairs. This novel application of imaging flow cytometry represents a major advance in dendritic cell function and immunological synapse research as it facilitates quantitative, high throughput analysis of the interaction between live, ex vivo DC and T cells.
抗原特异性T细胞与抗原呈递细胞（APC）之间免疫突触的形成涉及细胞骨架的重组（丝状肌动蛋白的聚合）和黏附分子的募集（例如淋巴细胞功能相关抗原-1、细胞间黏附分子-1）。这种相互作用对于特异性免疫反应的产生至关重要。直到最近，对这些相互作用进行定量、高通量测量才成为可能。相反，以往的评估依赖于活细胞的定性显微镜观察，通常是将APC固定在表面，悬浮的T细胞需要迁移以促进突触形成。虽然这种方法可以证明突触形成的能力，但它无法对大量相互作用的细胞对进行定量，也不允许在测试条件之间进行统计学上可靠的比较。我们开发了一种利用成像流式细胞术评估纯化的体外树突状细胞（DC）与反应性抗原特异性CD4(+) T细胞之间免疫突触形成的方法，使我们能够量化DC/T细胞对之间界面处的淋巴细胞功能相关抗原-1和丝状肌动蛋白的重排。成像流式细胞术的这种新应用代表了树突状细胞功能和免疫突触研究的一项重大进展，因为它有助于对活的体外DC和T细胞之间的相互作用进行定量、高通量分析。
