Center for Infectious Medicine, Department of Medicine, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden.
J Immunol Methods. 2011 Mar 7;366(1-2):106-18. doi: 10.1016/j.jim.2011.01.014. Epub 2011 Feb 2.
An early step in immunosurveillance by cytotoxic lymphocytes is leukocyte functional antigen (LFA)-1-dependent adhesion to target cells, which is promoted by inside-out signals from receptors such as the T cell receptor and a variety of natural killer (NK) cell activating receptors. Inside-out signals induce a conformational change in LFA-1, resulting in an extension of the extracellular domain of the receptor. Here, we have evaluated several mAbs that specifically detect the extended conformation of LFA-1 and detail a protocol for flow cytometric quantification of β2-integrin activation in human peripheral blood cytotoxic T cells and NK cells in response to target cell recognition. By comparison to the markers of degranulation and chemokine synthesis, e.g. surface CD107a expression and intracellular MIP-1β expression, respectively, evaluation of LFA-1 conformational changes represent a sensitive and rapid parameter of primary cytotoxic lymphocyte activation that can facilitate isolation of viable cells. Potentially, combined with other read-outs of cytotoxic lymphocyte function, this technique is applicable to the assessment of various clinical conditions, including for the diagnosis of primary immunodeficiency syndromes and for evaluating the efficacy of tumor targeting by donor lymphocytes.
细胞毒性淋巴细胞免疫监视的早期步骤是白细胞功能抗原（LFA）-1 依赖于靶细胞的黏附，这是由受体（如 T 细胞受体和各种自然杀伤（NK）细胞激活受体）的内信号促进的。内信号诱导 LFA-1 的构象变化，导致受体的细胞外结构域延伸。在这里，我们评估了几种专门检测 LFA-1 延伸构象的 mAb，并详细介绍了一种流式细胞术定量检测人外周血细胞毒性 T 细胞和 NK 细胞对靶细胞识别反应中β2-整合素激活的方案。与脱颗粒和趋化因子合成的标志物（例如，表面 CD107a 表达和细胞内 MIP-1β 表达）相比，LFA-1 构象变化的评估代表了细胞毒性淋巴细胞激活的敏感和快速参数，可促进活细胞的分离。潜在地，与细胞毒性淋巴细胞功能的其他读出相结合，该技术适用于各种临床情况的评估，包括原发性免疫缺陷综合征的诊断和评估供体淋巴细胞对肿瘤靶向的疗效。
