La Muraglia G M, O'Neil M J, Madariaga M L, Michel S G, Mordecai K S, Allan J S, Madsen J C, Hanekamp I M, Preffer F I
Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.
Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
J Immunol Methods. 2015 Dec;427:85-93. doi: 10.1016/j.jim.2015.10.005. Epub 2015 Oct 26.
In this study, we established a novel isotope-free approach for the detection of cell-mediated lympholysis (CML) in MHC defined peripheral blood mononuclear cells (PBMCs) using multiparameter flow and imaging cytometry. CML is an established in vitro assay to detect the presence of cytotoxic effector T-lymphocytes precursors (CTLp). Current methods employed in the identification of CTLp in the context of transplantation are based upon the quantification of chromium ((51)Cr) released from target cells. In order to adapt the assay to flow cytometry, primary porcine PBMC targets were labeled with eFluor670 and incubated with major histocompatibility complex (MHC) mismatched effector cytotoxic lymphocytes (CTLs). With this method, we were able to detect target-specific lysis that was comparable to that observed with the (51)Cr-based assay. In addition, the use of quantitative cell imaging demonstrates the presence of accessory cells involved in the cytotoxic pathway. This innovative technique improves upon the standard (51)Cr release assay by eliminating the need for radioisotopes and provides enhanced characterization of the interactions between effector and target cells. This technique has wide applicability to numerous experimental and clinical models involved with effector-cell interactions.
在本研究中，我们建立了一种全新的无同位素方法，用于使用多参数流式细胞术和成像细胞术检测MHC定义的外周血单核细胞（PBMC）中的细胞介导的淋巴细胞溶解（CML）。CML是一种既定的体外检测方法，用于检测细胞毒性效应T淋巴细胞前体（CTLp）的存在。目前在移植背景下鉴定CTLp所采用的方法基于对从靶细胞释放的铬（51Cr）的定量。为了使该检测方法适用于流式细胞术，将原代猪PBMC靶标用eFluor670标记，并与主要组织相容性复合体（MHC）不匹配的效应细胞毒性淋巴细胞（CTL）孵育。通过这种方法，我们能够检测到与基于51Cr的检测方法所观察到的相当的靶标特异性裂解。此外，定量细胞成像的使用证明了参与细胞毒性途径的辅助细胞的存在。这种创新技术通过消除对放射性同位素的需求改进了标准的51Cr释放检测方法，并提供了效应细胞与靶细胞之间相互作用的增强表征。该技术广泛适用于涉及效应细胞相互作用的众多实验和临床模型。
