Taguchi T, McGhee J R, Coffman R L, Beagley K W, Eldridge J H, Takatsu K, Kiyono H
Department of Oral Biology, University of Alabama, Birmingham 35294.
J Immunol Methods. 1990 Mar 27;128(1):65-73. doi: 10.1016/0022-1759(90)90464-7.
Although several sensitive and specific assays have been developed to quantify murine cytokines, these assays do not allow individual cells to be correlated with the specific cytokines they produce. The purpose of this study was to develop a sensitive and reproducible method for the detection of individual T cells which secrete either interferon-gamma (IFN-gamma) or interleukin-5 (IL-5). We have used an adaptation of the enzyme-linked immunospot (ELISPOT) assay in which monoclonal antibodies to IFN-gamma (R4-6A2) and to IL-5 (TRFK-5) were used to coat 96-well plates with a nitrocellulose base. Mouse splenic T cells, either nonstimulated or activated with concanavalin A (ConA) or phytohemagglutinin (PHA), were cultured in individual wells. Following incubation, the cells were removed, and the bound cytokines probed with either biotinylated mAb anti-IFN-gamma (XMG 1.2) or anti-IL-5 (TRFK-4) followed by avidin-peroxidase. The spots which developed with 3-amino-9-ethylcarbazole were discrete and enumerated with a dissecting microscope. Although unstimulated splenic T cells contained low numbers of cytokine-specific spot-forming cells (SFC), 24-72 h activation with mitogen was required to induce significant numbers of cytokine producing cells. When mitogen-stimulated splenic CD4+ T cells were assessed, approximately equal numbers of IFN-gamma and IL-5 SFC were seen. Approximately 20-30% of all mitogen-activated splenic T cells produced at least one of these two cytokines. Pre-incubation of biotinylated anti-IFN-gamma with recombinant IFN-gamma (rIFN-gamma) or anti-IL-5 mAbs with rIL-5 completely inhibited cytokine-specific SFC. Further, use of nonrelevant antibodies did not result in spot formation, and treatment of mitogen-activated T cells with cycloheximide inhibited both IFN-gamma- and IL-5-specific SFC. A sensitive method has been developed which allows detection of individual T cells that produce either IFN-gamm or IL-5, and should be useful for detection of cytokine secretion at the single cell level.
尽管已经开发出几种灵敏且特异的检测方法来定量小鼠细胞因子，但这些检测方法无法将单个细胞与其产生的特定细胞因子相关联。本研究的目的是开发一种灵敏且可重复的方法，用于检测分泌干扰素-γ（IFN-γ）或白细胞介素-5（IL-5）的单个T细胞。我们采用了酶联免疫斑点（ELISPOT）检测法的一种改良方法，其中用抗IFN-γ（R4-6A2）和抗IL-5（TRFK-5）的单克隆抗体包被带有硝酸纤维素基质的96孔板。将未刺激的或用刀豆球蛋白A（ConA）或植物血凝素（PHA）激活的小鼠脾T细胞培养在各个孔中。孵育后，去除细胞，并用生物素化的抗IFN-γ单克隆抗体（XMG 1.2）或抗IL-5单克隆抗体（TRFK-4）探测结合的细胞因子，随后用抗生物素蛋白-过氧化物酶处理。用3-氨基-9-乙基咔唑显色的斑点清晰可辨，并用解剖显微镜计数。尽管未刺激的脾T细胞中细胞因子特异性斑点形成细胞（SFC）数量较少，但需要用丝裂原激活24 - 72小时才能诱导出大量产生细胞因子的细胞。当评估丝裂原刺激的脾CD4 + T细胞时，观察到IFN-γ和IL-5 SFC的数量大致相等。所有丝裂原激活的脾T细胞中约20 - 30%产生这两种细胞因子中的至少一种。用重组IFN-γ（rIFN-γ）预孵育生物素化的抗IFN-γ或用rIL-5预孵育抗IL-5单克隆抗体可完全抑制细胞因子特异性SFC。此外，使用无关抗体不会导致斑点形成，用放线菌酮处理丝裂原激活的T细胞可抑制IFN-γ和IL-5特异性SFC。已经开发出一种灵敏的方法，可用于检测产生IFN-γ或IL-5的单个T细胞，并且应该有助于在单细胞水平检测细胞因子分泌。
