Detection of low-frequency antigen-specific IL-10-producing CD4+ T cells via ELISPOT in PBMC:: cognate vs. nonspecific production of the cytokine
Detection of low-frequency antigen-specific IL-10-producing CD4+ T cells via ELISPOT in PBMC:: cognate vs. nonspecific production of the cytokine
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DOI：
10.1016/s0022-1759(03)00240-0
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发表时间：
2003-08-01
影响因子：
2.2
通讯作者：
Schwander, SK
中科院分区：
文献类型：
--
作者：
Guerkov, REM;Targoni, OS;Schwander, SK
单细胞分辨率细胞因子ELISPOT测定越来越多地用于深入了解体内1型和2型效应T细胞群体的克隆大小。然而，允许监测调节性IL-10产生T细胞的ELISPOT测定迄今尚未建立。与在PBMC上进行的IFN-γ、IL-2、IL-4和IL-5测定不同，其中回忆抗原诱导的细胞因子点是T细胞衍生的，我们在此表明，在此类测定中IL-10主要是单核细胞衍生的。T细胞产生的IL-10斑点大小为80 × 10（3）mum（2），比单核细胞产生的斑点大7倍，而B细胞产生的斑点甚至更小。基于斑点大小设门和使用B细胞作为APC，我们建立了允许测量低频抗原特异性T细胞产生同源IL-10的测试条件。在结核病患者中检测到产生IL-10的PPD特异性CD 4（+）T细胞的频率与分泌IFN-γ的CD 4（+）T细胞的频率相当，但在未感染的健康对照个体中没有检测到。相比之下，在健康个体中，不能检测到对一组回忆抗原特异性的IL-10分泌型CD 4（+）T细胞的频率>1/100，000，这些健康个体的CD 4（+）细胞对这些抗原产生1型或2型细胞因子，频率范围为1：100，000 -1：1000。因此，产生IL-10的T细胞的诱导似乎受到比Th 1/Th 2细胞更严格的控制，显然局限于慢性免疫刺激的状态。对产生IL-10的T细胞进行低频免疫监测将为调节性T细胞在健康和疾病中的作用提供新的见解。(C)2003 Elsevier B. V.保留所有权利。
Single-cell resolution cytokine ELISPOT assays are increasingly used to gain insights into clonal sizes of type 1 and type 2 effector T cell populations in vivo. However, ELISPOT assays permitting monitoring of regulatory IL-10-producing T cells have so far not been established. Unlike IFN-gamma, IL-2, IL-4, and IL-5 assays performed on PBMC in which the recall antigen-induced cytokine spots are T cell-derived, we show here that in such assays IL-10 is primarily monocyte-derived. T cell-derived IL-10 spots were 80 x 10(3) mum(2) in size, seven times larger than spots produced by monocytes, and B cells produced even smaller spots. Based on spot size gating and the use of B cells as APC, we have established test conditions that permit measurement of cognate IL-10 production by low-frequency antigen-specific T cells. IL-10-producing PPD-specific CD4(+) T cells were detected in frequencies comparable to IFN-gamma-secreting CD4(+) T cells in tuberculosis patients, but not in uninfected healthy control individuals. In contrast, IL-10-secreting CD4(+) T cells specific for a panel of recall antigens could not be detected in frequencies >1/100,000 in healthy individuals whose CD4(+) cells responded to these antigens with type 1 or type 2 cytokine production in the 1:100,000-1:1000 frequency range. Therefore, the induction of IL-10-producing T cells seems to be under tighter control than that of Th1/Th2 cells, apparently confined to states of chronic immune stimulation. Access to low-frequency immune monitoring of IL-10-producing T cells will provide new insights into the role of regulatory T cells in health and disease. (C) 2003 Elsevier B.V. All rights reserved.