Guerkov Robert E M, Targoni Oleg S, Kreher Christian R, Boehm Bernhard O, Herrera Maria T, Tary-Lehmann Magdalena, Lehmann Paul V, Schwander Stephan K
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-4943, USA.
J Immunol Methods. 2003 Aug;279(1-2):111-21. doi: 10.1016/s0022-1759(03)00240-0.
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) microm(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.
单细胞分辨率细胞因子ELISPOT检测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，迄今为止尚未建立能够监测产生调节性白细胞介素-10（IL-10）的T细胞的ELISPOT检测方法。与在PBMC上进行的干扰素-γ（IFN-γ）、白细胞介素-2（IL-2）、白细胞介素-4（IL-4）和白细胞介素-5检测不同，在这些检测中回忆抗原诱导的细胞因子斑点是T细胞来源的，我们在此表明，在这种检测中IL-10主要来源于单核细胞。T细胞来源的IL-10斑点大小为80×10³微米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。基于斑点大小门控以及将B细胞用作抗原呈递细胞（APC），我们建立了允许测量低频抗原特异性T细胞产生的同源IL-10的检测条件。在结核病患者中，检测到产生IL-10的结核菌素纯蛋白衍生物（PPD）特异性CD4⁺T细胞的频率与分泌IFN-γ的CD4⁺T细胞相当，但在未感染的健康对照个体中未检测到。相比之下，在CD4⁺细胞以1:100,000 - 1:1000频率范围产生1型或2型细胞因子来响应这些抗原的健康个体中，未能检测到频率>1/100,000的针对一组回忆抗原的分泌IL-10的CD4⁺T细胞。因此，产生IL-10的T细胞的诱导似乎比Th1/Th2细胞受到更严格的控制，显然局限于慢性免疫刺激状态。能够对产生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。
