Ott Patrick A, Berner Beate R, Herzog Bernhard A, Guerkov Robert, Yonkers Nicole L, Durinovic-Bello Ivana, Tary-Lehmann Magdalena, Lehmann Paul V, Anthony Donald D
Department of Pathology, School of Medicine, Case Western Reserve University, BRB 930, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
J Immunol Methods. 2004 Feb 15;285(2):223-35. doi: 10.1016/j.jim.2003.12.007.
The frequencies of antigen-specific memory cells are often low in chronic disease states related to infection and autoimmunity, making detection of such populations difficult, even with high sensitivity assays such as the cytokine enzyme-linked immunospot (ELISPOT). The spectrum and function of antigen presenting cells (APC) in the peripheral compartment can differ considerably from the inflamed target organ. In order to approximate the costimulatory environment of the target organ, we measured T cell responses with and without the addition of agonistic anti-CD28 antibody in the ELISPOT assay. CD4 and CD8 IFN-gamma responses to viral (hepatitis C) and autoimmune antigens (islet cell) were tested in 10 hepatitis C and 8 type 1 diabetic as well as healthy control subjects. IFN-gamma responses to tetanus toxoid, mumps and cytomegalovirus (CMV) protein antigen, as well as Epstein-Barr virus and CMV peptides were also measured in healthy control subjects. We found higher frequencies of T cells reactive to protein and peptide antigens when anti-CD28 antibody was present, often detecting responses only in the presence of anti-CD28 antibody. These results demonstrate that anti-CD28 antibody signal enhanced ELISPOT assays can facilitate the identification of low precursor frequency T cells in chronic infectious and autoimmune disease states where suboptimal costimulatory environment may exist in the periphery. The use of such costimulation may also enable a more quantitative assessment of circulating memory effector T cell frequency.
在与感染和自身免疫相关的慢性疾病状态下，抗原特异性记忆细胞的频率通常较低，这使得即使使用细胞因子酶联免疫斑点法（ELISPOT）等高灵敏度检测方法来检测这类细胞群体也很困难。外周区室中抗原呈递细胞（APC）的谱系和功能可能与炎症靶器官有很大差异。为了模拟靶器官的共刺激环境，我们在ELISPOT检测中测量了添加和不添加激动性抗CD28抗体时的T细胞反应。在10名丙型肝炎患者、8名1型糖尿病患者以及健康对照者中检测了CD4和CD8 IFN-γ对病毒（丙型肝炎）和自身免疫抗原（胰岛细胞）的反应。还在健康对照者中测量了IFN-γ对破伤风类毒素、腮腺炎和巨细胞病毒（CMV）蛋白抗原以及爱泼斯坦-巴尔病毒和CMV肽的反应。我们发现当存在抗CD28抗体时，对蛋白质和肽抗原反应的T细胞频率更高，通常仅在存在抗CD28抗体时才能检测到反应。这些结果表明，抗CD28抗体信号增强的ELISPOT检测可以促进在慢性感染和自身免疫疾病状态下识别低前体频率的T细胞，在这些疾病状态下外周可能存在共刺激不足的环境。使用这种共刺激还可以更定量地评估循环记忆效应T细胞的频率。
