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Allergen-specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression

机译：通过CD154和细胞内细胞因子表达监测对免疫疗法的过敏原特异性T细胞反应

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Background A sensitive measurement of low numbers of intracellular cytokine-expressing antigen-specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD 154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy.Objective To evaluate the applicability of this method for measuring changes in cytokine production by allergen-specific T cells in a clinical trial setting.Methods Ex vivo ragweed-specific CD 154 and intracellular cytokine expression were evaluated using a subset of subjects in an environmental chamber study of allergic rhinitis immunotherapy. PBMC were collected and cryopreserved from Amb a 1-immunostimulatory oligodeoxynucleotide conjugate (AlC)-treated (w=17) and placebo-treated (n=15) ragweed-allergic subjects both after pre- and post-treatment ragweed exposures. In vitro allergen-stimulated CD3~+CD4~+CD154~+ Tcell intracellular IL-4, IL-5, IL-13, andlFN-gamma expression were evaluated by flow cytometry.Results Compared with the T helper type 2 (Th2) cytokine expression measured after pre-treatment ragweed exposures, placebo-treated subjects demonstrated a significantly elevated ragweed- and Amb a 1-specific T cell IL-4 and IL-13 co-expression (P = 0.005 and P = 0.022, respectively) and a significantly elevated ragweed-specific IL-5 expression (p<0.001) following post-treatment ragweed exposures. In contrast, AIC-treated subjects demonstrated no increases in allergen-specific Th2 cytokine expression following post-treatment ragweed exposures. IFN-gamma expression remained low and un-changed in both groups. Subject reported total nasal symptom scores demonstrated modest but significant correlations with Amb a 1- and ragweed-stimulated intracellular Th2 cytokine responses. Conclusion Combined CD 154 and intracellular cytokine staining in PBMC can be used to sensitively monitor changes in antigen-specific T cell subset frequencies in clinical studies. Antigen-specific cytokine expression moderately correlated with the reported levels of allergic symptoms. Trial Registration NCT00537355
机译：背景技术使用CD 154作为最近激活的T细胞的标志物，可以灵敏地测量来自外周血单核细胞（PBMC）的少量细胞内表达细胞因子的抗原特异性T细胞。该技术可能具有监测外周血T细胞对免疫疗法反应的潜力。目的评估该方法在临床试验环境中测量过敏原特异性T细胞产生的细胞因子变化的适用性。方法离体豚草特异性CD 154和在变应性鼻炎免疫疗法的环境试验室研究中，使用一部分受试者评估了细胞内细胞因子的表达。从豚草暴露前和治疗后的1-免疫刺激性寡脱氧核苷酸缀合物（AlC）处理（w = 17）和安慰剂处理（n = 15）豚草过敏受试者中收集PBMC并冷冻保存。流式细胞术检测体外变应原刺激的CD3〜+ CD4〜+ CD154〜+ T细胞内细胞内IL-4，IL-5，IL-13和1FN-γ的表达。结果与2型T辅助细胞（Th2）相比豚草暴露于治疗前的表达水平，安慰剂治疗的受试者表现出豚草和Amb 1特异性T细胞IL-4和IL-13的共表达显着升高（分别为P = 0.005和P = 0.022）和处理豚草暴露后豚草特异性IL-5表达显着升高（p <0.001）。相反，经过豚草暴露后，经AIC治疗的受试者表现出的过敏原特异性Th2细胞因子表达没有增加。两组中的IFN-γ表达均保持较低且不变。受试者报告的总鼻部症状评分显示与Amb a 1和豚草刺激的细胞内Th2细胞因子反应适度但显着相关。结论PBMC中CD 154和细胞内细胞因子结合染色可用于临床研究中灵敏地监测抗原特异性T细胞亚群频率的变化。抗原特异性细胞因子的表达与过敏症状的报道水平适度相关。试用注册NCT00537355

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