Infectious Bacterial Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States.
Food Safety and Enteric Pathogens Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, United States.
Front Immunol. 2020 Sep 2;11:1944. doi: 10.3389/fimmu.2020.01944. eCollection 2020.
Bovine brucellosis, cause by infection with , causes reproductive failure in cattle, has a major economic impact to producers, and as a zoonoses, it is a disease of public health concern. Characterization of the protective immune response against infection is important to our understanding of disease pathogenesis and for the development of diagnostic assays and vaccines. Most of the knowledge regarding protection against comes from studies in the murine model, but less is known about the immune responses in cattle. Assessment of antigen-specific T cell frequency and functional phenotype are critical to understand the immune status of the host, characterize mechanisms of protective immunity and immunopathology, and to predict immune protection. The frequency of circulating T cells specific for a particular pathogen is often very low, making analysis of such responses difficult. Our goal was to develop a flow-cytometry based approach to better track -specific T cell responses. Using peripheral blood mononuclear cells (PMBC) from strain RB51-vaccinated cattle, we optimized an stimulation protocol based on a combination of antigen and pan-T cell stimulation. We then assessed RB51-specific T cell responses by concurrently measuring proliferation and cytokine production using flow-cytometry. This methodology enhances the detection of peripheral, -specific responses in cattle following RB51 vaccination. This protocol is versatile in that it can be modified to fit other stimulation systems and additional functional or phenotypic parameters can be added for flow cytometric detection and characterization of antigen-specific T cells.
牛布鲁氏菌病由 感染引起，可导致牛繁殖失败，对生产者造成重大经济影响，而且作为一种人畜共患病，它是一个公共卫生关注的疾病。对 感染的保护性免疫反应的特征对于我们了解疾病发病机制以及开发诊断检测和疫苗非常重要。大多数关于针对 感染的保护的知识来自于鼠模型的研究，但对于牛的免疫反应知之甚少。评估抗原特异性 T 细胞频率和功能表型对于了解宿主的免疫状态、表征保护性免疫和免疫病理学的机制以及预测免疫保护至关重要。针对特定病原体的循环 T 细胞的频率通常非常低，使得对这些反应的分析变得困难。我们的目标是开发一种基于流式细胞术的方法来更好地跟踪 -特异性 T 细胞反应。使用 RB51 疫苗接种的牛的外周血单核细胞 (PMBC)，我们基于抗原和全 T 细胞刺激的组合优化了 刺激方案。然后，我们通过同时使用流式细胞术测量增殖和细胞因子产生来评估 RB51 特异性 T 细胞反应。该方法增强了 RB51 疫苗接种后牛外周血中针对 RB51 的特异性反应的检测。该方案具有通用性，可以进行修改以适应其他 刺激系统，并添加其他功能或表型参数，用于流式细胞术检测和表征抗原特异性 T 细胞。
