Wittenborn Thomas R, Hagert Cecilia, Degn Søren E
Department of Biomedicine, Aarhus University.
Department of Biomedicine, Aarhus University;
J Vis Exp. 2019 Apr 11(146). doi: 10.3791/59397.
Autoimmune diseases present a significant health burden. Fundamental questions regarding the development and progression of autoimmune disease remain unanswered. One requirement for advancements in our understanding of the underlying disease mechanisms and cellular dynamics is the precise coupling of the microanatomical location of cell subsets with downstream molecular or functional analyses; a goal that has traditionally been difficult to achieve. The development of stable photoactivatable biological fluorophores and their integration into reporter strains has recently enabled precise microanatomical labeling and tracking of cellular subsets in murine models. Here, we describe how the ability to analyze autoreactive lymphocytes from single germinal centers may help to provide novel insights into autoimmunity, using the combination of a novel chimeric model of autoimmunity with a photoactivatable reporter as an example. We demonstrate a procedure for generating mixed chimeras with spontaneous autoreactive germinal centers populated by lymphocytes carrying a photoactivatable green fluorescent protein reporter. Using in vivo labeling strategies, single germinal centers can be visualized in explanted lymphoid tissues and their cellular constituents photoactivated by two-photon microscopy. Photoactivated lymphocytes from single germinal centers can then be analyzed or sorted flow cytometrically, as single cells or in bulk, and may be subjected to additional downstream molecular and functional analyses. This approach may directly be applied to provide renewed insights in the field of autoimmunity, but the procedure for generating bone marrow chimeras and the photoactivation procedure may additionally find broad application in studies of infectious diseases and tumor metastases.
自身免疫性疾病带来了沉重的健康负担。关于自身免疫性疾病的发生和发展的基本问题仍未得到解答。要在我们对潜在疾病机制和细胞动态的理解上取得进展，一个要求是将细胞亚群的微观解剖位置与下游分子或功能分析精确耦合；这一目标传统上一直难以实现。稳定的光激活生物荧光团的开发及其整合到报告菌株中，最近使得在小鼠模型中能够对细胞亚群进行精确的微观解剖标记和追踪。在这里，我们以一种新型的自身免疫嵌合模型与光激活报告基因相结合为例，描述了分析来自单个生发中心的自身反应性淋巴细胞的能力如何有助于为自身免疫提供新的见解。我们展示了一种生成混合嵌合体的方法，其中自发的自身反应性生发中心由携带光激活绿色荧光蛋白报告基因的淋巴细胞组成。使用体内标记策略，可以在外植淋巴组织中可视化单个生发中心，并通过双光子显微镜对其细胞成分进行光激活。然后，可以通过流式细胞术对来自单个生发中心的光激活淋巴细胞进行分析或分选，作为单个细胞或批量进行，并且可以进行额外的下游分子和功能分析。这种方法可以直接应用于为自身免疫领域提供新的见解，但生成骨髓嵌合体的方法和光激活方法可能还会在传染病和肿瘤转移研究中得到广泛应用。
