Klein Christophe, Devi-Marulkar Priyanka, Dieu-Nosjean Marie-Caroline, Germain Claire
Center of Cellular Imaging and Cytometry, Institut National de la Santé et de la Recherche Médicale (INSERM) UMRS 1138, Cordeliers Research Center, Paris, France.
Sorbonne University, UMRS 1138, Cordeliers Research Center, Paris, France.
Methods Mol Biol. 2018;1845:47-69. doi: 10.1007/978-1-4939-8709-2_4.
Tertiary lymphoid structures (TLS) are considered as genuine markers of inflammation. Their presence within inflamed tissues or within the tumor microenvironment has been associated with the local development of an active immune response. While high densities of TLS are correlated with disease severity in autoimmune diseases or during graft rejection, it has been associated with longer patient survival in many cancer types. Their efficient visualization and quantification within human tissues may represent new tools for helping clinicians in adjusting their therapeutic strategy. Some immunohistochemistry (IHC) protocols are already used in the clinic to appreciate the level of immune infiltration in formalin-fixed, paraffin-embedded (FFPE) tissues. However, the use of two or more markers may sometimes be useful to better characterize this immune infiltrate, especially in the case of TLS. Besides the growing development of multiplex labeling approaches, imaging can also be used to overcome some technical difficulties encountered during the immunolabeling of tissues with several markers.This chapter describes IHC methods to visualize in a human tissue (tumoral or not) the presence of TLS. These methods are based on the immunostaining of four TLS-associated immune cell populations, namely follicular B cells, follicular dendritic cells (FDCs), mature dendritic cells (mDCs), and follicular helper T cells (T), together with non-T T cells. Methodologies for subsequent quantification of TLS density are also proposed, as well as a virtual multiplexing method based on image registration using the open-source software ImageJ (IJ), aiming at co-localizing several immune cell populations from different IHC stainings performed on serial tissue sections.
三级淋巴结构（TLS）被视为炎症的真正标志物。它们在炎症组织或肿瘤微环境中的存在与局部活跃免疫反应的发展相关。虽然TLS的高密度与自身免疫性疾病或移植排斥反应中的疾病严重程度相关，但在许多癌症类型中它与患者更长的生存期相关。在人体组织中对其进行有效的可视化和定量分析可能代表了帮助临床医生调整治疗策略的新工具。一些免疫组织化学（IHC）方案已在临床上用于评估福尔马林固定、石蜡包埋（FFPE）组织中的免疫浸润水平。然而，使用两种或更多种标志物有时可能有助于更好地表征这种免疫浸润，特别是在TLS的情况下。除了多重标记方法的不断发展，成像也可用于克服在用几种标志物对组织进行免疫标记过程中遇到的一些技术困难。本章描述了在人体组织（无论是否为肿瘤组织）中可视化TLS存在的IHC方法。这些方法基于对四种与TLS相关的免疫细胞群体进行免疫染色，即滤泡B细胞、滤泡树突状细胞（FDC）、成熟树突状细胞（mDC）和滤泡辅助性T细胞（T），以及非T细胞。还提出了后续定量TLS密度的方法，以及一种基于使用开源软件ImageJ（IJ）进行图像配准的虚拟多重标记方法，旨在将在连续组织切片上进行的不同IHC染色中的几种免疫细胞群体共定位。
