Advancement of Techniques for Precise Visualization and Quantification of Tertiary Lymphoid Structure-Associated Immune Cells in Tissue Samples

Tertiary Lymphoid Structures (TLS) are considered as genuine markers of inflammation. Their presence within inflamed tissues or 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 and more recently with positive responses to anti-PD-1 immunotherapy. Their efficient visualization and quantification within human tissues may represent new tools for helping clinicians in adjusting their therapeutic strategy. In clinical settings, the use of single-marker immunohistochemistry (IHC) protocols prevails in immune cell infiltration in formalin-fixed, paraffin-embedded (FFPE) tissues. In contrast, the development of automated multiplex immunofluorescence markings, i.e., 40-plex, requires very costly investments in equipment and analysis stations. Yet, employing two or more markers can enhance the characterization of immune infiltrates, particularly in the context 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 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_FH), together with non-T_FH 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.