Methods for analysis of tertiary lymphoid structures and immune activity by multiplex immunofluorescence histology

Tertiary lymphoid structures (TLS) are ectopic lymphoid aggregates that are correlated with improved patient outcomes in several solid cancers, including melanoma. Multiplex immunofluorescent histology (mIFH) has been used in numerous studies to identify and characterize TLS. However, detailed studies evaluating immune cell subsets and markers of immune activity at TLS sites have been limited. Here, we introduce multiplex immunofluorescence histology methods to identify TLS, their associated immune cell components, and markers of immune activity. We outline two mIFH panels for evaluating and quantifying TLS, and markers of immune activity, offering methodologies that can be used to gain a more nuanced understanding of the role and immunological activity of TLS in cancer prognosis and therapy.