Sequential chromogenic immunohistochemistry: spatial analysis of lymph nodes identifies contact interactions between plasmacytoid dendritic cells and plasmablasts

Recent clinical observations highlight the importance of the spatial organization of immune cells into lymphoid structures for the success of cancer immunotherapy and patient survival. Sequential chromogenic immunohistochemistry (scIHC) supports the analysis of multiple biomarkers on a single tissue section thus providing unique information about relative location of cell types and assessment of disease states. Unfortunately, widespread implementation of scIHC is limited by lack of a standardized, rigorous guide to the development of customized biomarker panels and by the need for user-friendly analysis pipelines able to streamline the extraction of meaningful data. Here, we examine major steps from classical IHC protocols and highlight the impact they have on the scIHC procedure. We report practical examples and illustrations of the most common complications that can arise during the setup of a new biomarker panel and how to avoid them. We described in detail how to prevent and detect cross- reactivity between secondary reagents and carry over between detection antibodies. We developed a novel analysis pipeline based on non-rigid tissue deformation correction, Cellpose-inspired automated cell segmentation and computational network masking of low-quality data. The resulting biomarker panel and pipeline was used to study regional lymph nodes from head and neck cancer patients. We identified contact interactions between plasmablasts and plasmacytoid dendritic cells in vivo. Given that TLR receptors, which are highly expressed in plasmacytoid dendritic cells play a key role in vaccine efficacy, the significance of this cell-cell interaction decisively warrants further studies. In conclusion, this work streamlines the development of novel biomarker panels for scIHC, which will ultimately improve our understanding of immune responses in cancer.