In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization

• DOI: 10.3389/fimmu.2021.683396
• Publication Date: 2021-06-09T14:41:12Z
• Authors: Eirini Moysi; Perla M. Del Rio ...; Fernanda Torres-Ruiz; Gustavo Reyes-Terán; Richard A. Koup; Constantinos Petr...
• Keywords: Follicular dendritic cells; Immunofluorescence

CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper cells, particularly those residing in germinal centers known as follicular (Tfh), provide critical help to B-cells promote their survival, isotype switching selection high affinity memory B-cells. On the other hand, important role Tfh for maintenance HIV reservoir is well documented. Thus, interrogating better understanding tissue specific micro-environment subsets that contribute optimal cell differentiation function designing successful prevention cure strategies. Here, we describe development optimization eight multispectral confocal microscopy immunofluorescence panels designed depth characterization immune-profiling relevant formalin-fixed paraffin-embedded human samples. We a comprehensive library antibodies use CD4+ T-cells -including regulatory T-cells- CD8 T-cells, B-cells, macrophages dendritic discuss how resulting datasets can be quantitatively dissected using HistoCytometry pipeline collect information about relative frequencies spatial distributions. Cells harboring actively transcribed virus analyzed an in-situ hybridization assay mRNA positive combination with additional protein markers (multispectral RNAscope). The application this methodology tissues offers means interrogate multiple targets simultaneously at increased resolution reproducible manner guide T-cell studies