Abstract
CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.
Highlights
To understand the tissue specific mechanisms that govern successful clinical outcomes in infection and vaccination, for example pathogen clearance or the production of neutralizing antibodies, it is important to understand the context in which these outcomes arise
Within the B-cell lineage, Bcl-6 expression is mainly confined to germinal center (GC) B cells where it acts to promote the selection of B-cells by silencing the antiapoptotic molecule Bcl-2, enhancing the ability of GC B cells to tolerate DNA damage, preserve B cell identity and fine-tune BCR mediated responsiveness [19] Bcl-6 is expressed in CD4+ Tcells and has been shown to direct Tfh lineage commitment [32]
We used the marker CD57, an epitope expressed in a subset of CD4 T cells that are positive for CD69 and CD45RO and detected in approximately 15-25% of tonsil CXCR5+ CD4 T cells [33], to track two distinct GC-Tfh populations (CD57+ and CD57-) that have been shown to possess divergent immunophenotypes [24]
Summary
To understand the tissue specific mechanisms that govern successful clinical outcomes in infection and vaccination, for example pathogen clearance or the production of neutralizing antibodies, it is important to understand the context in which these outcomes arise. The study of immune cell dynamics in lymphoid tissues has been performed using single-cell methodologies such as flow cytometry and more recently mass cytometry (i.e. CyTOF) and single-cell or bulk RNAseq after dissociating the cells from the tissue. Such methodologies have greatly enriched our understanding of circulating and tissueresident immunophenotypes, their transcriptional programs and population kinetics at different life stages or in pathology [8,9,10,11]. Methodologies employing cell suspensions do not interrogate by design the microanatomical segregation or positioning of immune subsets relative to each other despite the importance of these two parameters for mechanism and function [12]
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