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

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.

Human T follicular helper clones seed the germinal center-resident regulatory pool

“…Although the follicular dendritic cell lattice attracts Tfh and Tfr cells by maintaining a CXCL13 gradient ( 31,32 ), not all CXCR5 expressing T helper cells are located within GCs. Prior analysis of human lymph nodes indicate most human Tfr cells accumulate within follicles but clearly outside GC borders ( 20,33 ). To identify the precise locations of Tfr cells in human tonsillar tissue we employed CODEX to sequentially stain and image a DAPI-stained donor tonsil with a panel of 23 oligo-conjugated antibodies.…”

Imaging dendritic cell functions*

“…114 This method also allowed insight into the DC positioning within human tissue and their interactions with T and B cells. 81,115,116 The need for staining with several antibodies to label a particular DC subpopulation was partially alleviated by the generation of transgenic mice that express fluorescent markers specifically in DCs. Several such mouse strains are currently available, each with its strengths and weaknesses (Table 2).…”

“…130 A similar approach was recently applied to describe the distribution of cDCs (and pDCs) in human tonsils. 116 Another visualization method that can match flow cytometry in the number of simultaneously detected markers is mass cytometry imaging (MCI), a method that uses either imaging mass cytometry or multiplexed ion beam imaging. 5 The basic principle of MCI is that antibodies are conjugated to stable isotopes instead of fluorophores.…”

“…More recently, confocal microscopy visualized interactions of interfollicular DCs with fibroblastic reticular cells important for their homeostatic proliferation and survival 114 . This method also allowed insight into the DC positioning within human tissue and their interactions with T and B cells 81,115,116 …”

“…All those DC subpopulations are predominantly associated with lymph node blood vessels but locate to different parts of vascular trees so that there is little local mixing of cells 130 . A similar approach was recently applied to describe the distribution of cDCs (and pDCs) in human tonsils 116 …”