The Intricate Network of Immune Cells Revealed by Multiplex Tissue Microscopy
Introduction

The Intricate Network of Immune Cells Revealed by Multiplex Tissue Microscopy

Researchers investigate the physical locations and relationships of immune cells in healthy and inflamed tissues.

In our daily lives, just by eating or breathing, we are constantly exposing our tissues to microbes and particulate matter that can elicit low level inflammatory responses. The impact of this inflammation over time is not well understood.

Mononuclear phagocyte cells (MPs) are key regulators in the inflammatory process. They are present in every tissue and are very diverse with multiple origins. One of the goals in Prof. Andreas Schlitzer’s lab at the Life and Medical Sciences Institute (LIMES) of the University of Bonn, Germany, is to understand the distribution, function and origin of MPs in healthy tissues and during acute or chronic inflammation. His lab is using a multiplex tissue microscopy system with the Akoya Biosciences solution and the ZEISS Axio Observer microscope to characterize the physical locations and relationships of immune cells in these tissues.

Dr. David Bejarano, Life and Medical Sciences Institutes (LIMES), University of Bonn, Germany

Although there were several options in the market, we selected the ZEISS Axio Observer microscope to integrate with the Akoya Biosciences solution as it offered the best sensitivity and an optimal speed of acquisition. We can acquire the full left murine lung stained with 40 antibodies in less than 32 hours.

Dr. David Bejarano

Postdoctoral Researcher, Schlitzer Lab at the Life and Medical Sciences Institute (LIMES), University of Bonn, Germany

The Complexity of the Immune System in the Context of the Tissue

Visualized with Multiplex Microscopy using ZEISS Axio Observer

1 µm section of a formalin fixed paraffin-embedded human lymph node. Section was stained with a 14 CODEX antibody panel. Image shows some essential markers to distinguish mononuclear phagocytes (CD68 in red, HLA-DR in green, CD11c in magenta). To delineate major anatomical structures, such as blood vessels, CD31 is shown in cyan. Proliferation marker Ki67 (yellow) delineates germinal centers. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.
1 µm section of a formalin fixed paraffin-embedded human lymph node. Section was stained with a 14 CODEX antibody panel. Image shows some essential markers to distinguish mononuclear phagocytes (CD68 in red, HLA-DR in green, CD11c in magenta). To delineate major anatomical structures, such as blood vessels, CD31 is shown in cyan. Proliferation marker Ki67 (yellow) delineates germinal centers. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

1 µm section of a formalin fixed paraffin-embedded human lymph node. Section was stained with a 14 CODEX antibody panel. Image shows some essential markers to distinguish mononuclear phagocytes (CD68 in red, HLA-DR in green, CD11c in magenta). To delineate major anatomical structures, such as blood vessels, CD31 is shown in cyan. Proliferation marker Ki67 (yellow) delineates germinal centers. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

1 µm section of a formalin fixed paraffin-embedded human lymph node. Section was stained with a 14 CODEX antibody panel. Image shows some essential markers to distinguish mononuclear phagocytes (CD68 in red, HLA-DR in green, CD11c in magenta). To delineate major anatomical structures, such as blood vessels, CD31 is shown in cyan. Proliferation marker Ki67 (yellow) delineates germinal centers. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

Multiplex Imaging Brings New Understanding

Previously, the lab's findings were mainly based on high dimensional flow cytometry and single-cell transcriptomics. Although both approaches allow a deep understanding of immune responses at a single cell level, they lack spatial resolution and context.

The incorporation of a highly multiplexed imaging technique with ZEISS Axio Observer allows visualizing at a single-cell level more than 40 cell markers in record time without compromising tissue morphology or image quality has brought new understanding to their research. They can now visualize major tissue morphological changes induced by different conditions, localize cells of interest, characterize the surrounding of these cells, look at their interaction partners and determine how the local environment shapes the function of a cell.

Exploring the Spatial Distribution and Diversity of Myeloid Cells

Visualized with Multiplex Microscopy using ZEISS Axio Observer

5 µm section of a formalin fixed paraffin-embedded mouse small intestine swiss roll. Section was stained with a 14 CODEX antibody panel designed to visualize intestinal stem and enteroendocrine cells (Lysozyme in red, villin in green, MHCII in gray, vimentin in cyan, Ki67 in magenta, and Olfm4 in yellow). Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.
5 µm section of a formalin fixed paraffin-embedded mouse small intestine swiss roll. Section was stained with a 14 CODEX antibody panel designed to visualize intestinal stem and enteroendocrine cells (Lysozyme in red, villin in green, MHCII in gray, vimentin in cyan, Ki67 in magenta, and Olfm4 in yellow). Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

5 µm section of a formalin fixed paraffin-embedded mouse small intestine swiss roll. Section was stained with a 14 CODEX antibody panel designed to visualize intestinal stem and enteroendocrine cells (Lysozyme in red, villin in green, MHCII in gray, vimentin in cyan, Ki67 in magenta, and Olfm4 in yellow). Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

5 µm section of a formalin fixed paraffin-embedded mouse small intestine swiss roll. Section was stained with a 14 CODEX antibody panel designed to visualize intestinal stem and enteroendocrine cells (Lysozyme in red, villin in green, MHCII in gray, vimentin in cyan, Ki67 in magenta, and Olfm4 in yellow). Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

Studying Macrophages and Dendritic Cells in the Small Intestine

Dr. Schlitzer's team has now incorporated multiplex tissue microscopy to explore the spatial distribution and diversity of myeloid cells. In one example, they have visualized macrophages and dendritic cells in the longitudinal axis of the murine intestine and found that there is a gradient in their distribution. They are currently focusing on the characterization of macrophage populations that they observe in the different layers of the intestinal wall and would like to use spatial-omics in fate mapping mouse models to track their origin.

3 µm frozen section of the left mouse lung 3 days after intranasal stimulation with LPS. Section was stained with a 37 CODEX antibody panel. Image shows the canonical markers of mononuclear phagocytes (CD11c in red, MHCII in green, F4/80 in cyan, CD11b in magenta). To delineate major anatomical structures, such as lung airways and blood vessels, EpCAM (yellow) and SMA (gray) are displayed. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.
3 µm frozen section of the left mouse lung 3 days after intranasal stimulation with LPS. Section was stained with a 37 CODEX antibody panel. Image shows the canonical markers of mononuclear phagocytes (CD11c in red, MHCII in green, F4/80 in cyan, CD11b in magenta). To delineate major anatomical structures, such as lung airways and blood vessels, EpCAM (yellow) and SMA (gray) are displayed. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

3 µm frozen section of the left mouse lung 3 days after intranasal stimulation with LPS. Section was stained with a 37 CODEX antibody panel. Image shows the canonical markers of mononuclear phagocytes (CD11c in red, MHCII in green, F4/80 in cyan, CD11b in magenta). To delineate major anatomical structures, such as lung airways and blood vessels, EpCAM (yellow) and SMA (gray) are displayed. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

3 µm frozen section of the left mouse lung 3 days after intranasal stimulation with LPS. Section was stained with a 37 CODEX antibody panel. Image shows the canonical markers of mononuclear phagocytes (CD11c in red, MHCII in green, F4/80 in cyan, CD11b in magenta). To delineate major anatomical structures, such as lung airways and blood vessels, EpCAM (yellow) and SMA (gray) are displayed. Blue signals correspond to DAPI nuclear staining. Acquisition was done with a ZEISS Axio Observer microscope and processing with the Akoya CODEX Processor.

Spatial Analysis with Multiplex Microscopy Brings New Insights

In another project, they are studying the effect of low-grade inflammation in the lung. After stimulation, there is a rapid and pronounced influx of a subpopulation of alveolar macrophages that have a pro-inflammatory profile that is kept for several weeks and enhances the response to subsequent challenges. Interestingly, when pulmonary fibrosis is induced in mice that had been stimulated, the disease progression is slower and survival rates improve. Besides doing a detailed characterization of these cells and the genes controlling this phenotype, they used a 40-plex CODEX antibody panel to visualize the lung architecture and determine if major structural changes occur.

In agreement with their flow cytometry analyses, the images showed an increase of monocytes and monocyte-derived alveolar macrophages. They also revealed the absence of major structural changes. Nonetheless, in the aerial spaces, such as bronchioles, infiltrates of cells were observed. These infiltrates were composed predominantly of the macrophage/monocyte populations they had identified, but also other myeloid cells and even lymphoid cells were present.

 

We would like to exploit the high dimensionality of multiplex tissue microscopy to study the diversity of myeloid cells in multiple tissues and identify non-described tissue-specific populations. There is so much we still don’t understand and we see great potential in leveraging such findings to eventually lead to new and better targeted treatments for multiple health conditions.

Dr. David Bejarano

Postdoctoral Researcher, Schlitzer Lab at the Life and Medical Sciences Institute (LIMES), University of Bonn, Germany


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