Dr. Rainer Glauben and Adrian Huck, recipients of one of the Scipio bioscience grant prizes, have spent the past few years trying to elucidate the role of an enigmatic, dense group of cells located in the wall of the small intestine: Peyer’s Patches. Now, with the Asteria scRNA-seq sample preparation technology and sequencing costs covered by the grant, they can dig for information further than ever before.
Peyer’s Patches are lymphoid follicles suspected to play an important role in downplaying our food being treated as foreign matter by our body (such as would-be pathogens) and activating our immune system every time we eat. For patients suffering from inflammatory bowel diseases (IBD) such as Crohn’s disease, the immune cells in those patches might be dysfunctional.
For Dr. Glauben, the project started years ago in partnership with Prof. Ulrich Steinhoff (Philipps-Universität Marburg, Germany) investigating nutritional-antigen-activated T-cells in the gut, particularly in the Peyer’s Patches. Their collaboration showed that mice with an antigen-free, protein-free diet reacted very differently to stimuli than control mice. Their study highlighted how specific T-cells in the Peyer’s patches are activated by nutritional antigen, triggering apoptosis – programmed cell death – leading to the expression of IL-10, thus reducing inflammation downstream. Using human samples, they showed that there were fewer of those specific T-cells in patients suffering from Crohn’s disease and fewer apoptosis as well, which could explain the inflammatory symptoms suffered by IBD patients.
Now they plan to investigate further into those Peyer’s Patches to figure out the exact role of those T-cell lymphocytes.
Single-cell RNA sequencing to provide cellular information other techniques couldn’t
Due to their size and their collagen-rich micro-environment, human Peyer’s Patches are particularly tricky samples to harvest: they are hard to see and hard to extract from gut tissues. With a limited amount of biopsy samples (that would eventually not always include the desired patches), the range of relevant analytical assays available for the team remained narrow.
“It made it really difficult to do proper flow cytometry as we usually do,” explained Dr. Glauben. “We came to the idea of doing histological assays, but then the numbers of colors and markers available were limited. Then we thought imaging mass cytometry would be perfect for our project.”
Imaging mass cytometry is a relatively new field, and pipelines and programs are evolving all the time, but even then Peyer’s Patches remained a tough nut to crack. The tissue is so dense and so packed that it makes it very difficult for analysis software to distinguish lymphocyte subpopulations based on surface markers of neighboring cells, even with nuclei staining. The team pushed on and made progress over the years, but the situation is still far from ideal.
“It is still really difficult to harvest Peyer’s Patches directly, particularly in older patients,” continues Dr. Glauben. “So we thought to extract them ex vivo and we were kind of successful, but not enough for our flow cytometry purposes. We then realized we really needed to know which cells belonged to the Peyer’s Patches and which cells belonged to the surrounding lamina propria, and this is where single-cell sequencing comes into play.”
By comparing samples including confirmed Peyer’s Patches and control samples of surrounding lamina propria, the team is aiming to sort out which immune cells, particularly lymphocytes, belong to each tissue. They also aim to generate new biomarkers for Peyer’s Patches lymphocytes to refine future high-dimensional flow cytometry analysis and new rounds of imaging mass cytometry.
Aiming for simplicity: scRNA-seq on the go
With patient endoscopy taking place in-house at the university hospital with experienced enterologists, the team has access to fresh samples from which PhD student Adrian Huck can extract Peyer’s Patches directly on the bench.
“It would be to our advantage if we could do everything on the bench,” assesses Dr. Glauben. “With the technology from Scipio bioscience, we can actually do the single-cell sample preparation directly on the bench in our lab, and not have to go to another core facility, in another location in Berlin. This would help us a lot because the isolation already takes some time, and we really would have everything processing very fast to get a proper image of the gene expression of the cells.”
The transcriptome being dynamic and reacting to external stimuli, it is important for fresh cells to be processed as fast as possible to limit the appearance of artifacts in the gene expression. This is even truer for immune cells to retain the activation state they had in physiological conditions and preserve the expression of specific biomarkers. Moreover, one does not want to compromise with those precious samples and require freezing or fixing methodologies prior to the single-cell sample preparation step.
With the RevGel™ technology from the Asteria™ kits, the team will be in an optimal position to characterize the lymphocytes from Peyer’s Patches and shed new light on their role in nutrition-based inflammation pathways.