Quake Lab Utilizes RNA Sequencing Tool For Food Allergy Diagnostics

By Benjamin Ross

January 24, 2019 | Researchers in the Stanford lab of Stephen Quake are using single cell RNA sequencing (scRNA-seq) in an effort to provide insight into food allergies and the antibodies that cause them. The results from their research were recently published in Science (DOI: 10.1126/science.aau2599).

The study focuses on the effect of Immunoglobulin E (IgE) antibodies, which, while providing a defense against parasite infections, are also the source of many food allergies.

Derek Croote, a PhD candidate at Stanford and a co-author of the study, told Bio-IT World that he and Stephen Quake began discussing the possibility of a project like this years ago. The application of scRNA-seq was something that fascinated Croote.

One issue that arises when researchers target IgE antibodies is coming up with a way to accurately identify cells producing them, Croote said. “These cells are extremely rare, and on top of that there are also other cells that look like them but aren’t. So what [scRNA-seq] allows you to do is capture cells with high sensitivity and not worry about specificity as much.”

The method took a while to develop, Croote says. When it comes to blood sample tests, optimization took time to get right, and breaking blood down to individual cells was a challenge.

Croote and his colleagues collected as many IgE cells as they could from the blood of six children with severe food allergies, and because each potential IgE antibody was isolated according to its single-cell, they were able to analyze them individually and with high sensitivity. This allowed the team to “characterize each cell’s gene expression, splice variants, and heavy and light chain antibody sequences,” according to the published paper.

“It was a big moment to go from a process that was fraught with challenges around scarcity and mimicry and end up with something that captured a lot of cells, and then we were able to locate the IgE cells and not really care about the others,” Croote said.

Having isolated the IgE antibodies within the blood samples, Croote and his team discovered cross-reactive antibodies that were also high-affinity. Croote says this could be the reason why a reaction to a food allergy may be more severe than another.

“We’re moving forward in evaluating whether this will be efficacious across a large group of people, and determining whether or not these antibodies can inhibit artificial reactions,” said Croote.

Setting up artificial reactions in a test tube can allow researchers at the Quake lab to take cells that release histamines, causing allergic reactions, and observe how they react to certain proteins and antibodies, said Croote.

“We can take this test that’s normally diagnostic in value and add our antibodies to the cocktail to see if they will inhibit a reaction,” Croote said.

Using scRNA-seq can also provide insight into a population’s susceptibility for other diseases as well. “Insight into convergent evolution of high affinity antibodies in unrelated individuals can guide vaccine design and lead to strategies for population-level passive immunity; it is also a process that has been argued to occur in response to a number of pathogens such as influenza, HIV, Streptococcus pneumoniae,” Croote and his co-authors write in the paper.

A Broader Application

Though results are still early, Croote says there is a potential to develop a therapeutic to treat food allergies from lab’s research. The Quake lab has already filed intellectual property claims, according to Croote.

“We’re years away, but what a potential treatment could look like is we take these antibodies that we know bind peanuts very strongly and then modify them,” says Croote. “So when we reintroduce [these antibodies] into people, they still bind peanuts but no longer have the potential to cause a severe allergic reaction.”

And they aren’t the only ones looking to develop a solution for food allergies. Partnerships between  AllerGenis and Luminex, as well as developments by Thermo Fisher Scientific, have been progressing novel diagnostic technology for food allergies.

For instance, Thermo Fisher Scientific’s ImmunoCAP test helps predict whether patients may have a sensitization to Ara h 6, a protein component in peanuts that can cause allergic reactions. The test also includes assays for cross-reactive carbohydrate determinants (CCD) and profilins, which are both allergens contained in various plants, pollens, and foods.

“Advanced diagnostic capabilities for peanut allergies can contribute to better outcomes for allergy sufferers, especially young children,” a representative from Thermo Fisher Scientific wrote in a press release.

For Croote, scRNA-seq holds the key to dive deeper into the nature of food allergies. Why are patients allergic to peanuts but not tree nuts, or why might they have an allergy for some tree nuts but not others? ScRNA-seq’s novel capabilities allow Croote and the Quake lab to find answers to these questions due to its unbiased capture of cells secreting IgE antibodies.

“Therefore, if an individual is allergic to tree nuts, for example, we would expect to capture B-cells producing IgE antibodies that bind tree nuts,” Croote said in an email interview. “We could then perform a process analogous process to what we did in our paper—we could test each antibody against a panel of tree nuts to understand how monoclonal antibody binding relates to an individual's allergies.”