When your immune system is exposed to a vaccine, an allergen, or an infectious microbe, subsets of T cells that can recognize a foreign intruder jump into action. Some of these T cells are prepared to kill infected cells, while others serve as memory cells that circulate in the body and take care that the intruder reappears.
MIT researchers have now found a way to identify T cells that share a specific goal through a process known as high-throughput single-cell RNA sequencing. This type of profiling can reveal the unique functions of these T cells by determining which genes they turn on at a particular time. In a new study, researchers used this technique to identify T cells that cause the inflammation seen in peanut allergy patients.
In the current work, the researchers are using this method to investigate how patients' T cells respond to oral immunotherapy for peanut allergies to determine whether the therapy works for a particular patient. Such studies could also help researchers develop and test new therapies.
"Food allergies affect about 5 percent of the population, and there is no clear clinical intervention other than prevention that can cause a lot of stress for families and patients themselves," said J. Christopher Love, Raymond A. and Helen E. St Laurent Professor of Chemical Engineering and member of the Koch Institute for Integrative Cancer Research at MIT. "Understanding the underlying biology that drives these reactions is still a really critical question."
Greetings and Alex K. Shalek, Associate Professor of Career Development at Pfizer-Laubach at MIT, Associate Professor of Chemistry, core member of the MIT Institute for Medical Technology and Science (IMES) and Associate Member of the Koch Institute, are the lead authors of the study that appears today in Nature Immunology. The main authors are PhD student Ang Andy Tu and former postdoc Todd Gierahn.
The researchers' new method builds on their previous work and develops techniques for quickly performing single cell RNA sequencing on large cell populations. By sequencing messenger RNA, scientists can find out which genes are expressed at a specific point in time and gain insights into the functions of individual cells.
Performing RNA sequencing on immune cells such as T cells is of great interest because T cells play so many different roles in the immune response. However, previous sequencing studies have failed to identify populations of T cells that respond to a particular target or antigen determined by the sequence of the T cell receptor (TCR). This is because in single cell RNA sequencing, usually only one end of each RNA molecule is labeled and sequenced. Most of the variation in the T cell receptor genes is at the opposite end of the molecule that is not sequenced.
"People have been describing T cells and their transcriptome using this method for a long time, but without knowing what type of T cell receptor the cells actually have," says Tu. "When this project started, we thought about how we can try to recover this information from these libraries so that the single cell resolution of these records is not compromised and we do not have to dramatically change our sequencing workflow and platform. "
In a single T cell, the RNA encoding T cell receptors makes up less than 1 percent of the cell's total RNA. The MIT team therefore developed a method to amplify these specific RNA molecules and then remove them from the entire sample. They could be fully sequenced. Each RNA molecule is labeled with a barcode to identify which cell it is from so that researchers can align the T cell's targets with their RNA expression patterns. In this way, they can determine which genes are active in populations of T cells that target specific antigens.
"To put the function of T cells in context, you have to understand what they are trying to recognize," says Shalek. "With this method, you can use existing RNA sequencing libraries for single cells and extract relevant sequences that you may want to characterize. In essence, the approach is a simple strategy for extracting some information that is hidden in genome-wide expression profile data."
Another advantage of this technique is that it does not require expensive chemicals, relies on equipment that is already available in many laboratories, and can be applied to many previously processed samples, the researchers said.
Analysis of allergies
In Nature Immunology, the researchers showed that this technique can be used to identify mouse T cells that were active against the human papillomavirus after the mice were vaccinated against the virus. They found that although all of these T cells responded to the virus, the cells had different TCRs and were in different stages of development - some were very active in killing infected cells, others focused on growth and division.
The researchers then analyzed T cells from four peanut allergy patients. After exposure to peanut allergens, the cells were able to identify T cells that were active against these allergens. They also showed which subgroups of T cells were most active and found some that produced inflammatory cytokines that are usually associated with allergic reactions.
"We can now begin to layer the data to identify the most important cells that we could not previously identify with RNA sequencing alone," Tu said.
Love's Lab is now working with Massachusetts General Hospital researchers to use this technique to track the immune responses of people undergoing oral immunotherapy for peanut allergies - a technique that consumes small amounts of the allergen, thereby building tolerance to the immune system it can.
Clinical studies have shown that this technique works in some, but not all, patients. The MIT / MGH team hopes that their study will help identify factors that could be used to predict which patients respond best to treatment.
"One would certainly like to know better as early as possible whether an intervention will be successful or not," says Love.
This strategy could also be used to develop and monitor immunotherapy treatments for cancer, such as: B. CAR-T cell therapy can be used, in which a patient's own T cells are programmed so that they target a tumor. Shalek's laboratory is also actively using this technique, along with staff from the Ragon Institute at MGH, MIT and Harvard, to identify T cells that help fight infections such as HIV and tuberculosis.
The research was funded by the Koch Institute Support (core) Grant of the National Institutes of Health, the bridge project of the Koch Institute Dana-Farber / Harvard Cancer Center, the Food Allergy Science Initiative of the Broad Institute of MIT, and Harvard, Arnold and Harvard Mabel Beckman Foundation, a Searle Scholar Award, a Sloan Research Fellowship in Chemistry, the Pew-Stewart Scholars Program and the National Institutes of Health.
Ang A. Tu, Todd M. Gierahn, Brinda Monian, Duncan M. Morgan, Naveen K. Mehta, Bert Ruiter, Wayne G. Shreffler, Alex K. Shalek, J. Christopher Love. TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures. Nature Immunology, 2019; 20 (12): 1692 DOI: 10.1038/s41590-019-0544-5