Scientists at the UCLA Broad Stem Cell Research Center have developed a technique able to more efficiently isolate specific T-cells — immune cells involved not only in the fight against infections and cancer but also in autoimmune diseases such as multiple sclerosis (MS) — for research.
In particular, the new method may help to identify and characterize the specific T-cells participating in the abnormal immune responses against myelin that characterize MS, and to discover new ways of suppressing their activity. Myelin is the protective sheath surrounding nerve fibers that is damaged in MS.
The technique and its potential applications were described in the study, “Droplet-based mRNA sequencing of fixed and permeabilized cells by CLInt-seq allows for antigen-specific TCR cloning,” published in the journal Proceedings of the National Academy of Sciences.
T-cells are a type of white blood cell that recognizes fragments of molecules, called antigens, through specific membrane receptors (TCRs). Each of these cells has unique TCRs that recognize a specific antigen.
This antigen can be, for instance, a fragment of a particular virus or bacterium, a specific type of cancer cell, or, in the case of autoimmune diseases, a particular molecule of a person’s own body promoting inadequate attacks against it.
When a T-cell encounters the antigen its receptor recognizes, it produces many copies of itself and instructs other immune cells to attack the same antigen.
“They’re both the effectors and organizers of the body’s adaptive immune response, which means they can be used as therapeutics and studying their dynamics can shed light on overall immune activity,” Pavlo Nesterenko, the study’s first author and a graduate student at UCLA, said in a university press release.
As such, increasing efforts have been made to develop methods to efficiently isolate specific T-cells, as this may help to better understand certain diseases and develop new therapeutic approaches.
“Once you know the sequence of a T-cell receptor of interest, you can use that information to develop therapies that either make more of that cell in the case of fighting cancer and viruses or introduce regulatory T-cells with this receptor sequence to curb an overactive immune response in a given area,” Nesterenko said.
Of note, regulatory T-cells (Tregs) are a type of T-cell that instead of mounting immune attacks against a particular antigen, work by dampening such antigen-specific responses. These Tregs are thereby key for maintaining immune tolerance to the body’s own molecules.
In MS, Tregs with a TCR that specifically recognizes a myelin fragment could be expanded in the lab and delivered to MS patients, or could be specifically activated within the body through several strategies, including non-inflammatory vaccines.
However, isolating specific T-cells has proven a challenging task, as they “are found in very low numbers” in blood and tissue samples, said Owen Witte, the study’s senior author and founding director of the UCLA Broad Stem Cell Research Center.
Part of the reason this process is inefficient is that when researchers attempt to isolate T-cells with specific TCRs, they end up capturing other immune cells activated by such T-cells and which are found in larger numbers; these are known as bystander cells.
Witte, who also holds the presidential chair of developmental immunology and is a member of the UCLA Jonsson Comprehensive Cancer Center, noted that “existing methods for capturing and identifying these T-cells are labor-intensive and need improvement.”
The new method, called CLInt-Seq, incorporates a technique that enables researchers to more efficiently distinguish T-cells with receptors of interest from most bystander cells, followed by single-cell TCR sequencing through a method called droplet-based sequencing.
CLInt-Seq involves the identification of T-cells of interest through staining of specific immune messenger molecules and others within cells, which was found to be more effective than current isolation approaches based on molecules in the cell’s membranes.
“The innovation of this system is that it combines an improved method that identifies T-cell receptors with more specificity with a chemical adaptation that makes this process compatible with droplet-based mRNA sequencing,” Witte said, noting that it “addresses challenges at the heart of finding T-cell receptors for treating cancer and other diseases.”
Moreover, the new method readily and successfully identified Tregs and T-cells with TCRs that recognize fragments of the Epstein-Barr virus, which causes mononucleosis, supporting that CLInt-Seq “could be used to discover TCRs reactive to any antigen of interest,” the researchers wrote.
Witte’s team is now using this technology to address a number of scientific questions, including identifying TCRs that react to the virus that causes COVID-19 and developing T-cell therapies for prostate cancer.
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