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Immunotherapies Could Benefit From New Method to Interrogate T Cell Receptor Sequence and Affinity


NEW YORK (GenomeWeb) – Researchers from the University of Texas at Austin have developed a technique that combines single-cell next-generation sequencing with the measurement of T cell receptor affinity to better understand the function of T cells. The method — dubbed iTAST for in situ TCR affinity and sequence test —sequences T cell receptors from single cells and determines their affinity to specific antigens. Since T cell receptor affinity influences theactivation and function of T cells, the method could be used to identify T cells to use for immunotherapy, to monitor vaccine response, or to better understand how individuals fight infection.

The researchers have filed a patent application on the method and are looking to commercialize it, Jenny Jiang, senior author of the study and an assistant professor of biomedical engineering at the University of Texas, told GenomeWeb.

The method, which was described this week in Science Translational Medicine, combines a common immunology test known as an adhesion assay, which is used to study the interaction between T cells and their antigens, with single-cell sequencing to get both information about the T cell receptor's affinity and its sequence.

In the study, the researchers applied the approach to understand the immune response of healthy individuals to hepatitis C virus, identifying and sequencing the T cell receptors specific to the virus, but ultimately the technique could be used to characterize and understand T cell response to any antigen, including viruses and even cancer cells.

Jian Han, a faculty investigator at the HudsonAlpha Institute for Biotechnology, who was not involved with the study, told GenomeWeb that he thought the method could be important to the field because it "combined several high throughput technologies to address T cell function" at the single-cell level.

The idea is to identify specific cells from a subclass of T cells known as CD8+ T lymphocytes, which kill cancer cells and pathogen-infected cells by recognizing the peptides that bind to the major histocompatibility complex (MHC). T cell receptors' ability to recognize the pathogen peptides depends on their sequence.

The gold standard method for studying T cell receptor affinity has been surface plasmon resonance (SPR), which is "a labor intensive process" involving protein purification and cloning, Jiang said. Other types of cell-based affinity tests are not as reliable because they rely on obtaining a large number of cells of the same type by cell proliferation, but some cells cannot be grown, she said. In addition, those methods are also low in throughput and do not identify both the T cell receptor's  affinity and its sequence.

With iTAST, the CD8+ T cells are first enriched using streptamer and then sorted out from the blood via flow cytometry. The streptamer is removed and affinity is measured with an adhesion assay. Essentially, peptide bound to the MHC is introduced so that affinity between the peptide and the T cell receptor can be measured.

In this study, the researchers looked at a peptide associated with HCV with the goal of identifying the T cell precursors that would respond to the virus if the individual was exposed. They did this by introducing the HCV peptide to the sample, isolating the specific T cell that bound to that peptide and sequencing the T cell receptor.

As expected, in samples from healthy individuals, these cells were rare, having a frequencies of between two per million and 18 per million CD8+ T cells, but they found a great deal of heterogeneity between the strength of affinity for the peptide between individuals.

The T cells that recognize the viral antigen in individuals who have not been exposed to the virus are "like the safety net of our immune system," Jiang said. In addition, "to our surprise, the affinities of the T cell receptors of these cells [had] a large dynamic range." That was something that was not previously known, because existing technologies had been unable to measure it.

To further study the heterogeneity between individuals, the researchers evaluated 12 samples from nine people. Five samples were from individuals 33 years of age or younger, while seven were from persons who were 49 or older.

They found that individuals from the younger cohort had higher numbers of the high-affinity antiviral T cells than those from the older cohort.

"What that means is that as you get older, you are losing the best cells that help you fight infection," Jiang said.

"This reduced frequency of high-affinity virus-specific T cells could be a contributing factor toward the increased risk of infection and diminished vaccine response in the elderly," the authors wrote.

Older individuals tend to be at a higher risk for infection by seasonal influenza and are less likely to respond to vaccines than younger individuals. One reason could be that they have fewer high-affinity T cells that recognize viral antigens, Jiang said.

While such conclusions are very preliminary and would have to be validated in larger numbers of individuals, Jiang said that she thinks the iTAST method could be used to do just that.

In addition, of the researchers are looking at possibilities for commercializing the method, potentially by forming a spinout company. The iTAST method itself could potentially be developed into a product, she said, andthe researchers are still working on developing and refining the protocol.

Eventually, she said, the method could have implications for the development of immunotherapies for both infectious disease and cancer. For instance, T cells with high affinity TCRs could be engineered and given back to patients who lack them, she said.