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UC Santa Cruz Team Devises Approach for High-Throughput T Cell Profiling


NEW YORK – A team led by researchers at the University of California, Santa Cruz, has developed an approach for high-throughput T cell profiling.

Described in a paper published this week in Nature Communications, it could allow scientists to screen populations of T cells against peptide antigens at large scale, enabling them to better study what T cells are involved in leading the immune response against different diseases and how differences in patient T cell repertoires may be linked to, for instance, disease progression and outcomes.

The researchers have been pursuing the work primarily for applications in cancer and cancer immunotherapeutic research but have also begun to explore its usefulness in studying SARS-CoV-2, said Nikolaos Sgourakis, assistant professor of chemistry and biochemistry at UC Santa Cruz and senior author on the study.

The key advance presented in the paper is the ability to load peptides of interest on the major histocompatibility complex proteins that the body uses to present foreign antigens to the immune system, Sgourakis said. These MHC proteins display these antigens on the surface of cells, activating the body's T cell response, through which the immune system kills malfunctioning or infected cells.

The ability to express antigens in high-throughput fashion would be a boon for immunologists as it could, for instance, allow them to more rapidly and comprehensively profile patient responses to antigens linked to cancer or different infectious diseases. In the case of SARS-CoV-2, for example, researchers could load MHC proteins with peptides comprising the full complement of the virus' proteins and look at which peptides were most important in prompting the T cell response or how T cell repertoires varied depending on the severity of infection or patient outcome, Sgourakis noted.

A major challenge for this sort of immune profiling has been the difficulty of producing stable versions of the MHC proteins with the antigen peptides of interest.

"It is a notoriously difficult biochemical task," Sgourakis said.

Traditionally, he said, researchers would do this by expressing the individual MHC components and combining them with the peptides of interest, a process that would take several days and multiple sample processing steps.

"You could do this with a couple of peptides, but if you wanted to look at 100, you can imagine the workload that would entail," he said.

This process was streamlined somewhat by use of a workflow that produced MHCs bound to standard placeholder peptides instead of the particular peptide of interest, which allowed researchers to produce MHC-antigen peptide complexes in bulk. The placeholder peptides were bound to the MHC via a photosensitive bond that could be disrupted by applying UV light, allowing researchers to remove the placeholders and replace them with the actual peptides of interest when it was time to perform T cell profiling.

This method had its challenges, though. The placeholder peptides were expensive and because they were bound to the MHCs via photosensitive bonds, much of the preparation had to be done in the dark.

More recently, researchers have developed a method in which they modified MHC molecules with a disulfide bond in a way that allows them to generate stable, unbound MHC molecules that can then be loaded with peptides of interest. The process is not very efficient, however, in terms of the amount of viable unbound MHC produced, and there is also the question of whether the disulfide bond modifies T cell interactions.

To address these issues, Sgourakis and his colleagues developed an approach using the protein TAPasin Binding Protein Related (TAPBPR), a chaperone protein that binds to MHCs to maintain their stability and also facilitates the exchange of peptides bound to the MHC.

"A reasonable question was, well, if the empty [MHC] molecule is so unstable and doesn't really exist in solution, how does a cell do it?" Sgourakis said. "And the way the cell does it is by using these molecular chaperone proteins. And this particular chaperone nicely shelters the MHC peptide binding groove and opens it so peptide exchange can occur."

"We can use this system to load, at will, empty MHC complexes with high affinity peptides," he said.

By combining this peptide-loading system with DNA barcoding to track the peptide antigens and sequencing to identifying the T cells that bind them, the researchers are able to investigate T cell-antigen binding at large scale.

"I think now that we have broken the bottleneck, which was preparing these [MHC] molecules, people are going to start doing larger, higher-throughput experiments with all sorts of interesting immunology waiting to be discovered," Sgourakis said.

He cited as an example of such work a recent study he and his colleagues published as a bioRxiv preprint where they took the SARS-CoV-2 genome and modeled the 439 9mer and 279 10mer predicted epitopes that could be produced by the virus. These peptides can then be used to profile the T cell repertoires of COVID-19 patients.

This could allow researchers to investigate which virus peptides are most important in eliciting T cell response as well as differences in the T cell repertoire between patients, Sgourakis said. "We know this disease varies significantly from one individual to the next. Is there a signature in the CD8 repertoire that allows us to diagnose, monitor, and predict which individuals can handle the disease and which cannot?"

He said that his lab is also collaborating with researchers at the Children's Hospital of Philadelphia, two of whom co-authored the Nature Communications paper, on applying the method to studying neuroblastoma.

The University of California has applied for a patent for the technique with Sgourakis as inventor.