A cell’s secrets might be divulged by its surface, decorated with tens to tons of of 1000’s of molecules that help immune cells determine friend from foe. A few of those protruding molecules are antigens that trigger the immune system to attack, but it will probably be difficult for scientists to discover those antigens, which frequently vary across individuals, within the molecular forest.

A team of Stanford scientists led by Polly Fordyce, an Institute Scholar at Sarafan ChEM-H, has developed a recent method to faster and more accurately predict which antigens will result in a robust immune response. Their approach, which was reported in Nature Methods on Sept. 5, could help scientists develop simpler cancer immunotherapies.

T cells, a category of immune cells, crawl along and squish past other cells as they patrol the body, using T cell receptors to molecularly read peptides, or short pieces of proteins – that are cradled inside larger proteins called major histocompatibility complexes (pMHCs) that project from cell surfaces. Healthy host cells display an array of pMHCs that don’t trigger an immune response, but once T cells recognize disease-indicating peptides, they turn into activated to seek out and kill cells bearing these foreign signatures. Understanding how T cells sensitively distinguish these antigenic peptides from host peptides to avoid mistakenly killing host cells has long been a mystery.

“A T cell can detect a single antigenic peptide amongst a sea of 10,000 or 100,000 non-antigenic peptides being displayed on cell surfaces,” said Fordyce, assistant professor of bioengineering and of genetics.

The important thing to selectivity is within the T cell crawl. T cells’ sliding puts stress on the bonds between receptors and peptides, and generally, that extra stress is sufficient to break that bond. But sometimes, it has the other effect. Chris Garcia, co-author of the study and professor of molecular and cellular physiology and of structural biology, and others had already shown that essentially the most antigenic peptides are those whose interactions with T cell receptors grow stronger in response to sliding.

“It’s form of like a Chinese finger trap,” said Fordyce. “While you pull a bit on the receptor-antigen interaction, the binding actually lasts longer.”

Cellular mimicry

Identifying the most effective antigen-receptor pairs requires concurrently applying that sliding, or shear, force between a peptide and a T cell and measuring T cell activation, ideally 1000’s of times to get repeatable data for a lot of possible peptide/T cell receptor pairs. But existing methods are time-intensive and may end up in measuring just one peptide with tons of of T cells in a day.

The study’s first writer, postdoctoral scholar Yinnian Feng, developed a trick that permits the team to measure 20 unique peptides interacting with 1000’s of T cells in lower than five hours.

To make a simplified system that mimics cells with dangling peptides, they constructed small spherical beads from a cloth that expands upon heating and attached a number of molecules of a given peptide-studded pMHC to their surfaces. After depositing a T cell atop each bead and waiting long enough for receptors to bind to the peptides, they then very barely heated the bead. The bead’s expansion increases the gap between tether points, and the corresponding stretching of the T cell mimics the force it will experience sliding along cells within the body. After exerting that force, the team then measured how lively the T cells were.

They might do tons of of individual experiments in parallel through the use of beads which are each labeled with a singular color, making it possible to trace multiple different pMHCs. They took two sets of images tiling across each slide after each run: one set that tells them which pMHC a given bead is displaying and one other that tells them how lively each T cell atop that bead is. Cross-referencing those images tells them which antigens led to the strongest T cell responses.

On this demonstration of their platform, the team showed, with 21 unique peptides, that their results confirmed known activating and non-activating peptides for one T cell receptor and uncovered a previously unknown antigen that induced a robust T cell response. Working with the Garcia lab, they’ve also already begun to handle a challenge in immunotherapy: the T cell receptors that form the best affinity interactions with antigens within the lab are sometimes also activated by non-antigenic peptides within the body, a dangerous side effect that results in the killing of healthy cells. Using their technology, the team characterised T cell receptors engineered to specifically recognize tumor antigens without off-target reactivity. In future work, they plan to construct libraries of over 1,000 peptides to uncover novel antigens.

They hope that this approach, which is quick and requires few cells, or an optimized type of it could sooner or later be used to enhance personalized immunotherapies.

“This platform may also help improve efforts to engineer T cells that specifically goal cancer cells, in addition to determine which antigens are able to potently activating a patient’s own T cells to more effectively goal cancer cells,” said Fordyce.

Source:

Journal reference:

10.1038/s41592-022-01592-2