Novel cryo-EM findings could revolutionize T cell immunotherapy design

One of the most exciting advances in cancer treatments in the past decade is the development of T cell immunotherapies, in which a patient's own immune system is trained to recognize and attack dangerous cells. Yet a full understanding of how they actually work has eluded researchers. That's been a significant limitation, because while T cell immunotherapies are highly effective for certain subtypes of cancers, they're ineffective for the majority of them-and the reasons why are unclear. Understanding their modus operandi could bring their benefits to a much broader group of cancer patients. 

Now researchers at The Rockefeller University have revealed key details about the T cell receptor (TCR), which is embedded in the cell membrane and essential to T cell therapies. Using cryo-EM to image the protein in a biochemical environment that replicates its native milieu, researchers from the Laboratory of Molecular Electron Microscopy have discovered that the receptor is a sort of jack-in-the-box that springs open when it's presented with an antigen or similarly suspect particle. This discovery is contrary to all previous cryo-EM studies of the complex.

The novel finding, published in Nature Communications, has the potential to refine and expand T cell therapies. 

"This new fundamental understanding of how the signaling system works may help re-engineer that next generation of treatments," says first author Ryan Notti, an instructor in clinical investigation in Walz's lab and a special fellow in the Department of Medicine at Memorial Sloan Kettering Cancer Center, where he treats patients with sarcomas, or cancers that arise in soft tissue or bone.

"The T cell receptor is really the basis of virtually all oncological immunotherapies, so it's remarkable that we use the system but really have had no idea how it actually works-and that's where basic science steps in," says Walz, a world expert in cryo-EM imaging. "This is some of the most important work to ever come out of my lab."

Activating T cells

Walz's lab specializes in visualizing macromolecular complexes, particularly cell membrane proteins, which mediate interactions between the cell interior and exterior. The TCR is one such complex. This intricate, multi-protein structure allows T cells to recognize and respond to antigens presented by human leukocyte antigen (HLA) complexes of other cells. It's this response that T cell therapies have capitalized on to enlist a patient's own immune system in the cancer fight.

But while the components of the TCR have been known for decades, the earliest steps of its activation have remained unknown. As a physician-scientist, Notti was frustrated by this knowledge gap: Many of his sarcoma patients were not reaping the benefits of T cell immunotherapies, and he wanted to understand why.

Determining that would help us understand how the information gets from outside the cell, where those antigens are being presented by HLAs, to the inside of the cell, where signaling turns on the T cell."

Ryan Notti, Rockefeller University

Notti, who received his Ph.D. in structural microbiology at Rockefeller before shifting his focus to oncology, proposed to Walz that they investigate it.

From custom membranes to improved immunotherapies and vaccines

Walz's group specializes in designing custom membrane environments that aim to mimic the native environment of specific membrane proteins. "We can change the biochemical composition, the thickness of the membrane, the tension and curvature, the size-all kinds of parameters that we know have an influence on the embedded protein," Walz says.

For the study, the researchers aimed to create a native-like environment for the TCR and observe how it behaved. To do so, they put the receptor into a nanodisc, which is a small disc-shaped patch of membrane that is kept in solution by a scaffold protein that wraps around the edge of the disc. It was no mean feat; "getting all eight of these proteins properly assembled into the nanodisc was challenging," Notti says.

All previous structural work on the TCR was performed in detergent, which tends to strip the membrane from the protein. This was the first study in which the complex was put back into a membrane, Walz notes. 

They then began cryo-EM imaging. These images revealed that in its resting state, the T cell receptor had a closed, compacted shape. Once activated by an antigen-presenting molecule, it opened up and extended, as if throwing its arms wide.

This came as a deep surprise. "The data that were available when we began this research depicted this complex as being open and extended in its dormant state," Notti explains. "As far as anyone knew, the T cell receptor didn't undergo any conformational changes when binding to these antigens. But we found that it does, springing open like a sort of jack-in-the-box."

The researchers suggest that combining two key methods made their new view possible. One, they concocted the correct membrane lipid cocktail to replicate the TCR's in vivo environment. And two, they returned the receptor to that membrane environment using nanodiscs prior to cryo-EM analysis. An intact membrane is key, they discovered, because it holds the TCR in place until activation. By removing the membrane via detergent, previous studies had inadvertently released the latch on the jack-in-the-box, prematurely springing it open.

"It was important that we used a lipid mixture that resembled that of the native T cell membrane," says Walz. "If we had just used a model lipid, we wouldn't have seen this closed dormant state either."

The researchers are excited about the potential their findings have for optimizing therapies based on T cell receptors. "Re-engineering the next generation of immunotherapies tops the charts in terms of unmet clinical needs," Notti says. "For example, adoptive T cell therapies are being used successfully to treat certain very rare sarcomas, so one could imagine using our insights to re-engineer the sensitivity of those receptors by tuning their activation threshold."

"This information may be used for vaccine design as well," Walz adds. "People in the field can now use our structures to see refined details about the interactions between different antigens presented by HLA and T cell receptors. Those different modes of interaction might have some implication for how the receptor functions-and ways to optimize it."