Novel cryo-EM findings could revolutionize the design of T cell immunotherapy

Novel cryo-EM findings could revolutionize the design of T cell immunotherapy

One of the most exciting advances in cancer treatment in the last decade is the development of T-cell immunotherapies, which train a patient's own immune system to recognize and attack dangerous cells. But researchers have so far been unable to gain a comprehensive understanding of how they actually work. This presented a significant limitation because while T-cell immunotherapies are highly effective in certain cancers, they are ineffective in most of them – and the reasons for this are unclear. You understandProcedurecould benefit a much larger group of cancer patients.

Now researchers at Rockefeller University have revealed key details about the T-cell receptor (TCR), which is embedded in the cell membrane and essential for T-cell therapies. Using cryo-EM to image the protein in a biochemical environment that replicates its natural environment, researchers at the Laboratory of Molecular Electron Microscopy have discovered that the receptor is a kind of grab bag that pops open when presented with an antigen or similarly suspicious particle. This discovery contradicts all previous cryo-EM studies of the complex.

The novel finding, published inNature communicationhas the potential to refine and expand T cell therapies.

“This new, fundamental understanding of how the signaling system works can help redesign the next generation of treatments,” says lead author Ryan Notti, clinical instructor in Walz’s lab and special associate professor 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 actually the basis of virtually all oncology immunotherapies, so it's remarkable that we use the system but have no idea how it actually works - and that's where basic science comes in," says Walz, a global expert in cryo-EM imaging. “This is one of the most important pieces of work that has ever come out of my laboratory.”

Activation of T cells

Walz's laboratory specializes in the visualization of macromolecular complexes, particularly cell membrane proteins, that mediate interactions between the cell interior and exterior. The TCR is one such complex. This complicated multiprotein structure allows T cells to recognize and respond to antigens presented by human leukocyte antigen (HLA) complexes of other cells. T cell therapies have taken advantage of this reaction to involve the patient's own immune system in the fight against cancer.

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

Determining this would help us understand how the information from outside the cell, where these antigens are presented by HLAs, gets to the inside of the cell, where signaling turns on the T cell.”

Ryan Notti, Rockefeller University

Notti, who received his Ph.D. He earned his doctorate in structural microbiology from Rockefeller before shifting his focus to oncology and suggested that Walz investigate it.

From tailored membranes to improved immunotherapies and vaccines

Walz's group specializes in developing tailored membrane environments that aim to mimic the natural environment of certain membrane proteins. “We can change the biochemical composition, the thickness of the membrane, the tension and curvature, the size – all sorts of parameters that we know have an influence on the embedded protein,” says Walz.

For the study, the researchers wanted to create a native environment for the TCR and observe how it behaves. To do this, they place the receptor in a nanodisc, a small disk-shaped piece of membrane that is held in solution by a scaffolding protein that wraps around the edge of the disk. It was not an easy task; “It was a challenge to properly integrate all eight of these proteins into the nanodisc,” says Notti.

All structural work on the TCR to date has been done in detergent, which tends to detach the membrane from the protein. This was the first study to reincorporate the complex into a membrane, notes Walz.

They then began cryo-EM imaging. These images showed that the T cell receptor had a closed, compact shape when resting. Once activated by an antigen-presenting molecule, it opened and stretched out, as if spreading its arms wide.

That was a deep surprise. “The data available at the beginning of this research showed that this complex was open and expanded in its resting state,” explains Notti. "As far as anyone knew, the T-cell receptor did not undergo any conformational changes when it bound to these antigens. But we found that it did pop open, popping open like some kind of jack-in-the-box."

The researchers suspect that the combination of two key methods enabled their new perspective. First, they assembled the right membrane lipid cocktail to reproduce the TCRsin vivoEnvironment. And second, they returned the receptor to this membrane environment using nanodiscs before cryo-EM analysis. They found that an intact membrane is key because it holds the TCR in place until activated. By removing the membrane with a cleaning agent, previous studies had accidentally released the jack-in-the-box's latch, causing it to pop open prematurely.

“It was important that we used a lipid mixture that was similar to that of the native T cell membrane,” says Walz. “If we had only used a model lipid, we would not have seen this closed resting state either.”

The researchers are excited about the potential of their findings for optimizing therapies based on T cell receptors. “Redesigning the next generation of immunotherapies is a priority in terms of unmet clinical needs,” says Notti. "For example, adoptive T cell therapies are successfully used to treat certain very rare sarcomas. So one could imagine using our findings to redesign the sensitivity of these receptors by adjusting their activation threshold."

“This information can also be used for vaccine development,” adds Walz. "People in this field can now use our structures to see refined details about the interactions between different antigens presented by HLA and T cell receptors. These different modes of interaction could have implications for how the receptor works - and ways to optimize it."

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