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Scientists can work out the detailed structure of proteins by turning them into a crystal – much like a salt crystal. These images show different protein crystals.

This entry is part 15 of 18 in the series Science Snaps

The emergence of immunotherapies to treat cancer has been hailed as a breakthrough.

And with good reason – even advanced cancers have responded well to immunotherapy in clinical trials, in some cases disappearing without trace and giving these patients years of life back. Immunotherapy is now a standard treatment for advanced skin cancer, and clinical trials across the world are testing it in other types of cancer.

But there are still big unanswered questions, like why immunotherapies can be such powerful weapons against tumours in some people, but not in others.

Most immunotherapies work by re-educating a type of white blood cell called a T cell. If the immune system was a game of chess, T cells would be the Queen, controlling the board. Alerting them to the danger cancer poses can turn them into ruthless leaders, unleashing an immune response and wiping the cancer cells out. So understanding the exact mechanics of immunity’s Queens could be pivotal in unlocking the secrets of immunotherapy.

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CD45 is much ‘taller’ than other key molecules on T cells. When a T cell spots something wrong and moves in to interrogate, CD45 gets ‘squeezed out’.

Now, after 15 years of painstaking work, a team of scientists* – including Cancer Research UK researchers Professor Christian Siebold and Professor E Yvonne Jones – has revealed the detailed structure of one of the molecules controlling a T cell’s fate.

And it turns out that the molecule, called CD45, has a key defining feature: its size.

Switching to ‘kill mode’

The study, led by Professor Simon Davis at the University of Oxford – published in the journal Nature Immunology in March – focuses on how CD45 controls the way T cells go about their important work.

T cells get ‘switched on’ by a small cluster of large proteins on their surface, known as the T cell receptor. A very small molecule (called a phosphate) can be added to the bit of the receptor that sits inside the cell, turning the receptor into its active state and switching the T cell into ‘kill mode’.

When a T cell recognises an enemy, more T cell receptors get a phosphate added. And once a threshold is reached, a cascade of signals is set off like fireworks.

But when a T cell is bumbling about its everyday business, it needs to be held in check somehow, otherwise it could attack us. And this is where CD45 first comes into play. When CD45 is next to the T cell receptor it steals away the phosphate, stopping the signals that switch the T cell into ‘kill mode’.

A giant amongst men

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Bombarding protein crystals with high energy X rays creates ‘diffraction patterns’, which scientists can use to work out the building blocks that make up the protein, revealing its precise structure.

The mystery that was puzzling scientists was how CD45’s control over the T cell is then released in the presence of danger, such as when a virus infects a nearby cell. What happens to CD45 when a T cell spots an enemy, what stops CD45 from phosphate-nabbing, allowing the T cell to be switched on?

To solve this puzzle, the Oxford team needed to know exactly what CD45 looks like – its chemical make-up, its size and its shape. The architectural plans of the molecule, if you like.

This turned out to be easier said than done.

Because molecules are extremely small, one of the best ways to reveal their structure is to make a crystal – like a salt crystal – then use X rays to study them. But CD45 is covered in sugar molecules, especially at its tip, which makes it very hard to get it to form crystals

The researchers managed to piece together the structure by removing the tip and chopping off most of the sugars in the lab, allowing it to form crystals that could be studied. Next, the researchers used an extremely powerful microscope to capture images of the whole of CD45, including the tip.

After 15 years of work, which involved making more than 100 different forms of CD45, they finally knew what CD45 looked like in detail.

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This is what the whole CD45 protein looked like down a very powerful microscope, with a doughnut-shaped blob of protein on the tip so the group could work out how long it is. It’s blurry because the protein is extremely small, but it gave researchers vital information about the shape of the protein with its tip.

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To study CD45 using X rays, the researchers had to chop off its tip, leaving the long straight backbone.

What did the study reveal?

The team found that CD45 was made up of four similar, small units in a rod-like arrangement, plus the extended “sugary” portion forming the tip of the molecule.

And it turned out to be quite tall and very rigid, standing straight up at the surface of the cell.

It turns out that this shape and arrangement of these parts of the molecule can explain how the CD45 is released to switch on the T cell.

When a T cell recognises something on another cell it doesn’t like the look of, the T cell receptors and other molecules stick to it, bringing the cells closer together.

All the other molecules involved at this point of contact we now know are quite a bit shorter than CD45, and the bigger molecule literally gets squeezed out. This physical separation of CD45 from the T cell receptors stops it removing the phosphate, firing the T cell into action.

All this happens very quickly – a T cell makes contact with another cell “in around 6 seconds”, according to Davis. And once contact is broken, CD45 can cosy up to the T cell receptor once more, shutting the signal off.

Once the T cell receptor has fired there are lots of other molecules that come into play – like PD-1, which is the target of some of the latest immunotherapy drugs. And it’s these molecules all working in combination that ultimately decide if the T cell goes into kill mode, or whether it was a false alarm. But CD45 being squeezed out of the way seems more likely than ever to be the important first step in switching the T cell on.

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Using the information from the microscope analysis, the team made an accurate map of CD45 with all its sugars attached (this shows the whole protein with the extra blob added at the tip).

‘Switching on’ T cells is a crucial way to alert the immune system to the threat cancer poses, so understanding the mechanics of this process could help scientists develop treatments that trigger the immune cells to better attack cancer.

Understanding the immune system

It’s too early to say if this research will help develop new drugs exploiting CD45, but with the floodgates open for immunotherapies, the more scientists understand about basic immunology the better.

Knowing exactly how T cells work might help researchers come up with new immunotherapies, or put together combinations of immunotherapies that boost the immune system. And it might help get to the bottom of why some cancers respond so well to immunotherapy, while others don’t.

“Understanding the signals controlling T cell behaviour is crucial to working out how we can exploit the immune system to treat cancer, and other diseases,” Davis tells us. “We may be able to switch T cells off as well as on.”

“There’s a world of exciting opportunities out there, and this kind of research into the fundamental workings of the immune system is the stepping stone to better treatments.”

Emma

*The research was funded by Wellcome Trust, Medical Research Council, the Royal Society and Cancer Research UK.

Our Cancer Immunology awards seek to catalyse research and build the UK’s research base in cancer immunology, by funding immunologists in non-cancer fields. For researchers interested in applying, please see our website (http://www.cancerresearchuk.org/funding-for-researchers/our-funding-schemes/cancer-immunology-project-awards).