ME/CFS Research Review

Simon McGrath explores the big biomedical stories

Tag: TCR

Dr Ron Davis’s big immune study is looking at HLA genes. Here’s why.

Dr Ron Davis has won a large NIH (US National Institutes of Health) grant for an immunology project with a strong focus on HLA genes. Which may have led some to wonder, ‘What are they?’

HLA (human leukocyte antigen) molecules play a critical role in the immune system, particularly by activating T cells. There is a huge amount of variation in the HLA genes that different people have, and Davis’s theory is that certain types of HLA genes could increase the risk of ME/CFS.

The following explanation of HLA molecules is taken from a piece I wrote a few years ago.

The short version

HLA molecules fire up T cells

HLA waiter1

HLA serves up an antigen, ready for a T cell receptor to recognise.

T cells play a key role in the immune system. Like antibodies, the receptors of T cells respond to very specific antigens (foreign proteins), much like a lock matching just one key.

However, while antibodies will recognise and bind to part of a whole protein, such as the protein coat of a virus, T cell receptors only recognise tiny fragments of proteins. And T cell receptors can’t respond to antigens unless they are presented in just the right way.

That’s where HLA molecules come in. At a very basic level, HLA molecules act like waiters, serving up the antigen on a plate. More precisely, HLA molecules – which sit on the cell surface – have a groove that cradles the small antigen, and the T cell receptor binds to the antigen and HLA molecule together.

If the T cell receptor recognises the antigen proffered by the HLA molecule (strictly speaking, several different molecules combine to make an HLA complex) then the T cell will snap into action. But without HLA molecules, T cells wouldn’t be able to take action against threats to the body.

HLA in ME/CFS and other diseases

We have six different types of HLA molecule that present to T cells, and there are many different versions of each of the six types. Ron Davis at Stanford believes that the version of HLA genes you have may influence the risk of getting ME/CFS, and certainly HLA gene variants have been linked to numerous diseases. One particular version of an HLA gene increases the risk of narcolepsy by 130 times. A version of another HLA gene conveys some protection against HIV developing into AIDS – though the same gene variant increases the risk of the autoimmune disease ankylosing spondylitis. In fact, HLA genes are linked to a number of autoimmune diseases.

More about the biology (it’s fun, really!)

HLA molecules help activate T cells

T cells are an important part of the immune system. T helper cells are ‘generals’, co-ordinating the immune system response. For instance, T helper cells play a critical role in firing up antibody-producing B cells and other types of T cell. Killer T cells are more like infantry, acting independently and killing off rogue cells such as those infected with a virus.

But T cells need to be activated first before they get going, and they are activated in part by antigens, which for T cells are peptides, short pieces of protein. And antigens are what HLA molecules are about: they sit on the cell surface and hold out the antigen morsels to the T cells.

If the T cell  receptor recognises the antigen –  in much the same way as an antibody recognises a specific antigen – then that will trigger activation of the T cell. In the case of killer T cells, that’s curtains for the cell doing the presenting – the cell has just signed its own death warrant, but that’s the whole idea.

By taking out the infected cell, the killer T cells are helping to contain an infection and protect other, healthy, cells.

Killer T cells also recognise cancerous cells. And when things go wrong, they can also recognise normal proteins as a threat, leading to autoimmune diseases.

‘Don’t kill me’: HLA class I and killer T cells

Apart from red blood cells and sperm, almost every cell in the body displays class 1 HLA molecules. HLA class I molecules acts as a kind of identity card: it’s the way a cell proves that it is healthy and should be left in peace by marauding killer T cells that will destroy any cell that doesn’t pass muster. You could see killer T cells as an internal security force that’s licensed to kill.

The whole system is pretty elegant and very hard to cheat as the key thing about HLA class I molecules is that they present little bits of whatever proteins the cell that bears them is producing. So if a cell is infected by a virus it will be manufacturing lots of viral proteins, and bits of these foreign proteins end up displayed on the HLA-I molecules. That’s ‘game over’ for the infected cell: once a killer T cell recognises that viral antigen it sends a signal to the infected cell to self-destruct.

So that’s HLA class I: cell-surface molecules that act as identity cards by displaying to killer T cells bits of whatever proteins the diseased cell is making. It’s a way for healthy cells to show that they are ‘clean’, so that the lethal killer T cells leave them alone, while infected cells mark themselves for destruction.

TCR HLA edit

A killer T cell in action against a cell infected by a virus. An HLA class I molecule offers up a viral antigen, and a T cell with a matching receptor binds to the HLA molecule and the antigen together. The T cell responds by killing the infected cell.

‘Here’s the threat, sort it out’: HLA class II

The other class of HLA molecules, class II, also display antigens, but while class I HLA are on almost all cells , class II are used by the ‘professionals’ – immune-system ‘antigen presenting cells’, such as macrophages, dendritic cells and B cells. Their role is to pick up foreign antigens and present them to other immune cells, particularly T helper cells to kickstart the adaptive immune response. (Adaptive immune responses are very specific, such as antibodies recognising a particular viral protein, as opposed to ‘innate’ responses that recognise classes of pathogens, such as recognising viral RNA or bacterial cells walls.)

Link to Mark Davis’s work on clonal expansion

Once they’ve been primed by class II HLA molecules, T helper cells don’t act directly against an infection. Instead, they help activate other immune cells, including the killer T cells we’ve just discussed and antibody-producing B cells. Killer T cells are the type of cells where Mark Davis has found clonal expansion. When killer T cells are activated (by both T helper cells and the antigen presenting cells) they copy themselves to form an army of clones ready to take on the threat: this is clonal expansion.

Back to the Ron Davis study: HLA and disease

There are three types of class I HLA molecules (HLA-A, HLA-B and HLA-C) – and three important types of class II HLA molecules (HLA-DP, HLA-DQ and HLA-DR). That makes six types, but there are huge numbers of different versions of each type.

Different versions of HLA are associated with increased risk (or even decreased risk) for certain diseases, particularly autoimmune diseases.

In 2014 Ron Davis reported that initial HLA profiling of 400 individuals indicated that patients had different versions of the genes that encode the HLA protein from healthy people – but that they needed to profile more people to confirm this finding. A new study that has just been announced should establish if particular versions of HLA molecules increase the risk of getting ME/CFS.

The project starts this month and is expected to complete by 2023. More information about the study at Health Rising.

(Image credits: Waiter – FreeStockPhotos.biz; HLA/TCR binding – adapted from Elemans et al.)

A plan to replicate Mark Davis’s remarkable findings of immune activation in ME/CFS

chris-ponting2

Chris Ponting

A team led by Edinburgh University’s Professor Chris Ponting has won funding for a PhD student who would follow up and expand on remarkable recent findings made at Stanford University, where Dr Mark Davis may have pinpointed a major issue in the immune system in ME/CFS.

Last year, Davis produced strong evidence of T cell clonal expansion, similar to that seen in illnesses including multiple sclerosis and acute Lyme disease. His discovery came from a new and sophisticated way of looking at the immune system in ME/CFS, which revealed the strongest evidence yet for immune activation in this disease. These results could indicate autoimmunity or a response to infection in ME/CFS, and could point to the core problem in the disease.

250px-Healthy_Human_T_Cell

Human T cell (photo: NIAID)

So if the results of the PhD work are as hoped then they could narrow down the cause of ME/CFS for some patients. The ultimate aim is for this research to form part of a concerted effort that provides affordable diagnostic tests and targets for treatment.

The PhD will be based at Ponting’s Computational and Disease Genomics lab in Edinburgh. The £90,000 cost of the PhD will be jointly funded by Action for ME and the Scottish Government’s Chief Scientist’s Office. Samples from patients will be provided by the UK ME/CFS Biobank, subject to a successful full application to the Biobank.

Ponting has drawn in impressive collaborators for this project: senior immunologist Professor Georg Höllander from Oxford University and molecular biologist Dr Lia Chappell from the prestigious Wellcome Sanger Institute. I am pleased to say that I have also been involved with this study from the outset and the successful PhD candidate will engage with more patients as the project gets underway.

The immune-system biology behind the study: T cells and their receptors

Many people have heard of B cells and the antibodies they produce; T cells are their cousins. T cells come in many forms, but the relevant ones here are called killer (CD8) T cells. These act like a security force, checking cells and killing off any that look sick because they are infected or cancerous. Much like with B cells and their antibodies, T cells are produced in countless varieties, each version specifically binding to just one molecule (called an antigen). It’s this specific binding that allows a T cell to pick off a cell infected with a particular pathogen (or affected by another problem) while avoiding collateral damage.

Because these varieties are produced randomly, most turn out to be completely useless. But all the T cells hang around, hoping to find a pathogen molecule (antigen) or tumour protein they happen to recognise, like a lock searching for the key that will open it. If a T cell finds its “key”, the T cell is activated and starts dividing to produce identical copies of itself – clones – in a process called clonal expansion. This new army of clones then heads off to target the infected or cancerous cells. And when things go awry with the immune system, T cells can end up targeting a person’s own molecules – “self” antigens – leading to autoimmunity.

Mark Davis’s study was comparing T cell clonal expansion in healthy people with that in several diseases, including multiple sclerosis (an autoimmune disease), and acute Lyme disease (an infection). At a late stage in the project, he won a grant to include samples from ME/CFS patients as well and discovered that clonal expansion in these patients was similar to that seen in these other diseases.

Measuring clonal expansion

But how do you measure clonal expansion? Every cell in a clone shares an identical T cell receptor (TCR) – the lock responsible for recognising the key of a specific molecule on the pathogen or tumour cell. So the process is to laboriously sequence the T cell receptor of every T cell and then count the matches. When a lot of T cells have matching TCRs then this means that there has been clonal expansion.

Mark Davis did this for six ME/CFS patients, and the results are shown in the figure below.

TCR Davis just mecfs

Image taken from a video of Davis’s presentation

The figure takes a little understanding, so here’s what the first pie chart shows. It’s for Patient L3-07. The white area of the chart shows that just over half of the tested cells were unique (no other cells had identical TCRs), indicating no clonal expansion for those cells. The coloured pie slices indicate the scale of clonal expansion. For example, there were two clones with more than 20 copies of the same TCR, as indicated by the two pie slices marked in red. The other coloured slices show the number of copies for clones with fewer copies.

The number underneath the chart shows that this patient’s sample contained 323 T cells.

After examining clonal expansion in ME/CFS, Davis then compared the results for ME/CFS with those for other illnesses. Examples from four patients with multiple sclerosis, ME/CFS, or a recent Lyme infection and from four healthy controls are shown below.

TCR Davis all diseases clearer

Image taken from a video of Davis’s presentation

The contrast between the disease patients – including the CFS patients – and the healthy group is striking. In the healthy individuals, about 90% of T cells are unique, with only a few expanded into clones. The ME/CFS patients look far more like patients with multiple sclerosis or Lyme. This is an astonishing result and, if replicated, could point firmly to the underlying disease process for at least some patients. It could also yield biomarkers.

Mark Davis is already trying to identify the antigen(s) that might be triggering clonal expansion, which could identify self-antigens (autoimmunity) or specific pathogens that have triggered the problem. That could pave the way to developing new treatments.

The new PhD project will use similar but improved technology. Because every cell must be sequenced separately, T cell sequencing is currently very expensive work, which effectively limits how many samples can be processed.

Dr Lia Chappell at Sanger will develop the method further in order to dramatically lower costs, allowing much bigger samples to be analysed, and the technique will be implemented by the PhD student. The goal is to sequence TCRs for 10,000 cells per patient and for many more patients. The initial target is 50 patients and 50 controls but the ultimate ambition is to eventually sequence the T cells for all samples in the UK ME/CFS Biobank, and possibly for other cohorts as well.

How the study came about

Chris Ponting is an old friend and I told him about Mark Davis’s remarkable study when I saw the video about it from last year’s Open Medicine Foundation symposium at Stanford. Chris also thought this work was exciting and important, though I hadn’t expected him to follow up on it with a research project. However, by a stroke of luck, Chris was part of a consortium at the Wellcome Sanger Institute that was looking to find a cheaper way to sequence T cell receptors. The consortium is headed by Höllander with Chappell developing the core technology and both agreed to collaborate with Chris on this new project. Chappell and Höllander are now adapting their technology to suit this new study.

Clonal expansion could be the smoking gun for what’s going wrong in ME/CFS. So if the findings of clonal expansion check out then this work could help to track down the cause of ME/CFS for at least some patients. The new technology being developed could also ultimately lead to an affordable way for doctors to measure clonal expansion, helping with diagnosis.

It’s impressive that Action for ME and the Scottish Government’s Chief Scientist’s Office are funding this important work. Replication is central to establishing robust results, and I can’t think of a more important finding to try to replicate than that of T cell clonal expansion.

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