Objective
The objective of this proposal is to develop a simple, blood test that can be measure autoantigen specific CD4+ T-cells. While is it clear that CD4+ T cells play a dominant role in guiding immune responses, including autoimmune responses, we are currently unable to routinely measure these responses. Here we propose to build upon our success in developing a simple whole blood-based test for responses to proinsulin, which we have shown works well in type 1 diabetes (T1D), to develop a similar test for multiple sclerosis (MS), systemic lupus erythematosus (SLE). We also plan to improve the test for T1D by testing antigens besides proinsulin that are implicated in T1D. Our goal is to identify a set of antigens that we can combine and use in our blood test. By combining several antigens, which we have shown to stimulate CD4+ T-cell responses only in people with the relevant autoimmune disease, we will have a stronger and more robust test. The more antigens that are recognized by autoantigen specific CD4+ T cells, the stronger the overall response and the more robust the assay.
Our proposal comprises three specific aims – one for each autoimmune disease. The first aim is to use our blood test to assess 20 antigens to find those that give a detectable CD4+ T-cell response in people with MS, but not in people who don’t have the disease. The antigens have been chosen because they have been reported to stimulate CD4+ T-cell responses in the blood of people with MS. However, based on our experience in T1D, we expect that some antigens will not stimulate any responses, some will stimulate responses in people with and without MS and only a few will stimulate responses only in people with MS. It is these antigens that stimulate responses only in MS that we will use to build our blood test for MS specific CD4+ T-cell responses.
Our second aim focuses on SLE. We will study blood samples from people with and without SLE. For this we have selected 15 antigens, based on an extensive survey of the published literature. We will use our assay to find the peptides which stimulate CD4+ T-cell responses only in the blood of people with SLE and build a pool of validated peptides for detecting CD4+ T-cell responses associated with SLE.
Our final aim is to expand on our work in T1D. Previously we have found that we can distinguish people with T1D from those without T1D by using our blood test to measure CD4+ T-cell responses to proinsulin. While proinsulin is considered to the principal antigen in T1D, there are several other important antigens. These include hybrid insulin peptides (known as ‘HIPs’) and glutamic acid decarboxylase (GAD), to cite just two examples. Our goal is to be able to measure CD4+ T-cell responses to the full spectrum of autoantigens seen by CD4+ T cells in T1D. This will give us a powerful tool for monitoring changes in the specificity of CD4+ T-cell responses during the development of the disease.
Our overarching goal is to create a common assay platform that can be used to monitor autoantigen specific CD4+ T-cell responses in SLE, MS and T1D. Future work will seek to expand this platform to other immune diseases and further evaluate autoantigen specific CD4+ T-cell responses in these autoimmune diseases.
Background Rationale
CD4+ T cells can be thought of as the “bosses” of the immune response. They control and direct how the immune system responds to infectious agents and cancers. Unfortunately, CD4+ T cells can equally direct an immune response against healthy tissues. When this happens, an autoimmune disease develops. The problem is that despite their central role in autoimmunity, we cannot currently routinely measure autoantigen-specific CD4+ T cells. This means that physicians are blind to the activity of the very cells that drive the disease they are trying to diagnose and treat. Scientists’ efforts are also frustrated: they are unable to answer important fundamental questions about how autoimmune diseases develop and progress in people. This has contributed to the very slow rate of progress in developing new therapies for autoimmune diseases and to continued suffering in the community.
In this application we outline a proposal to develop a simple whole-blood based test that can measure human autoantigen specific CD4+ T-cell responses. The great strength of our test is its simplicity and sensitivity – it is very easy to perform, yet it can detect very rare autoantigen specific CD4+ T-cell responses in a few milliliters of blood. In our view this makes it ideal for use in a clinical setting – unlike any currently available tests.
Other tests have been developed that can detect and measure autoantigen specific CD4+ T-cells responses. PI Mannering developed the CFSE-based proliferation assay for this purpose. However, none of the current tests, including the CFSE assay, are suitable for routine clinical use. This is because they are complex and time consuming to perform, require large volumes of blood and require expert staff to analyze and interpret the data.
The ability to routinely measure autoantigen specific CD4+ T-cell responses will reveal, for the first time, the dynamics of antigen-specific CD4+ T-cell responses in people with, or at risk of developing, autoimmune diseases. The consequences of this will be far-reaching. We anticipate that this will greatly facilitate diagnosis, particularly distinguishing closely related autoimmune diseases. This will in turn arm physicians with more information, empowering them to prescribe better treatments. Our assay will be a valuable tool for clinical trials, as it will allow us, for the first time, to monitor changes in antigen-specific CD4+ T-cell function induced by experimental therapies for autoimmune disease.
Description of Project
Autoimmune diseases develop when the immune system mistakenly attacks healthy parts of the body. Despite much research, we don’t understand how the immune system makes this ‘mistake’. Consequently, autoimmune diseases remain poorly treated. Autoimmune diseases share many immunological features which point towards a central role of a type of immune cell, called a CD4+ (aka helper) T cell. A CD4+ T cell’s job is to ‘manage’ the immune response by guiding the function of many other immune cells. Usually, CD4+ T cells guide our immune systems to protect us from infectious agents and cancer. However, when an autoimmune disease develops it is also the CD4+ T cells that lead the development of the autoimmune disease.
Despite the key role of the CD4+ T cells in guiding immune responses, it has been very difficult to measure them. Broadly speaking, each CD4+ T cell ‘sees’ a different target (which we call an antigen) and can only drive an immune response once they have ‘seen’ that antigen. Since we’re interested in autoimmune diseases, we want to measure only the CD4+ T cells that recognize antigens from healthy tissues (called ‘autoantigens’). We face two challenges: (i) CD4+ T cells that recognize autoantigens are scarce in the blood; and (ii) it’s not always clear which autoantigens are ‘seen’ the by CD4+ T cells that cause an autoimmune disease. While researchers have developed some tests which can detect autoantigen-specific CD4+ T cells, the tests are complex, technically demanding and require large volumes of blood. This makes them unsuitable for routine clinical use. For this reason, current tests focus on measuring antibodies that bind to autoantigens (aka autoantibodies). While autoantibody tests are useful, they do not give any insights into the underlying CD4+ T-cell responses driving autoimmune diseases. We have set out to address this capability gap.
We have developed a simple blood test, focusing for the moment on type 1 diabetes (T1D). This test is sensitive enough to detect the very rare autoantigen specific CD4+ T cells. Importantly, it has many advantages, it is: simple to perform, requires only a few milliliters of blood, logistically robust, and gives a measure of the number and activity of autoantigen-specific CD4+ T cells. Working with blood samples from people with, and without, T1D we have been able to optimize and validate this blood test. We can measure CD4+ T-cell responses to proinsulin, an important target in T1D. Importantly, this blood test can distinguish between people with and without T1D.
Our current goal is to use our new blood test to analyze autoantigen specific CD4+ T-cell responses in three autoimmune diseases: multiple sclerosis (MS), systemic lupus erythematosus (SLE) and T1D. We aim to test a panel of autoantigens implicated in MS and SLE to determine which antigens provoke a CD4+ T-cell responses in people with these diseases. In T1D we will build on our work to examine responses to autoantigens other than proinsulin.
Upon completion of this project, we will deliver a blood test that can measure autoantigen-specific CD4+ T-cell responses in MS, SLE and T1D. This will meet a long unmet need, enabling routine measurement in the clinic of autoantigen specific CD4+ T-cell responses in people with autoimmune diseases. This will allow, for the first time, the analysis of the autoimmune response which underlies the disease process. This will guide physicians in their diagnosis and care of people suffering from autoimmune diseases and be an invaluable tool for scientists seeking to understand autoimmune disease and develop safe and effective therapies to prevent or reverse these diseases.
Anticipated Outcome
Upon completion of this project, we will have demonstrated that our blood test is a suitable biomarker for SLE, MS and T1D. More specifically, we will have defined a short list of antigens that elicit CD4+ T-cell responses in the blood of people with, but not those without, the relevant autoimmune disease.
Our work in T1D is more advanced. We have already shown that components of proinsulin can elicit CD4+ T-cell responses in people with T1D, but not those without T1D. Upon completion of this project, we will have identified a larger panel of antigens that stimulate a CD4+ T-cell response in people with T1D, but not without. From this, we will formulate a mixture of antigens which will form the basis of our blood test to detect CD4+ T-cell responses in the blood of people with or without T1D. In future projects, armed with ourt panel of antigens we will examine when responses to different antigens develop as the autoimmune response that leads to T1D progresses.
Completion of this project will open the door to many important applications of our blood test. Specifically, we will be able to relate the CD4+ T-cell responses to a person’s symptoms. For example, do particular CD4+ T-cell responses occur in people of a certain age, gender or ethnicity? Does it correlate with their responses to therapies or the severity of their disease? Is there a link between their genes and the responses we measure? In SLE and MS, we will seek to identify further autoantigens, since these have not been well studied in these diseases. Identifying other antigens in these diseases will allow us to build a more informative and robust assay, as we are doing for T1D. Hence, we are confident that our blood test will become an essential clinical and research tool which will greatly aid efforts to improve therapies for the autoimmune diseases, SLE, MS and T1D.
Relevance to T1D
This project is directly relevant to T1D as well as SLE and MS. Here we directly address a long-standing and vexing problem – the inability to routinely measure autoantigen specific CD4+ T-cell responses in people.
Our new blood test will facilitate that analysis of CD4+ T-cell responses in all three diseases. However, our work is more advanced in T1D. We have already shown that it works very well for responses to proinsulin. Now we seek to expand the pool of antigens beyond proinsulin. While proinsulin is considered by most in the field to be the most important antigen, we know that CD4+ T-cell responses to several other antigens contribute to destruction of the body’s insulin-producing cells. Adding other antigens will have two important benefits. First, it will make responses easier to detect, because we will be measuring the responses of multiple CD4+ T cells at the same time. Second, it will give us the opportunity to map when, during the development of T1D, responses to different antigens develop. This information will be essential for improved diagnosis of T1D, by allowing more accurate prediction of when an individual will progress to stage 3 T1D. It will also identify antigens which may be used as antigen-specific therapies to prevent or reverse T1D.