Objective
This project comprises three aims. The first aim is to develop and validate a simple blood test that can measure human T cell responses to beta cell antigens using a small (2.0-5.0mL) sample of blood. The second aim is to modify the assay so it can also measure regulatory T-cell responses against beta-cell antigens. It is the job of the regulatory T cells (Treg) to ‘turn off’ unwanted or potentially damaging immune responses. The third aim is to apply this new assay to measuring beta cell antigen specific responses in people without T1D and at the different stages of the progression of type 1 diabetes (stages I to III). Each aim is described in more detail below.
In Aim 1 we will finish optimising the conditions for the assay. Specifically, we’ll confirm the following parameters for the assay: lowest volume of blood required, which antigens (peptides), concentration of antigens, and whether we can pool different antigens together. In addition, we’ll work out how much the results vary when we test the same sample repeatedly and when we test the same individual repeatedly. These are important parameters for the application of the assay.
In Aim 2 we will modify the assay to see if we can detect when the immune system is sending a ‘stop’ message, or what we call a regulatory T-cell response. Regulatory T cells work by suppressing the function of neighbouring T cells. Here we’ll try two approaches (i) to measure cytokines (messenger molecules of the immune system) to see if we can detect those that are sending a ‘stop’ signal. The second approach is to measure the inhibition of responses to antigens that people are exposed to by vaccination or natural infection.
In Aim 3 we’ll use our now standardized assay to measure T cell responses to beta cell antigens in people without T1D and at different stages of type 1 diabetes. That is, people who have islet autoantibodies, but no metabolic impact (stage I), islet autoantibodies and metabolic impairment (stage II) and clinical type 1 diabetes (stage III). This will have two important outcomes. First it will be a further validation and evaluation of the assay, specifically how easy it is to use in a more clinical setting. Second it will allow us to ‘map’, in a cross-sectional manner, how the autoimmune T-cell responses to beta-cell antigens change as diabetes progresses. Finally, we’ll ask our colleagues from different institutions to use the assay. They will take the blood from people with and without T1D, and stimulate it with our antigen mixture. Then they’ll harvest the plasma (the liquid part of blood), and send it to us frozen. We will analyse the plasma, without first knowing the T1D status of the individual, and predict who we believe to have T1D, based on the assay results. This will give us important baseline data for future trials and form the start of a longitudinal study.
Background Rationale
This project addresses the lack of a simple and robust assay to measure the T cells that attack insulin-producing beta cells and drive type 1 diabetes. The lack of such a ‘T-cell assay’ is a major impediment to advancing therapies for treating or preventing T1D. The assays currently available that can detect T cells specific for beta cell antigens in peripheral blood are too complex, require too large sample volumes, are too technically demanding, too expensive, too time consuming and require overly complex informatic data analysis to be useful in a clinical setting.
This project will develop a test for T-cell function that uses a very small volume of whole blood, making the process simple and applicable for clinical use. This test is carried out by incubating a sample of whole blood with antigens from beta cells. This stimulates the rare T cells present in blood that react to beta cell antigens to release messenger chemicals (cytokines), but at very low levels. Measuring these signals is now possible because we now have very sensitive analytical techniques that allow us to routinely detect cytokines at concentrations that were previously undetectable.
We propose methods to distinguish between responses that contribute to autoimmune beta cell destruction (i.e., effector T cell responses, Teff) and those that protect the beta cells (i.e., regulatory or Treg responses). We anticipate that being able to measure both ‘sides’ of the immune responses against beta cells will be very powerful.
By developing this clinically suitable assay, we could more accurately monitor changes in T cell-mediated autoimmunity against the insulin-producing beta cells. We anticipate that this would allow us to predict more accurately when someone who is at-risk of developing T1D (e.g.: someone who has autoantibodies against their beta cells) would become clinically diabetic and require insulin.
Promising new immune therapies are currently being developed that aim to ‘turn off’ the autoimmune response that causes T1D. However, clinical trials to test these emerging therapies are long and complex because we have no direct way of measuring their impact on the beta-cell specific autoimmune T-cell responses. Currently we can only measure beta cell function (by C-peptide levels) and the onset of diabetes. An assay that can directly measure both the quantity (that is, how many) and the quality (that is, what they are doing) of autoimmune T-cells targeting beta cells would let us identify and refine effective disease-altering therapies much more rapidly. The goal of this project is to develop such a test and validate it across groups of people without T1D, at-risk of developing T1D, and those with clinical disease.
Description of Project
Type 1 diabetes (T1D) develops when the immune system’s T cells destroy the insulin producing beta cells found in the pancreas. While we have known for several decades that it is the T-cell arm of the immune system that causes T1D it has not been possible, for technical reasons, to easily measure them. Instead, we have had to rely on measuring another part of the immune system, namely auto-antibodies that target the beta cells. These auto-antibodies indicate that an immune response against the beta cells is underway, but don’t directly cause beta-cell destruction and clinical T1D. Currently, auto-antibodies against different beta cell components are routinely measured and are very useful for identifying people whose immune systems have started attacking their beta cells. Measuring T-cell responses, on the other hand is more difficult. While techniques exist for studying beta cell specific T cells that have been useful research tools, they require too much blood and are too laborious to be suitable for higher through-put applications.
We have developed a blood test that can measure beta cell specific T cell responses. This project has three aims. First, to finish optimising our blood test. Second, to adapt the assay so it can measure regulatory T cell responses that ‘turn off’ the damaging immune responses. Finally, to use this blood test to examine how both effector (damaging in T1D) and regulatory (protective in T1D) T cell responses to beta cells change at the different stages of the development of T1D.
The assay works by ‘showing’ T cells different targets (or antigens) that are known to be recognized by T cells in people with T1D. Here we are focusing on preproinsulin, since we know this is a central target antigen in type 1 diabetes. When a T cell encounters an antigen that it ‘recognizes’ it produces soluble proteins, known as cytokines, that the immune system uses to communicate. Broadly speaking, each cytokine delivers a different message to the other immune cells. We then measure how much of a particular cytokine is produced in response to beta-cell antigens which gives us a readout of the ‘strength’ of the immune response against the antigen of interest.
Our new blood-based assay has several important advantages. First, we will use unmanipulated blood (known as ‘whole blood’) directly from the person. Normally, the immune cells are separated from the red blood cells and other components of blood. Using whole blood makes the assay very quick and simple to set up. The second advantage is that we are using new technologies that allow very low concentrations cytokines to be measured. By bringing together these two innovations we will create a simple, but very sensitive, blood test to easily measure the T cell component of the immune response against beta cells. The final strength of this assay is that we can add different beta-cell antigens, or pools of different antigens. We have already shown that this assay can detect responses in the blood from people with T1D, but rarely in people without T1D.
Upon completion, we will have a simple, robust, and thoroughly validated assay for measuring human beta cell antigen-specific T cell responses. This assay will be a powerful new tool for: (i) documenting the dynamics of antigen specific T-cell responses during the natural history of T1D; (ii) identifying individuals at risk of developing T1D and predicting when they will develop clinical T1D; and (iii) monitoring changes in beta cell antigen-specific T cell responses in clinical trials for emerging antigen-specific therapies for T1D.
Anticipated Outcome
Upon completion, we will have a fully validated T cell assay that will allow us to measure both pathogenic and protective immune responses against beta cell antigens. We will then use this assay to document T cell responses to beta cell antigens at the different stages of type 1 diabetes.
Upon completion of the project we will:
1. Have a thoroughly optimized and validated T cell assay for monitoring pathogenic and protective immune responses against beta cells.
2. Have collected baseline data on the T cell responses to preproinsulin peptides from subjects at all the different stages of T1D, from people without evidence of T1D through to people with long-standing type 1 diabetes.
We anticipate that this assay will be a powerful and versatile tool that will be widely adopted by both researchers, clinical trialists and clinicians who care for people with, or at risk of, T1D.
Relevance to T1D
This work is extremely relevant to T1D. It directly addresses a long-term need within the field – an assay to monitor changes in beta cell specific T cell responses. We anticipate that it will be an essential tool for developing immune therapies to prevent, delay or reverse T1D and research projects that seek to measure autoimmune T cell response against beta cell antigens.
Currently individuals at greater risk of developing T1D can be identified by the presence of antibodies against their beta cells. However, the analysis of these autoantibodies gives little insight into when an individual will develop clinical T1D, and when immune therapies intended to prevent T1D should be administered. We suggest that the whole blood assay developed here will allow people at risk of T1D to be more closely managed and therefore benefit from T1D-preventing immune therapies. This assay will also be an invaluable tool that facilitates the rapid testing of emerging antigen specific therapies intended to restore immune tolerance to beta cell antigens.