Molecular determinants of immune tolerance in Graves' disease

Autoimmune thyroid diseases are the most prevalent of the autoimmune conditions, with Graves' disease (GD) affecting close to 2% of women over a lifetime and impacting 7.5 million worldwide. In GD, the immune system generates antibodies that stimulate the thyroid gland leading to excessive thyroid hormone production (hyperthyroidism). Excess thyroid hormone affects multiple systems within the body including the heart, circulation, nervous system, skin and metabolic regulation, causing diverse symptoms such as elevated heart rate, irritability, sweating and weight loss. Thyroid eye disease, a complication of GD, also causes facial disfigurement with protuberance of the eyes, double vision and occasionally loss of vision.

The current treatment for GD has remained largely unchanged since 1951, when the antithyroid drug carbimazole was introduced. Following a 6 month or longer course of carbimazole tablets about 50% of people will have a durable remission of their condition, whereas the other 50% will relapse, which is the rule in younger people. Relapsing patients may ultimately require radio-iodine treatment or surgical intervention to remove the thyroid gland, both leading to lifelong dependence on thyroid hormone replacement, with attendant monitoring costs.

Graves' disease runs strongly in families and 80% of the risk has been shown to be owing to genetic factors. In addition, the exact cause of the thyroid overactivity is known: antibodies that mimic the shape of thyroid stimulating hormone (TSH) which persistently stimulate the TSH-receptor. Given the central role of these anti-TSH receptor antibodies (TRAbs) in causing GD, we need to understand more about why these antibodies are produced by the antibody-producing arm of the immune system (also known as the humoral immune system). Bone marrow-derived B lymphocytes, and their derivative cell types, plasmablasts and plasma cells are responsible for making these TRAb antibodies. My fellowship aims to understand what is different about the humoral immune system in GD patients who sustain a prolonged remission with disappearance of TRAbs following antithyroid drugs, compared to those whose TRAbs persist and who immediately relapse following the same medication.

The study will involve a group of 120 adult GD patients who are about to stop taking the usual antithyroid drug therapy. I will compare many factors between the half who will remain cured of their thyroid disease (remission group) and those whose thyroid overactivity is about to come back (relapse group). Firstly, several blood markers of B lymphocyte production rate and development will be measured, including TRAbs, circulating B cell growth factors (eg. BAFF) and small DNA loops known as KRECs which indicate the rate of B lymphocyte production. Then the quantity of different gene-products in purified B cells from each group will also be examined using the powerful technique of RNA-sequencing. This will allow me to identify a B cell molecular fingerprint that distinguishes people entering disease remission vs those headed for relapse. Lastly, we will look at the genetic variations underlying these differences in gene expression and use this to confirm an inherited (genomic) underpinning for the relapse or remission phenomena. This will provide a catalogue of molecular differences (both RNA and genomic) in B cells between people with sustained remission and those who relapsed, along with functional information about their humoral immune response. This will give novel insights into how the immune system is able to become tolerant again following an autoimmune disease, which will help to advance the development of new treatments. Understanding the factors that determine the response to therapy will also give prognostic information and allow treatment to be tailored to the individual. Many other autoimmune diseases have a relapsing and remitting course, so this work may have widespread relevance.

Autoimmune thyroid diseases are the most prevalent of the autoimmune diseases (AID), with Graves' disease (GD) affecting close to 2% of women over a lifetime. Standard antithyroid drug (ATD) therapy results in remission in 50% of cases, which provides a tractable model to investigate the molecular determinants of restoration of immune tolerance. TSH receptor stimulating antibodies (TRAbs) play a pivotal role in the immunopathogenesis of GD, but the humoral immune response that drives this autoimmune process is poorly understood. My fellowship aims to identify how the humoral immune system in GD patients who sustain a prolonged remission following ATD therapy differs from those who relapse.

The phenotype of remission or relapse one year after treatment will be determined in 120 GD patients who are about to stop thionamide therapy and used to categorise for my analyses, aiming for 50 or more patient samples available per group.

Concentrations of B cell growth factors (BAFF, APRIL, CXCL13) and TRAbs will be measured by ELISA along with KRECs (qPCR) to determine whether plasma cells, the activity of existing B cells, or the generation of new B cells is independently driving the remission/relapse process. RNAseq of CD19+ B cells will allow a high-fidelity survey of cell-specific gene expression and identify those transcripts that are most differentially fold-changed between relapsing and remitting patients. The genomic underpinning of this differential expression will be explored using SNPs known to tag eQTLs in the original RNAseq patient dataset, and confirmed in a wider sample of available GD patient DNAs.

This in depth exploration of B lymphocyte biology in treated GD will allow a molecular understanding of factors determining relapse or remission, will provide important prognostic information to stratify patient therapy and could lead to critical insights into the mechanisms behind long-term restoration of immune tolerance relevant to other AIDs.

Developing a more detailed understanding of the molecular determinants of the clinical course of Graves' disease (GD), and advancing our understanding of individual response to antithyroid drug therapy which ultimately leads to relapse or remission, could potentially lead to treatment stratification, as well as identifying potential targets for innovative therapies.

Health and wellbeing
The individuals who stand to benefit most from this research are young people with GD who are most likely to relapse using standard treatment. An understanding of why some patients respond to therapy and how immune tolerance is generated will help to:

1. Stratify treatment to the individual with a greater understanding of their likely prognosis.
2. Reduce the duration of antithyroid drug exposure and associated adverse events in patients who would not ultimately respond to antithyroid drug therapy.
3. Identify candidate genes to assist in the development of novel therapies, ultimately aiming to stop patients from being hypothyroid in the longer term after stopping antithyroid drugs, which requires lifelong surveillance and thyroid hormone treatment, impacting the patient's quality of life.

NHS and clinical profession
There is the potential for economic benefit for the NHS with reduced need for surgical interventions, radio-iodine therapy and lifelong monitoring in primary care. Other beneficiaries include endocrinologists (nationally and internationally) who manage young people with GD, general practitioners who manage patients on thyroid hormone replacement, and support groups such as the British Thyroid Foundation.

Dissemination
The results of these trials will be profiled at the British Endocrine Society meetings, European Society for Paediatric Endocrinology meetings, the annual American Endocrine Society meeting and the European Thyroid Association meeting. Academic papers will be submitted to high-impact endocrinology and immunology journals. Any research papers would also be reported in The British Thyroid Foundation quarterly newsletter in a digestible lay summary.

Exploitation
We may identify a transcriptomic profile (or a derivative of this) that predicts remission and this may have exploitable clinical potential.