Objective
The overarching goal of this proposal is to enhance the tools for diagnosis of adult onset T1D (AO-T1D) through expansion of the repertoire of known autoantibodies characteristic of autoimmune diabetes in general, and of AO-T1D in particular.
Specifically, we will:
(1) Characterize the antibody populations in vicinity to islets and pancreatic lymph nodes of AO-T1D patients, and identify those which are most likely to participate in beta cell destruction. This will be achieved using a novel method for spatial RNA sequencing, which facilitates analysis of B- and T- cell receptor sequences from histological sections; Combined with application of advanced computational tools for BCR sequence analysis. The B cell receptor (BCR) is the protein used by B cells to identify their cognate target, and in its secreted form it is known as “antibody”. Each B cell generates a unique BCR, and upon interaction with its target it proliferates and generates a clone – multiple cells with identical BCRs. By sequencing these clones from islets and lymph nodes of patients, we will obtain the structure of the antibodies associated with the disease.
(2) Reveal the identity of the cognate antigens of these autoantibodies. Knowing the sequence of an antibody does not tell us its target. Rather, we will use the sequence of the antibodies produced in the diabetic pancreas to generate identical antibodies in vitro, and then use the synthetic antibodies to “fish” for the antigen they bind. This will be performed by incubating the antibody with beta cells and analyzing the identity of the proteins it binds using a mass spectrometer.
(3) Determine which of the identified autoantibodies may serve as a marker to improve AO-T1D diagnosis. We will survey blood samples of patients with T1D, AO-T1D and T2D for the presence of autoantibodies against the antigens we discovered, and examine which of the autoantibodies can distinguish the latter two from each other.
In a set of preliminary experiments, we used islets from NOD mice to streamline the proposed pipeline for novel antibody discovery. Our successful discovery of a novel mouse antigen provides a strong proof for our ability to identify novel antigens associated with T1D in general, and with AO-T1D in particular. The success of the proposed study is expected to yield a wider clinical study, to determine the diagnostic value of the autoantibodies we discover, and specifically their contribution to distinction between AO-T1D and T2D.
Background Rationale
Type 1 diabetes (T1D) is often associated with children (hence its past name – “juvenile diabetes”), but it can actually occur at any age. However, in older people its progress is usually slower than at childhood and its symptoms milder. This often leads to its confusion with type 2 diabetes (T2D), which indeed occurs at older age and is much more prevalent. While the major manifestation of both T1D and T2D is disregulation of the body’s consumption of glucose, their underlying causes are different. T1D is caused by an autoimmune attack which leads to specific destruction of insulin producing beta cells in the pancreatic islets of Langerhans, while T2D is a metabolic disorder with little involvement of the immune system. Thus, misdiagnosis can lead to improper medication, and hampers the use of novel immune modulatory drugs which may slow – or even prevent - the progression of the autoimmune disease. At young age T1D diagnosis is assisted by its association with genetic predisposition, familial occurrence and – most effectively – the presence of autoantibodies against peptides (antigens) expressed in the pancreatic islet. Unfortunately, the association of these factors with adult onset T1D (AO-T1D) is weaker and their role as markers – limited. However, occurrence of some autoantibodies in AO-T1D suggests that the antigens targeted at old age differ from those in young patients, and therefore better characterization of the antigens targeted in AO-T1D may yield novel diagnostic tools.
Description of Project
Type 1 diabetes (T1D) is often associated with children (previously named – “juvenile diabetes”), but it actually occurs at any age. However, in older people its progression is usually slower than at childhood and its symptoms milder. This often leads to its confusion with type 2 diabetes (T2D), which indeed occurs mostly at older age and is much more prevalent. While the major manifestation of both T1D and T2D is dysregulation of the body’s consumption of glucose, their underlying causes are different. T1D is caused by an autoimmune attack which leads to specific destruction of insulin producing beta cells in the pancreatic islets of Langerhans, while T2D is a metabolic disorder with little involvement of the immune system. Thus, misdiagnosis can lead to improper medication, and hampers the use of novel immune modulatory drugs which may slow the progression of the autoimmune disease. At young age T1D diagnosis is assisted by its association with genetic predisposition, familial occurrence and – most effectively – the presence of autoantibodies against peptides (antigens) expressed in the pancreatic islet. Unfortunately, the association of these factors to adult onset T1D (AO-T1D) is weaker and their role as markers – limited. However, occurrence of some autoantibodies in AO-T1D suggests that the antigens targeted at old age differ from those in young patients, and therefore better characterization of the antigens targeted in AO-T1D may yield novel diagnostic tools.
The overarching goal of this proposal is to enhance the tools for diagnosis of adult onset T1D (AO-T1D) through expansion of the repertoire of known autoantibodies characteristic of autoimmune diabetes in general, and of AO-T1D in particular. Specifically, we will:
(1) Characterize the antibody populations in vicinity to islets and pancreatic lymph nodes of AO-T1D patients. This will be achieved using state-of-the-art tools for spatial RNA sequencing, which facilitate analysis of B cell receptor (BCR) sequences from histological sections; Combined with advanced computational tools for BCR sequence analysis. BCRs are the proteins used by B cells to identify their cognate target. Each B cell generates a unique BCR, and upon interaction with its target it may proliferate and generate a clone (multiple cells with identical BCRs) or initiate secretion of the BCRs as antibodies. By sequencing these clones from islets and lymph nodes of patients, we will obtain the structure of the antibodies associated with the disease.
(2) Reveal the identity of the cognate antigens of these autoantibodies. Knowing the sequence of an antibody does not tell us its target. Rather, we will use the sequence of the antibodies to generate identical antibodies in vitro, and then use the synthetic antibodies to “fish” for the antigen they bind. This will be performed by incubating the antibody with beta cells and analyzing the identity of the proteins it binds using a mass spectrometer.
(3) Determine which of the identified autoantibodies may serve as a marker to improve AO-T1D diagnosis. We will survey blood samples of patients with T1D, AO-T1D and T2D for the presence of autoantibodies against the antigens we discovered, and examine which of the autoantibodies can distinguish the latter two from each other.
In a set of preliminary experiments, we used islets from NOD mice to streamline the proposed pipeline for novel antibody discovery. Our successful discovery of a novel mouse antigen provides a strong proof for our ability to identify novel antigens associated with T1D in general, and with AO-T1D in particular. The success of the proposed study is expected to yield a wider clinical study to determine the diagnostic value of the autoantibodies we discover, and specifically their contribution to distinction between AO-T1D and T2D.
Anticipated Outcome
The proposed study is expected to yield novel antigens previously unknown to elicit an autoimmune response in T1D in general, and in AO-T1D in particular. A follow up study on a large cohort of patients will determine which of the autoantibodies we discover has a true clinical value in diagnosis of T1D.
Relevance to T1D
The presence of autoantibodies – antibodies that target proteins produced by the person’s own body, is a hallmark of T1D, and their presence in a patient’s blood serves as a powerful diagnostic tool. However in adults, autoantibodies seem to target somewhat different antigens, which limits our ability to rely on them as a tool for AO-T1D diagnosis. Our discovery of novel autoantibodies and their targets is expected to harness the power of this well proven diagnostic tool to the distinction between AO-T1D and T2D, and promise each case its proper medication. Hoping that the autoantibodies we discover prove to be specific to patients suffering from autoimmune diabetes, they may also facilitate population-wide screens to identify people at risk for AO-T1D – just as being done today in juveniles. Overall, our research is expected to open a path to a point where T1D can be as accurately diagnosed in adults as in juveniles.