Assays for t-cell responses

Abstract

The present invention relates to methods for detecting and monitoring type 1 diabetes (T1D) in individuals in need thereof, and / or who have been identified as being at risk of T1D. In particular, the present invention relates to methods for detecting and monitoring T1D in individuals in need thereof, the method comprising determining T cell responses in the individual.

Description

10062250371Assays for T-cell responsesField of the invention

[0001] The present invention relates to methods for detecting and monitoring type 1 diabetes (T1D) in individuals in need thereof, and I or who have been identified as being at risk of T1D.Cross reference to earlier application

[0002] This application claims priority to Australian provisional applicationno. 2024903912, the entire content of which is hereby incorporated by reference in its entirety.Background of the invention

[0003] Type 1 diabetes (T1D) is a T-cell-mediated autoimmune disease arising from destruction of pancreatic insulin-producing β-cells. β-cell antigen-specific CD4+ and CD8+ T cells collaborate to mediate β-cell destruction. Despite much effort, there is currently no assay suitable for routine clinic-based monitoring of autoantigen-specific cells, which only occur at low frequency.

[0004] Anti-β-cell autoimmunity is typically detected by measuring β-cell specific autoantibodies, which are believed to reflect an underlying pathogenic autoimmune T-cell response. Currently, autoantibodies to four β-cell proteins are routinely measured: insulin, glutamic acid decarboxylase 65kDa (GAD-65), islet tyrosine phosphatase (IA-2) and zinc Transporter 8 (ZnT8). These are collectively referred to as ‘islet autoantibodies’ (lAAb). While islet autoantibodies are a useful marker for identifying individuals at high risk of developing T1D, they are not believed to have a direct role in the pathogenesis of T1D. They are also poor predictors of when an individual will progress from normal glucose tolerance with multiple islet autoantibodies (Stage 1) to dysglycemia (Stage 2 T1 D) and ultimately insulin dependent, clinical T1 D (Stage 3 T1 D).

[0005] As such, there is a need for new methods of detecting and monitoring T1 D in individuals who have been identified as being at risk of T1D.10062250372

[0006] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0007] In one aspect, the present invention provides a method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof indicates the individual has type 1 diabetes.

[0008] In one aspect, the present invention provides a method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof, to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the polypeptide or pool of polypeptides indicates the individual has type 1 diabetes.10062250373

[0009] In one aspect, the present invention provides a method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof indicates the individual is displaying early signs or symptoms of type 1 diabetes.

[0010] In one aspect, the present invention provides a method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:- contacting a whole blood sample from an individual with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the polypeptide or pool of polypeptides indicates the individual is displaying early signs or symptoms of type 1 diabetes.

[0011] In one aspect, the present invention provides a method of determining whether an individual has or is at risk of type 1 pre-diabetes, the method comprising:10062250374- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof indicates the individual has or is at risk of type 1 pre-diabetes.

[0012] In one aspect, the present invention provides a method of determining whether an individual has or is at risk of type 1 pre-diabetes, the method comprising:- contacting a whole blood sample from an individual with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with polypeptide or pool of polypeptides indicates the individual has or is at risk of type 1 pre-diabetes.

[0013] In another aspect, the present invention provides a method for detecting and / or monitoring effector T cell (Teff) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample;10062250375wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof detects and / or monitors effector T cell (Teff) responses in the individual.

[0014] In another aspect, the present invention provides a method for detecting and / or monitoring effector T cell (Teff) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the polypeptide or pool of polypeptides detects and / or monitors effector T cell (Teff) responses in the individual.

[0015] In another aspect, the present invention provides a method for detecting and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes, the method comprising:- contacting a whole blood sample from the individual at a first time point with at least one T-cell autoantigen or fragment thereof to form a first assay sample; - contacting a whole blood sample from the individual at a second time point with at least one T-cell autoantigen or fragment thereof to form a second assay sample;wherein the second time point is a period of time after the first time point;10062250376- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, a cytokine in the second assay sample compared to the first assay sample indicates progression of type 1 diabetes in the individual.

[0016] In another aspect, the present invention provides a method for detecting and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual at a first time point with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form a first assay sample;- contacting a whole blood sample from an individual at a second time point with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form a second assay sample;wherein the second time point is a period of time after the first time point;- determining the presence of, or amount of, a cytokine in the first and second assay samples;10062250377wherein presence of, or increase in amount of, a cytokine in the second assay sample compared to the first assay sample indicates progression of type 1 diabetes in the individual.

[0017] In one aspect, the present invention provides a method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual has type 1 diabetes.

[0018] In one aspect, the present invention provides a method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides indicates the individual has type 1 diabetes.10062250378

[0019] In one aspect, the present invention provides a method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual does not have type 1 diabetes.

[0020] In one aspect, the present invention provides a method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides indicates the individual does not have type 1 diabetes.

[0021] In one aspect, the present invention provides a method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:10062250379- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual is displaying early signs or symptoms of type 1 diabetes.

[0022] In one aspect, the present invention provides a method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof, to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides indicates the individual is displaying early signs or symptoms of type 1 diabetes.

[0023] In one aspect, the present invention provides a method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample;100622503710wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual is not displaying early signs or symptoms of type 1 diabetes.

[0024] In one aspect, the present invention provides a method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof, to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides indicates the individual is not displaying early signs or symptoms of type 1 diabetes.

[0025] In another aspect, the present invention provides a method of determining whether an individual has or is at risk of type 1 pre-diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual is has or is at risk of type 1 pre-diabetes.100622503711

[0026] In another aspect, the present invention provides a method of determining whether an individual has or is at risk of type 1 pre-diabetes, the method comprising: - contacting a whole blood sample from an individual with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides indicates the individual is has or is at risk of type 1 pre-diabetes.

[0027] In another aspect, the present invention provides a method of determining whether an individual has or is at risk of type 1 pre-diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual does not have or is not at risk of type 1 pre-diabetes.

[0028] In another aspect, the present invention provides a method of determining whether an individual has or is at risk of type 1 pre-diabetes, the method comprising: - contacting a whole blood sample from an individual with a driver antigen and:100622503712(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides indicates the individual does not have or is not at risk of type 1 pre-diabetes.

[0029] In another aspect, the present invention provides a method for detecting and / or monitoring regulatory T cell (Treg) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof detects and / or monitors regulatory T cell (Treg) responses in the individual.

[0030] In another aspect, the present invention provides a method for detecting and / or monitoring regulatory T cell (Treg) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof, or100622503713(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides detects and / or monitors regulatory T cell (Treg) responses in the individual.

[0031] In another aspect, the present invention provides a method for detecting and / or monitoring regulatory T cell (Treg) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof detects and / or monitors regulatory T cell (Treg) responses in the individual.

[0032] In another aspect, the present invention provides a method for detecting and / or monitoring regulatory T cell (Treg) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,100622503714to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and polypeptide or pool of polypeptides detects and / or monitors regulatory T cell (Treg) responses in the individual.

[0033] In any aspect or embodiment, the individual may not have previously been diagnosed with type 1 diabetes.

[0034] In any aspect or embodiment of the disclosure, the driver antigen stimulates production of a cytokine, preferably any cytokine expressed by an effector T cell (such as IL-2, or any other cytokine described herein). In some embodiments, the driver antigen includes an antigen that is used in vaccination. Examples of such antigens include influenza haemagglutinin, influenza neuraminidase, the spike protein of SARS-CoV-2, Diptheria toxoid, tetanus toxoid, inactivated polio virus, hepatitis B antigen, or human papilloma virus antigen. The skilled person is aware that a number of different driver antigens may be used, including multivalent vaccines that include multiple vaccine-derived epitopes, and that the examples provided are not limiting. In one embodiment, the driver antigen may be tetanus toxoid.

[0035] In another aspect, the present invention provides a method for detecting and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual at a first time point with a driver antigen and at least one T-cell autoantigen or fragment thereof to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with a driver antigen and at least one T-cell autoantigen or fragment thereof to form a second assay sample;wherein the second time point is a period of time after the first time point;100622503715- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, a cytokine in the second assay sample compared to the first assay sample indicates progression of type 1 diabetes in the individual.

[0036] Preferably, the at least one T-cell autoantigen or fragment thereof used to contact the whole blood sample at a first time point is the same at least one T-cell autoantigen or fragment thereof used to contact the whole blood sample at the second time point.

[0037] In another aspect, the present invention provides a method for detecting and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual at a first time point with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with a driver antigen:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,100622503716to form a second assay sample;wherein the second time point is a period of time after the first time point;- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, a cytokine in the second assay sample compared to the first assay sample indicates progression of type 1 diabetes in the individual.

[0038] As outlined above, there are methods of the present disclosure which involve where an assay sample is formed by contacting a whole blood sample with at least one T-cell autoantigen or fragment thereof, and aspects of the present disclosure which involve where an assay sample is formed by contacting a whole blood sample with at least one T-cell autoantigen or fragment thereof and a driver antigen. Both methods may be performed (also referred to herein as a “combined assay” or “combined method”) and the outcome of both methods when considered together may be indicative for:- determining whether an individual has type 1 diabetes;- determining whether an individual has type 1 pre- diabetes;determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease;- detecting and / or monitoring effector T cell (Teff) responses in an individual; - detecting and / or monitoring regulatory T cell (Treg) responses in an individual, preferably regulatory CD4+or CD8+T cells;- detecting and / or monitoring and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes; or100622503717- monitoring changes in immune responses associated with type 1 diabetes, for example, changes in immune function induced by therapies for reversing, preventing, or delaying the onset of type 1 diabetes.

[0039] The at least one T-cell autoantigen or fragment thereof that is used in the aspects which involve forming an assay sample by contacting a whole blood sample with at least one T-cell autoantigen or fragment thereof, may differ from the at least one T-cell autoantigen or fragment thereof used in other aspects of the present disclosure which involve forming assay sample by contacting a whole blood sample with at least one T-cell autoantigen or fragment thereof and a driver antigen.

[0040] For example, the combined assay or combined method may determine an individual has type 1 diabetes:- when, in the first method or assay, the presence of, or increase in amount of, the cytokine in the assay sample following contact with a first at least one T-cell autoantigen or fragment thereof indicates the individual has type 1 diabetes; and- when, in the second method or assay, presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and a second at least one T-cell autoantigen or fragment thereof indicates the individual has type 1 diabetes.

[0041] For example, the combined assay or combined method may determine whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease:- when, in the first method or assay, the presence of, or increase in amount of, the cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof indicates the individual displaying early signs or symptoms of the disease; and- when, in the second method or assay, presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual displaying early signs or symptoms of the disease.100622503718

[0042] In any aspect or embodiment, the individual may be, or have been identified as, at risk of developing type 1 diabetes. For example, the individual may have one or more risk factors, including risk factors as described herein. Examples of risk factors include having a family history of type 1 diabetes, such as a parent or sibling with type 1 diabetes, having HLA alleles associated with increased risk of type 1 diabetes, and having evidence of autoimmunity, such as having autoantibodies to beta cell proteins. In one embodiment, the individual has increased genetic risk for type 1 diabetes. For example, the individual may be identified as having a genetic risk score indicating that they are at risk of developing type 1 diabetes.

[0043] In any aspect or embodiment, the individual may be pre-symptomatic or symptomatic for type 1 diabetes.

[0044] In any aspect or embodiment, the individual may have, or have been identified as having, recent-onset type 1 diabetes. For example, the individual may have one or more signs or symptoms of recent-onset type 1 diabetes, including signs or symptoms as described herein.

[0045] In any aspect or embodiment, the method further comprises detecting and / or monitoring beta cell autoimmunity in individuals who may be pre-symptomatic or symptomatic for type 1 diabetes, have recent-onset diabetes, or be at risk of developing type 1 diabetes.

[0046] In any aspect or embodiment, the method further comprises detecting and / or monitoring of the efficacy of therapeutics administered to an individual to prevent or treat type 1 diabetes. For example, the method may comprise comparing the efficacy of a therapeutic administered to an individual with pre-symptomatic type 1 diabetes to prevent progression to symptomatic type 1 diabetes, to the efficacy of a placebo administered to an individual with pre-symptomatic type 1 diabetes. In another example, the method may comprise comparing the efficacy of a therapeutic administered to an individual to prevent or treat pre-clinical type 1 diabetes, to the efficacy of a placebo administered to an individual with pre-clinical type 1 diabetes. In one embodiment, the monitoring of the efficacy of therapeutics informs management of individuals receiving the therapeutics.100622503719

[0047] In another aspect, the present invention provides a method for monitoring autoimmune (T cell) responses in an individual involved in a clinical trial of an immune modulating therapy the prevention or treatment of type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual at a first time point with at least one T-cell autoantigen or fragment thereof to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with at least one T-cell autoantigen or fragment thereof to form a second assay sample; wherein the second time point is a period of time after the first time point;- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, or no difference in the amount of the cytokine in the second assay sample compared to the first assay sample indicates the individual is not responding to the immune modulating therapy; orwherein absence of, or decrease in amount of, a cytokine in the second assay sample compared to the first assay sample indicates the individual is responding to the immune modulating therapy.

[0048] In another aspect, the present invention provides a method for monitoring autoimmune (T cell) responses in an individual involved in a clinical trial of an immune modulating therapy the prevention or treatment of type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual at a first time point with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,100622503720to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form a second assay sample;wherein the second time point is a period of time after the first time point;- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, or no difference in the amount of the cytokine in the second assay sample compared to the first assay sample indicates the individual is not responding to the immune modulating therapy; orwherein absence of, or decrease in amount of, a cytokine in the second assay sample compared to the first assay sample indicates the individual is responding to the immune modulating therapy.

[0049] In another aspect, the present invention provides a method for monitoring the response of an individual to a treatment for type 1 diabetes, the method comprising: - contacting a whole blood sample from an individual at a first time point with a driver antigen and at least one T-cell autoantigen or fragment thereof to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with a driver antigen and at least one T-cell autoantigen or fragment thereof to form a second assay sample;100622503721wherein the second time point is a period of time after the first time point, and the individual has been provided with a treatment for type 1 diabetes after the first time point but before the second time point,- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, or no difference in the amount of the cytokine in the second assay sample compared to the first assay sample indicates the individual is not responding to the treatment for type 1 diabetes, orwherein absence of, or decrease in amount of, the cytokine in the second assay sample compared to the first assay sample indicates the individual is responding to the immune modulating therapy.

[0050] In another aspect, the present invention provides a method for monitoring the response of an individual to a treatment for type 1 diabetes, the method comprising: - contacting a whole blood sample from an individual at a first time point with a driver antigen and:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with a driver antigen:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or100622503722(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin or a fragment thereof,to form a second assay sample;wherein the second time point is a period of time after the first time point, and the individual has been provided with a treatment for type 1 diabetes after the first time point but before the second time point,- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, or no difference in the amount of the cytokine in the second assay sample compared to the first assay sample indicates the individual is not responding to the treatment for type 1 diabetes, orwherein absence of, or decrease in amount of, the cytokine in the second assay sample compared to the first assay sample indicates the individual is responding to the immune modulating therapy.

[0051] In one embodiment, the individual may express the HLA alleles DQ8, DQ2, DR3 and / or DR4. Preferably, the individual expresses the HLA alleles DQ2, DQ8, or DQ2 and DQ8. The DQ8 allele may be HLA-DQA1 *03:01 or HLA-DQB1*03:02. The DQ2 allele may be DQA1*05:01 or DQB1*02:01. The DRB1 allele may be DRB1*04:0X.

[0052] In any aspect or embodiment, the individual is an adult.

[0053] In any aspect or embodiment, the individual is a child.

[0054] In any aspect or embodiment, the individual is male.

[0055] In any aspect or embodiment, the individual is female.

[0056] In any aspect or embodiment, the individual aged from 2 years old to 70 years old, from 2 years old to 60 years old, from 2 years old to 50 years old, from 2 years old to 40 years old, from 2 years old to 30 years old, from 2 years old to 20 years old, from 10 years old to 70 years old, from 10 years old to 60 years old, from 10 years old to 50100622503723years old, from 10 years old to 40 years old, from 10 years old to 30 years old, or from 10 years old to 20 years old. Also contemplated is an individual with an age of, or between any two ages of, 2 years old, 10 years old, 20 years old, 30 years old, 40 years old, 50 years old, 60 years old, 70 years old, or any other age described herein, including in Tables 2 and 3.

[0057] In any aspect or embodiment, the individual aged from about 2 years old to about 70 years old, from about 2 years old to about 60 years old, from about 2 years old to about 50 years old, from about 2 years old to about 40 years old, from about 2 years old to about 30 years old, from about 2 years old to about 20 years old, from about 10 years old to about 70 years old, from about 10 years old to about 60 years old, from about 10 years old to about 50 years old, from about 10 years old to about 40 years old, from about 10 years old to about 30 years old, or from about 10 years old to about 20 years old. Also contemplated is an individual with an age of, or between any two ages of, about 2 years old, about 10 years old, about 20 years old, about 30 years old, about 40 years old, about 50 years old, about 60 years old, about 70 years old, or about any other age described herein, including in Tables 2 and 3.

[0058] In any aspect or embodiment, the individual aged from 2 years old to 20 years old, from 2 years old to 17 years old, from 2 years old to 11 years old, from 2 years old to 8 years old, from 2 years old to 6 years old, from 2 years old to 4 years old, from 4 years old to 20 years old, from 6 years old to 20 years old, from 8 years old to 20 years old, from 11 years old to 20 years old, from 17 years old to 20 years old, from 4 years old to 17 years old, from 6 years old to 17 years old, from 8 years old to 17 years old, from 11 years old to 17 years old, or from 3 years old to 11 years old. Also contemplated is an individual with an age of, or between any two ages of, 2 years old, 3 years old, 4 years old, 6 years old, 8 years old, 11 years old, 17 years old, 20 years old, or any other age described herein, including in Table 3.

[0059] In any aspect or embodiment, the individual aged from about 2 years old to about 20 years old, from about 2 years old to about 17 years old, from about 2 years old to about 11 years old, from about 2 years old to about 8 years old, from about 2 years old to about 6 years old, from about 2 years old to about 4 years old, from about 2 years old to about 3 years old, from about 4 years old to about 20 years old, from about 6 years old to about 20 years old, from about 8 years old to about 20 years old, from100622503724about 11 years old to about 20 years old, from about 17 years old to about 20 years old, from about 4 years old to about 17 years old, from about 6 years old to about 17 years old, from about 8 years old to about 17 years old, from about 11 years old to about 17 years old, or from about 3 years old to about 11 years old. Also contemplated is an individual with an age of, or between any two ages of, about 2 years old, about 3 years old, about 4 years old, about 6 years old, about 8 years old, about 11 years old, about 17 years old, about 20 years old, or any other age described herein, including in Table 3.

[0060] In any aspect or embodiment, the whole blood is peripheral blood. Preferably, the whole blood sample includes an anti-coagulant (e.g. heparin). Preferably the anticoagulant is not EDTA. In one embodiment, the whole blood is heparinized. In one embodiment, the only treatment of the whole blood sample after being obtained from an individual and before being contacted with at least one T-cell autoantigen or fragment thereof or polypeptide or pool of polypeptides in a method of the disclosure is heparinization.

[0061] In any aspect or embodiment, the whole blood is untreated or unpurified.

[0062] In any aspect or embodiment, the whole blood sample is contacted with the peptides or pool of peptides present in a tube or tissue culture plate, for example a polypropylene tube. In one embodiment, the tube is a blood collection tube.

[0063] In any aspect or embodiment, the cytokine is a cytokine that is known to be associated with T1D pathogenesis and / or that is expressed by a T cell in response to a Beta cell antigen. Cytokines that are associated with T1 D pathogenesis and / or that are expressed by a T cell in response to a Beta cell antigen include cytokines that play an anti-inflammatory role such as IL-10, TGF-β, and type 2 cytokines, and cytokines that play a pro-inflammatory role, such as IL-1, IL-6, TNF-α, IFN-α, IL-17, GM-CSF, and IL-21. Cytokines that are associated with T1D pathogenesis and / or that are expressed by a T cell in response to a Beta cell antigen also include cytokines that have pleiotropic effects, such as IL-2, IFN-γ, or IL-15. Cytokines that are associated with T1D pathogenesis and / or that are expressed by a T cell in response to a Beta cell antigen are well known to the skilled person in the art, and are not limited to the examples provided.100622503725

[0064] In any aspect or embodiment, the cytokine is a cytokine associated with T cell responses to an antigen in T1D. In an embodiment, the cytokine is a cytokine associated with effector T cell response. In a preferred embodiment, the effector T cell response is a CD4+ effector T cell response. In another embodiment, the cytokine is a cytokine associated with regulatory T cell response. In another preferred embodiment, the regulatory T cell response is a regulatory CD4+ T cell response or a CD8+ T cell response.

[0065] In any aspect or embodiment, the cytokine is IL-2 or a fragment thereof.

[0066] In any aspect or embodiment, the at least one T-cell autoantigen or fragment thereof comprises a polypeptide that binds to T cells in an individual that has, or is at risk of, type 1 diabetes.

[0067] In any aspect or embodiment, the polypeptide or pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, comprises a polypeptide that binds to T cells in an individual that has, or is at risk of, type 1 diabetes.

[0068] In any aspect or embodiment, the at least one T-cell autoantigen or fragment thereof is any one described herein or any polypeptide described herein, such as in the Examples or Figures.

[0069] In any aspect or embodiment, the at least one T-cell autoantigen or fragment thereof comprises, consists essentially of, or consists of an amino acid sequence as described in Table 1, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0070] In any aspect or embodiment, the at least one T-cell autoantigen or fragment thereof may be a single polypeptide, or part of a single polypeptide, or pool of polypeptides, for example a pool of T-cell autoantigens or fragments thereof. The pool of T-cell autoantigens or fragments thereof may comprise a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof. The pool of T-cell100622503726autoantigens or fragments thereof may also comprise polypeptides that do not comprise, consist essentially of, or consist of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof. Alternatively, the pool of T-cell autoantigens or fragments thereof may comprise combinations of polypeptides that do and do not comprise, consist essentially of, or consist of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof.

[0071] Also contemplated in this disclosure is a pool of T-cell autoantigens or fragments thereof in each instance “at least one T-cell autoantigen or fragment thereof” is mentioned.

[0072] In any embodiment of any aspect which does not involve contacting a whole blood sample with a driver antigen, the polypeptide that binds to T cells stimulates or increases cytokine expression by the T cells. Preferably, the polypeptide that binds to T cells stimulates or increases cytokine expression by CD4+and / or CD8+T cells.

[0073] In any embodiment of any aspect which involves contacting a whole blood sample with a driver antigen, the polypeptide that binds to T cells (i) stimulates or increases, or (ii) downregulates or reduces cytokine expression by the T cells.

[0074] In some embodiments of any aspect, the at least one T-cell autoantigen or fragment thereof comprises:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or (b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin, or a fragment thereof.

[0075] In preferred embodiments of any aspect which does not involve contacting a whole blood sample with a driver antigen, the at least one T-cell autoantigen or fragment thereof comprises:(a) a polypeptide comprising, consisting essentially of, or consisting of a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, or100622503727(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a B-chain, a C-peptide or an A-chain of proinsulin, or a fragment thereof.

[0076] In preferred embodiments of any aspect which involves contacting a whole blood sample with a driver antigen, the at least one T-cell autoantigen or fragment thereof comprises:(a) a polypeptide comprising, consisting essentially of, or consisting of a signal peptide of preproinsulin, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide of proinsulin or a fragment thereof.

[0077] In any aspect or embodiment, the polypeptide or pool of polypeptides comprising, consisting essentially of, or consisting of a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, comprises, consists essentially of, or consists of a signal peptide, a B-chain, a C-peptide or an A-chain of human preproinsulin, or a fragment thereof.

[0078] In any aspect or embodiment, the signal peptide consists essentially of or consists of the full length signal peptide of human preproinsulin. In one embodiment, the signal peptide consists essentially of or consists of an amino acid sequence of SEQ ID NO: 18.

[0079] In any aspect or embodiment, the B-chain consists essentially of or consists of the full length B-chain of human preproinsulin. In one embodiment, the B-chain consists essentially of or consists of an amino acid sequence of SEQ ID NO: 19. In another embodiment, the B-chain consists essentially of or consists of an amino acid sequence of SEQ ID NO: 38.

[0080] In any aspect or embodiment, the C-chain consists essentially of or consists of the full length C-chain of human preproinsulin. In one embodiment, the C-chain consists essentially of or consists of an amino acid sequence of SEQ ID NO: 20.

[0081] In any aspect or embodiment, the A-chain consists essentially of or consists of the full length A-chain of human preproinsulin. In one embodiment, the A-chain consists essentially of or consists of an amino acid sequence of SEQ ID NO: 21. In another100622503728embodiment, the A-chain includes an isoacyl bond between Thr8 and Ser9. In another embodiment, the A-chain does not include an isoacyl bond between Thr8 and Ser9.

[0082] In any aspect or embodiment, the signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0083] In any aspect or embodiment, the B-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 19 or 38, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0084] In any aspect or embodiment, the C-peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0085] In any aspect or embodiment, the A-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 21, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0086] In any aspect or embodiment, the fragment of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin as described herein may be at least 14, at least 15, at least 16, at least 17 or at least 18 amino acids in length, or may be 14, 15, 16, 17 or 18 amino acids in length.100622503729

[0087] In any aspect or embodiment, the fragment of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin as described herein may be at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9 amino acids in length, or may be 4, 5, 6, 7, or 8, or 9 amino acids in length.

[0088] In any aspect or embodiment, the fragment of a signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ I D NO: 1, 2 or 3, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0089] In any aspect or embodiment, the fragment of a signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 26, 21, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the fragment of a signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 30 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0090] In any aspect or embodiment, the fragment of a B-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8 9, or 52, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the fragment of a B-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 52 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least10062250373088%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0091] In any aspect or embodiment, the fragment of a C-peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 9, 10, 11, 12, 13 or 14, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0092] In any aspect or embodiment, the fragment of a A-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 14, 15, 16 or 17, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0093] In other embodiments of any aspect which does not involve contacting a whole blood sample with a driver antigen, the at least one T-cell autoantigen or fragment thereof comprises:(a) a polypeptide comprising, consisting essentially of, or consisting of a hybrid insulin peptide, non-insulin β cell antigen, or other T-cell autoantigen, or a fragment thereof, or(b) a pool of polypeptides comprising, consisting essentially of, or consisting of a hybrid insulin peptide, non-insulin β cell antigen, or other T-cell autoantigen or a fragment thereof.

[0094] ln preferred embodiments, the other T-cell autoantigen is the X-ID peptide. More preferably, the X-ID peptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%100622503731identical thereto. Most preferably, the X-ID peptide comprises or consists of the amino acid sequence of SEQ ID NO: 48.

[0095] In some embodiments, the hybrid insulin peptide, non-insulin beta cell antigen, or other T-cell autoantigen or fragment thereof comprises, consists essentially of, hybrid insulin peptides (HIP) 7 (HIP-7), HIP-8, HIP-13, Chromogranin-A (CHGA), Glutamic acid decarboxylase 65-kilodalton isoform (GAD65), Insulinoma-associated protein 2 (I-A2), Islet amyloid polypeptide (IAPP), Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), Zinc transporter 8 (ZnT8), or a fragment thereof.

[0096] In some embodiments, the CHGA, GAD65, I-A2, IAPP, IGRP, orZnT8, consists essentially of or consists of the full length human CHGA, GAD65, I-A2, IAPP, IGRP, orZnT8. In one embodiment, the CHGA, GAD65, I-A2, IAPP, IGRP, orZnT8 consists essentially of or consists of an amino acid sequence of any one of SEQ I D NOs: 53 to 58.

[0097] In some embodiments, the fragment of CHGA, GAD65, I-A2, IAPP, IGRP, or ZnT8 thereof comprises, consists essentially of, or consists of CHGA342-355, GAD121-140, GAD274-286, GAD555-567, I-A2318-342, I-A2752-775, IAPP5-13, IGRP228-236, GAD-65114-123, GAD113-132, GAD115-127, GAD265-284, GAD335-352, GAD521-535, I-A2198-216, I-A2293-311, I-A2449-488, I-A2523-536, I-A2545-562, I-A2654-674, I-A2709-736, I-A2854-872, I-A2955-975, IAPP65-84, IGRP23-35, IGRP226 -238, IGRP247 -259, ZnT81-27, ZnT8106-132, ZnT8120-146, ZnT8211-237, ZnT8244-258, ZnT8266-285, ZnT8267-293, ZnT8309-335, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the fragment of CHGA, GAD65, I-A2, IAPP, IGRP, orZnT8 thereof comprises, consists essentially of, or consists of CHGA342-355, GAD121-140, GAD274-286, GAD555-567, I-A2318-342, I-A2752-775, IAPP5-13, IGRP228-236, GAD-65114-123.

[0098] In some embodiments, the fragment of CHGA, GAD65, I-A2, IAPP, IGRP, or ZnT8 thereof comprises, consists essentially of, or consists of an amino acid sequence as described in Tables 7, 9, or 9.100622503732

[0099] In some embodiments, the hybrid insulin peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 39, 40, or 41, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0100] In some embodiments, the CHGA, GAD65, I-A2, IAPP, IGRP, ZnT8, or fragment thereof comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NOs: 53 to 58, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0101] In some embodiments, the fragment of CHGA comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 42, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0102] In some embodiments, the fragment of GAD65 comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 43 to 45, 51, or 59 to 63, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the fragment of GAD65 comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 43 to 45, 51, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.100622503733

[0103] In some embodiments, the fragment of I-A2 comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 46, 47, or 64 to 72, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the fragment of I-A2 comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 46, 47, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0104] In some embodiments, the fragment of IAPP comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 49, or 73, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the fragment of IAPP comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 49, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0105] In some embodiments, the fragment of IGRP comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 50, or 74 to 76, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the fragment of IGRP comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 50, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%,100622503734at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. In some embodiments, the fragment of ZnT8 comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 77 to 84, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0106] In any aspect or embodiment, the pool of polypeptides is two or more polypeptides or fragments thereof described herein. For example, the pool of polypeptides may be two or more of any polypeptide that comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NOs: 1 to 21 or 26 to 37. The pool of polypeptides may two or more polypeptides that comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NOs: 1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; 11 and 12; 13 and 14; 15 and 16, or 18 and 17. The pool of peptides may also be two or more polypeptides that comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NOs: 26 and 27; 28 and 29; 30 and 31; 32 and 33; 34 and 35; or 36 and 37.

[0107] In any aspect or embodiment, the pool of polypeptides may also comprise two or more polypeptides or fragments thereof of different peptides and proteins. For example, a pool may comprise, consist essentially of, or consist of any one of SEQ ID NOs: 20, 38, 39-41, 42-47, and 48 as described in Table 8. The pool may alternatively comprise, consist essentially of, or consist of any one of SEQ ID NOs: 29, 35, 36, 49, 50, 51, and 52 as described in Table 9.

[0108] In any aspect or embodiment of the invention where the whole blood sample is contacted with at least one T-cell autoantigen or fragment thereof and not a driver antigen, the change (e.g. increase or decrease) or lack thereof in amount of a cytokine may be measured in change in concentration and / or fold change in comparison to a whole blood sample not contacted with at least one T-cell autoantigen or fragment thereof. This sample may also be referred to herein as a control sample. Preferably, the whole blood sample, i.e. control sample, not contacted with at least one T-cell100622503735autoantigen or fragment thereof is derived from the same individual or from the same sample from the same individual to the whole blood sample which is contacted with at least one T-cell autoantigen or fragment thereof.

[0109] In any aspect or embodiment, the change in amount of a cytokine may be a change relative to the amount of a cytokine present in a whole blood sample where the whole blood sample has not been contacted with at least one T-cell autoantigen or fragment thereof as described herein (i.e. a control sample). Therefore, the method may further comprise a step of determining the amount of a cytokine in the whole blood sample (in this instance the sample is a control sample) and comparing the determined amount of the cytokine in the whole blood with the determined amount of the cytokine in the assay sample (e.g. may be referred to as a test sample). Typically, the whole blood sample is from the same individual as the whole blood sample used to form the assay sample.

[0110] In any aspect or embodiment of the invention where the whole blood sample is contacted with at least one T-cell autoantigen or fragment thereof and not a driver antigen, the change (e.g. increase or decrease) or lack thereof in amount of a cytokine may be measured in change in concentration and / or fold change in comparison to a reference value. Preferably, the reference value is derived from a whole blood sample not contacted with at least one T-cell autoantigen or fragment thereof from the same individual or from the same sample from the same individual to the whole blood sample which is contacted with at least one T-cell autoantigen or fragment thereof. Preferably, a reference value is derived from whole blood samples contacted with at least one T-cell autoantigen or fragment thereof from a population of individuals not known to have type 1 diabetes or not at risk of developing type 1 diabetes. Alternatively, a reference value is derived from whole blood samples contacted with at least one T-cell autoantigen or fragment thereof from a population of individuals known to have type 1 diabetes or at risk of developing type 1 diabetes.

[0111] In any aspect or embodiment, the change in amount of a cytokine may be a change relative to the amount of a cytokine present to a reference value. Therefore, in any method of the disclosure, the method further comprises a step of comparing the determined amount of the cytokine in the whole blood with the reference value.100622503736

[0112] In any aspect or embodiment of the invention where the whole blood sample is contacted with at least one T-cell autoantigen or fragment thereof and a driver antigen, the change (e.g. increase or decrease) or lack thereof in amount of a cytokine may be measured in fold change in comparison to a whole blood sample contacted with the driver antigen but not contacted with at least one T-cell autoantigen or fragment thereof. This sample may also be referred to herein as a control sample. Preferably, the whole blood sample not contacted with at least one T-cell autoantigen or fragment thereof is derived from the same individual or from the same sample from the same individual to the whole blood sample which is contacted with at least one T-cell autoantigen or fragment thereof.

[0113] In any aspect or embodiment, the increase in amount of a cytokine is an increase relative to the amount of a cytokine present in a whole blood sample contacted with the driver antigen but not contacted with at least one T-cell autoantigen or fragment thereof as described herein. Therefore, the method may further comprise a step of determining the amount of a cytokine in the whole blood sample, and comparing the determined amount of the cytokine in the whole blood with the determined amount of the cytokine in the assay sample. Typically, the whole blood sample is from the same individual as the whole blood sample used to form the assay sample.

[0114] In any aspect or embodiment of the invention where the whole blood sample is contacted with at least one T-cell autoantigen or fragment thereof and a driver antigen, the change (e.g. increase or decrease) or lack thereof in amount of a cytokine may be measured in fold change in comparison a reference value. Preferably, this reference value is derived from a whole blood sample not contacted with at least one T-cell autoantigen or fragment thereof but contacted with a driver antigen and is derived from the same individual or from the same sample from the same individual to the whole blood sample which is contacted with at least one T-cell autoantigen or fragment thereof. Preferably, this reference value is derived from whole blood samples contacted with a driver antigen and at least one T-cell autoantigen or fragment thereof from a population of individuals not known to have type 1 diabetes or not at risk of developing type 1 diabetes. Alternatively, a reference value is derived from whole blood samples contacted with a driver antigen and at least one T-cell autoantigen or fragment thereof100622503737from a population of individuals known to have type 1 diabetes or at risk of developing type 1 diabetes.

[0115] In any aspect or embodiment, the increase in amount of a cytokine is an increase relative to the amount of a cytokine present in a whole blood sample where the whole blood sample has not been contacted with at least one T-cell autoantigen or fragment thereof as described herein. Therefore, in any method of the disclosure, the method further comprises a step of determining the amount of a cytokine in the whole blood sample, and comparing the determined amount of the cytokine in the whole blood with the determined amount of the cytokine in the assay sample. Typically, the whole blood sample is from the same individual as the whole blood sample used to form the assay sample.

[0116] In any aspect or embodiment, the driver antigen used to generate a control sample or reference value is the same driver antigen used contact the whole blood sample from the individual in which type 1 diabetes ora symptom of type 1 diabetes is to be determined.

[0117] In any aspect or embodiment, the volume of the whole blood sample contacted with at least one T-cell autoantigen or fragment thereof is greater than 0.2 ml but equal to or less than 60 ml; greater than 0.2 ml but equal to or less than 50 ml; greater than 0.2 ml but equal to or less than 40 ml; greater than 0.2 ml but equal to or less than 30 ml; greater than 0.2 ml but equal to or less than 20 ml; greater than 0.2 ml but equal to or less than 10 ml; greater than 0.2 ml but equal to or less than 5 ml; greater than 0.2 ml but equal to or less than 2 ml; greater than 0.2 ml but equal to or less than 1 ml; equal to or greater than 0.5 ml but less than or equal to 5 ml; equal to or greater than 0.5 ml but less than or equal to 2 ml; or equal to or greater than 0.5 ml but less than or equal to 1 ml.

[0118] In any aspect or embodiment, the volume of the whole blood sample contacted with at least one T-cell autoantigen or fragment thereof is greater than 0.2 ml but equal to or less than about 60 ml; greater than 0.2 ml but equal to or less than about 50 ml; greater than 0.2 ml but equal to or less than about 40 ml; greater than 0.2 ml but equal to or less than about 30 ml; greater than 0.2 ml but equal to or less than about 20 ml; greater than 0.2 ml but equal to or less than about 10 ml; greater than 0.2 ml but100622503738equal to or less than about 5 ml; greater than 0.2 ml but equal to or less than about 2 ml; greater than 0.2 ml but equal to or less than about 1 ml; equal to or greater than about 0.5 ml but less than or equal to about 5 ml; equal to or greater than about 0.5 ml but less than or equal to about 2 ml; or equal to or greater than about 0.5 ml but less than or equal to about 1 ml.

[0119] In any aspect or embodiment, the volume of the whole blood sample contacted with at least one T-cell autoantigen or fragment thereof is 0.5 ml or 1 ml for each T-cell autoantigen or fragment thereof.

[0120] In one embodiment, the cells in the whole blood sample are not labelled, for example with a detectable label.

[0121] In any aspect or embodiment, the cells in the whole blood sample are not labelled with 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE).

[0122] In any aspect or embodiment, the whole blood sample and at least one T-cell autoantigen or fragment thereof are incubated for a period of time before determining the presence of, or amount of, a cytokine in the reaction mixture. The time period is sufficient to allow production of cytokines. Typically, the period of time is 8 hours; about 8 hours; greater than or equal to 8 hours but less than 7 days; 24 hours; about 24 hours; greater than or equal to 24 hours but less than 7 days.

[0123] In some aspects or embodiments, the whole blood sample, and driver antigen and / or at least one T-cell autoantigen or fragment thereof are incubated for a period of time before determining the presence of, or amount of, a cytokine in the reaction mixture. The time period is sufficient to allow production of cytokines. Typically, the period of time is 8 hours; about 8 hours; greater than or equal to 8 hours but less than 7 days; 24 hours; about 24 hours; greater than or equal to 24 hours but less than 7 days.

[0124] In any aspect or embodiment, the concentration of the at least one T-cell autoantigen or fragment thereof contacted with the blood sample is greater than or equal to 1 μM, greater than or equal to 2 μM, greater than or equal to 10 μM, greater than or equal to 1.0 μM, greater than or equal to 2.0 μM, greater than or equal to 10.0 μM, greater than or equal to 1 μM but less than or equal to 10 μM, greater than or equal100622503739to 1 μM but less than or equal to 2 μM, greater than or equal to 1.0 μM but less than or equal to 10.0 μM, or greater than or equal to 1.0 μM but less than or equal to 2.0 μM.

[0125] In some aspects or embodiments, the concentration of the driver antigen contacted with the blood sample is 0.01 - 300 pg / ml. Preferably, the concentration of the tetanus toxoid is 0.33 Lfu / ml (72 pg / ml).

[0126] In any aspect or embodiment, the presence of, or increase in amount of, a cytokine in the assay sample is determined by a method for analysing and detecting cytokines. Numerous methods for analysing and detecting cytokines are well known to the skilled person in the art, and include flow cytometry, quantitative real-time PCR (qPCR), enzyme linked immunosorbent assays (ELISA), multiplex arrays, or immunoassays, including electrochemiluminescence (ECL)-based immunoassays. The examples provided are not limiting, and the skilled person knows that many methods that can analyse and detect cytokines in assay samples may be used.

[0127] In a preferred embodiment, the presence of, or increase in amount of, a cytokine in the assay sample is determined by electrochemiluminescence (ECL)-based immunoassay.

[0128] In another aspect, the present invention provides a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 38.

[0129] In another aspect, the present invention provides a nucleic acid encoding a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 38.

[0130] In another aspect, the methods described herein can be used to identify peptides or epitopes which are recognised by T cell receptors present on T cells, i.e. T regulatory cells. These peptides can then be used to generate engineered T cell receptors such as chimeric antigen receptors (CARs) or used to identify endogenous T cell receptors from normal or diseased individuals that binds to such peptides.

[0131] In another aspect, the present invention provides a method of identifying if a candidate T-cell autoantigen or fragment thereof stimulates T cells, the method comprising:100622503740- contacting a sample comprising T cells with a driver antigen and the candidate T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and a candidate T-cell autoantigen or fragment thereof indicates the T cells have been stimulated.

[0132] In another aspect, the present invention provides a method of identifying if a candidate T-cell autoantigen or fragment thereof is recognised by T regulatory cells, the method comprising:- contacting a sample comprising T regulatory cells with a driver antigen and candidate T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and the candidate T-cell autoantigen or fragment thereof indicates the T cells recognised the candidate T-cell autoantigen or fragment thereof.

[0133] In some embodiments, the sample comprising T cells is a whole blood sample or a sample derived from whole blood. In some embodiments, the sample comprising T cells is a peripheral blood mononuclear cell (PBMC) sample, or a sample derived from PBMC. Preferably, the sample comprising T cells is obtained from an individual with an autoimmune disease, for example type 1 diabetes. Preferably, the sample comprising T cells comprises regulatory T cells.

[0134] In any aspect or embodiment of the invention where a sample comprising T cells is contacted with a candidate T-cell autoantigen or fragment thereof and a driver antigen, the change (e.g. increase or decrease) or lack thereof in amount of a cytokine may be measured in fold change in comparison a reference value. Preferably, this reference value is derived from a sample comprising T cells that is not contacted with a candidate T-cell autoantigen or fragment thereof but contacted with a driver antigen and is derived from the same individual or from the same sample from the same individual to100622503741the sample comprising T cells which is contacted with a candidate T-cell autoantigen or fragment thereof. Preferably, this reference value is derived from a sample comprising T cells contacted with a driver antigen and a candidate T-cell autoantigen or fragment thereof from a population of individuals not known to have autoimmune disease or not at risk of developing autoimmune disease. Alternatively, a reference value is derived from a sample comprising T cells contacted with a driver antigen and a candidate T-cell autoantigen or fragment thereof from a population of individuals known to have autoimmune disease or at risk of developing autoimmune disease.

[0135] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0136] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0137] Figure 1: IL-2 secretion in response to C-peptide: a comparison of BASTA and the CFSE-based proliferation assay. (A) Scheme for sampling peripheral blood from subjects with new-onset T1 D and non-diabetics for either PBMC isolation for CFSE-based proliferation assay, or direct stimulation with peptide and high sensitivity measurement of IL-2 in plasma. (B) Cell Division Index (CDI), derived from the CFSE-based proliferation assay, measuring 7-day proliferation of CD4+T cells in response to full-length C-peptide in PBMC samples processed in parallel from 18 donors with newly diagnosed T1D (less than 3 months prior to assay) or 8 HLA-matched (HLA-DR4-DQ8, or HLA-DR3-DQ2) non-diabetic subjects with 10 pM C-peptide, represented as foldchange to background proliferation levels in untreated PBMCs. Dotted line represents 3-fold over background. (C) Receiver operator characteristic (ROC) curve comparing the ability of (B) to distinguish samples from donors with new-onset T1 D from those without, determined via the area under the Receiver Operating Characteristic (AUROC). (D) IL-2 levels in plasma after 24 h incubation of whole-blood samples from the same participants and blood draw as (A), as a fold-change relative to untreated blood. (E)100622503742Receiver Operating Characteristic (ROC) and AUROC calculated from data in (D). (F) Direct paired comparison of C-peptide-induced IL-2 release versus CDI for newly diagnosed diabetic subject, or (G) subjects without type 1 diabetes. Medians are shown for each condition.

[0138] Figure 2: CFSE assay and cytokine detection (A) Example FACS plots showing gating and CFSE dye-dilution CD4+ T cell proliferation in untreated versus C-peptide- or tetanus toxoid (TT)-stimulated PBMC samples from one subject with newly diagnosed T1D (>3 months). (B) Multiplex Cytokine data for whole blood cytokine secretion (IL-2, IFN-γ, TNF, IL-6, IL-10, IL-13) in blood cultured without treatment, or in response to C-peptide, TT, or pooled positive control peptide epitopes CEFX. (C) Cytokine data from (B) for C-peptide and TT-induced cytokines represented as a foldchange to levels in unstimulated blood. (D) Comparison between C-peptide induced fold-change increase in IL-2 and IFN-γ for individual T1D donors.

[0139] Figure 3: T1D-association of IL-2 responses to peptides across preproinsulin. (A) 17 overlapping 14-18mer synthetic peptides from regions across the length of preproinsulin (PPI) were combined into 8 pools. Heatmap: IL-2 responses from whole blood of 9 non-diabetic subjects compared to 18 subjects with established T1 D stimulated with 8 peptide pools corresponding to preproinsulin regions across Signal Peptide (green), B-chain (blue), C-peptide (black) or A-chain (red)(junctions in grey not included). (B) Comparison of IL-2 responses to peptide pools in (A) to those induced by individual full-length peptides corresponding to the same preproinsulin region (Signal peptide, B-chain, C-peptide, or A-chain) by stimulated whole blood samples from 6 T1D subjects across a range of time points since diagnosis. (C) Responses to four individual full-length peptides from the four regions of preproinsulin from 19 non-diabetic subjects or 33 diabetic subjects across a range of time points since diagnosis. (D) Insulin B-chain was synthesised either containing the native sequence or with both Cysteine residues substituted with Serine (red). (E) IL-2 responses using BASTA to B-chain (native) or with C to S substitution were compared in 6 non-T 1 D subjects and 6 subjects with established T1D. (F) Differential responses in IL-2 fold-change induced by native or substituted B-chain were compared per donor. (G) Expanded validation cohort showing utility of BASTA with B-chain with C-to-S for discriminating non-T 1 D subjects (n = 13)100622503743from T1D subjects (n = 17). (H) ROC curve for data in (G). Dotted line represents 3-fold over background. Horizontal lines depict medians.

[0140] Figure 4: Assay optimization Comparing effect of: (A) Decreasing peptide concentration for full-length PPI Signal peptide, B-chain, and A-chain as stimuli for whole blood IL-2 release. (B) Peptide format: Cysteine- to-serine substitutions for A-chain (versus pool of native 14-18mers or full-length native A-chain synthesized as isoacyl form) and B-chain of insulin. (C) IL-2 responses from whole blood at 3 culture volumes (either 1 ml, 500 pl, or 250 pl) for each of the four PPI regions for 3 donors with established T1D. (D) IL-2 signal strength further compared as relative to 1 ml volume for samples in (C). (E) Blood cultures performed in triplicate comparing variance (%CV) for 500 pl and 250 pl culture volumes, using fold-change over %CV at 500 pl to compare samples and stimuli, (n = 8). (F) A combination of all four full-length peptide regions of PPI were tested as a single stimulus, and Cryovials versus 5 ml round bottom “Kahn” tubes as culture vessel, with blood from 3 subjects with established T1 D assayed in triplicate, and (G) %CV per donor compared between vessel type. Dotted line depicts assay cut-off of 3.

[0141] Figure 5: Optimized assay format reveals stability of disease-specific CD4+ T-cell responses to preproinsulin. (A) Responses to a pool of peptides from all four regions of preproinsulin as measured by IL-2 released in whole blood samples from non-diabetic subjects or diabetic subjects across a range of time points since diagnosis. (B) Receiver Operating Characteristic (ROC) curve for prediction of T1D status based on levels of IL-2 as measured in (A), area under the curve (AUC): 0.93. Dotted line represents 3-fold over background. Horizontal lines depict medians.

[0142] Figure 6: Preliminary assay validation (A) Responses to C-peptide alone in same cohort as 5A, with (B) corresponding ROC curve for C-peptide-induced IL-2 values. Dotted line depicts assay cut-off of 3.

[0143] Figure 7: Robustness of preproinsulin-induced IL-2 responses from whole blood across stimulation replicates and stability across multiple longitudinal measurements. (A) Assessment of intra-assay variability between duplicate stimulation tubes (black versus blue) from the same blood draw, and duplicate IL-2 measurements of plasma (individual dots). Right: Comparison of %CV as variance between stimulation100622503744replicates (blood tubes) versus IL-2 measurements from the same plasma (MSD plate wells). (B) Repeat measures of IL-2 responses to individual or pooled peptides from all regions of preproinsulin (Signal peptide, B-chain, C-peptide, A-chain, or all four together), or tetanus toxoid, from 4 subjects with established T1D (T1D#1-4), measured in triplicate on four occasions with intervals of 1-3 weeks between blood draws.

[0144] Figure 8: Inter- and intra-assay variability (A) Responses to individual full-length peptides from the four PPI regions alongside PPI and TT responses as per Fig.4B, measured in triplicate as shown by individual dots, connecting lines show means. (B) %CV calculated between triplicates for each stimulus at each visit in (A). (C) %CV of mean IL-2 across visits as a measure of inter-assay repeatability for (C), as well as concurrent responses to individual peptides. Lines at median are shown.

[0145] Figure 9: Antigen-reactivated CD4+ T cells are the major IL-2 secreting population in whole blood. (A) The effect of HLA-DP, -DQ, -DR, or a combination of all three, blocking mAbs on PPI-induced IL-2 release relative to PPI-stimulated blood alone (bars show median, n = 4 x T1 D). (B) FACS plots and proportion of IL-2+ cells as detected by IL-2 capture assay in CD4+ T cells isolated from untreated, PPI- or tetanus toxoid-stimulated whole blood following magnetic enrichment of IL-2 secretors. (C) Correlation between secreted IL-2 as detected via MSD immunoassay in plasma compared to the percentage CD4+, IL-2+ detected by the IL-2 capture assay. (D) Proportions of Tnaive (CD45RO-, CCR7+), TCM (CD45RO+, CCR7+) and Teff / em (CD45RO+, CCR7-) within the IL-2+ versus IL-2-, CD4+ T-cells in a PPI-stimulated whole blood assay as detected by capture assay. (E) Representative flow cytometry histograms and proportion of CD69+ cells within IL-2- and IL-2+ CD4+ populations as in D. Lines at median are shown. B-D show data from 5 individual subjects with T1D.

[0146] Figure 10: Analysis of the IL-2 producing cells (A) Effect on IL-2 release of blocking antigen presentation via HLA-A, -B, and -C using the monoclonal antibody, W6 / 32 pan -anti-HLA-class-l mAb (2 pg / ml) in PPI-stimulated whole blood, relative to PPI-stimulated blood alone. (B) mAb inhibition of TT-induced IL-2 as per7A. (C) Representative FACS gating for live CD3+CD4+ T cells and phenotyping using CD45RO and CCR7 for 4B-4D. n = 3-5 per condition, bars show median.100622503745

[0147] Figure 11: Cross-sectional analysis of IL-2 responses to combined and individual PPI-peptides in non-diabetics, or subjects with T1D using the optimized BASTA. (A) Individuals were categorized as either non-T 1 D (participants without T1 D and no family history (“low risk”), or AAB- and AAB+ pediatric subjects with a first-degree relative with T1 D), or T1 D, being clinically diagnosed with stage 3 T1 D within 100 days (New-Onset) or more than 100 days (Established T1D) prior to sampling. (B) Comparison of IL-2 release in response to the preproinsulin peptide pool from non-T1D and T1D samples (red circles = New-Onset T1D, filled circles = established T1D). (C) ROC and AUROC calculated from data in (B), to distinguish non-T1D from all T1D samples, with (D) comparing non-T1D to only New-Onset T1D. (E) IL-2 responses to C-peptide, with corresponding ROC and AUROC in (F) using C-peptide responses non-T1 D subjects from all with T1 D or (G) comparing only New-Onset T1 D to non-T 1 D. Dotted line represents 3-fold over background. Lines at median are shown.

[0148] Figure 12: Subgroup analysis of IL-2 responses to PPI peptides in pediatric cohort. IL-2 responses to individual or pooled full-length preproinsulin peptides stratified into five subgroups as described in Fig. 6A: low-risk, AAB-, AAB+, New-Onset T1 D, or established T1D, in response to: (A) Preproinsulin pool, (B) Signal peptide, (C) B-chain, (D) C-peptide (red circles = multi-AAB+ individuals), (E) A-chain. Dotted line represents 3-fold over background. Lines at median are shown.

[0149] Figure 13: IL-10 production, HLA and AAB stratification (A) IL-10 responses from lAAb- and IAAb+ at-risk pediatric donors from 9A, and subset of participants with New-Onset T1D. (B) Stratification of IL-2 responses in 9C according to H LA-type relative to high-risk HLA-class II alleles DQ2, DQ8, with non-T1D associated variants represented as X. (C) Stratification of IL-2 responses in 9C in at-risk subjects according to lAAb-, single IAAb+ (single-*-) or multi-IAAb+ (multi-*-) antibody status. Dotted line represents 3-fold over background. Lines at median are shown.

[0150] Figure 14: TT-suppression assay for measuring preproinsulin regulatory activity. (A) Scheme for TT-suppression assay using whole blood culture. The IL-2 concentrations in plasma of whole blood cultured with the driver antigen, tetanus toxoid (TT) plus synthetic peptides with sequences derived from preproinsulin (PPI) are presented as a percentage of IL-2 levels from TT alone with a negative control (solvent control and / or irrelevant peptides). (B) 17 overlapping 14-18mer synthetic peptides from100622503746regions across the length of preproinsulin (PPI) were combined into 8 pools. IL-2 concentrations in the plasma from the whole blood of individuals with T1D (n = 5) or without T1 D (n = 7) cultured with one of these 8 peptides plus TT were analysed as a percentage of IL-2 levels in blood taken in the same draw stimulated with TT and short (4mer) negative control peptides. (C) Pooled results from best candidate Pool 1 peptides (comprising two overlapping 18mers covering the sequence of PPI signal peptide) in original plus a second validation cohort of blood samples from non-T1D (n = 12) and T1D (n = 15) individuals. Right: Pooled results from PPI pool 1 responses in TT-suppression assay in both cohorts further analysed using receiver operator characteristic (ROC) curve, with an area under the ROC curve as 0.94. (D) Comparison of different synthetic peptides with sequences derived from the PPI signal peptide: 24mer (full length signal peptide sequence), pooled 18mers (PPI#1 and PPI#2), or each 18mer individually, in the TT-suppression assay using whole blood samples from non-T1 D (n = 12, performed in 4 separate sub-cohorts, each with a new lot of prepared stimuli) and T1 D (n = 3, performed alongside the first set of non-T 1 D samples).

[0151] Figure 15: The whole blood assay can detect antigen specific CD8+ T-cell responses to preproinsulin and influenza derived 9mer peptides. A series of overlapping 9mer peptides covering almost all of the 24 amino acids of preproinsulin signal peptide were used to stimulate 0.5mL duplicate cultures of heparinized whole blood taken from four subject with longstanding T1D (> 100-days since diagnosis, in this case donors all had T1 D for >4 years). Negative controls were: (i) no added peptide / antigen and (ii) peptide solvent alone (DMSO). Positive controls were: (i) an HLA-A2 restricted influenza matrix protein (influenza matrix protein (MP)58-66) epitope, (ii) tetanus toxoid, and (iii) a commercially available pool of influenza peptides (InfA Ultra, JPT, Berlin, Germany). Dotted line shows 3-fold IL-2 response above background.

[0152] Figure 16: X-ID peptide stimulates responses in whole blood from individuals with T1 D, but not those without T1 D (non-T 1 D) (A) CD4+T-cell responses from 7 individuals without T1 D and 14 with T1 D Whole blood was cultured with 10 pM X-l D peptide or an equal volume of solvent. All donors responded to tetanus toxoid, the positive control (not shown). Responses are fold change above the limit of detection of IL-2 in the whole blood, BASTA, assay. The difference between individuals with and without T1D is statistically significant, two-tailed Mann- Whitney test (p=0.0285) (B)100622503747Receiver-operator curve showing the specificity of responses to X-ID peptide for individuals with T1D.

[0153] Figure 17: A pool of p-cell antigen derived peptides stimulates responses in whole blood from individuals with T1 D, but not those without T1 D (non-T 1 D) (A) CD4+T-cell responses from 8 individuals without T1D and 15 with T1D. Whole blood was cultured with 2.5 pM X-ID peptide or an equal volume of solvent. All donors responded to tetanus toxoid, the positive control (not shown). Responses are fold change above the limit of detection of IL-2 in the whole blood, BASTA, assay. The difference between individuals with and without T1D is statistically significant, two-tailed Mann-Whitney test (p<0.05) (B) Receiver-operator curve showing the specificity of responses to peptide pool for individuals with T1 D.

[0154] Figure 18: Peptides recognized by CD8+T cells stimulate responses in whole blood, detectable with the whole blood assay, from individuals with T1D and individuals with islet autoantibodies. Each point represents a different donor. The red symbols indicate individuals who are HLA-A2+. T1D refers to individuals diagnosed with T1D (stage 3), IAAb+verefers to individuals who have multiple islet autoantibodies, but who have not yet developed clinical T1 D (stage 2 T1 D) and non T1 D refers to individuals who do not have a diagnosis of T1D. Peptides are derived from preproinsulin (PPI), islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), glutamic acid decarboxylase 65-kilodalton isoform (GAD65).

[0155] Figure 19: A pool of 7 peptides stimulates CD8+T-cell responses in individuals with islet autoantibodies (stage 2 T1 D), clinical T1 D (stage 3 T1 D), but not individuals without T1 D (non-T 1 D). peptide pools were tested at two concentrations: 5.0 and 1.0 pM (final concentration of each peptide within the pool).100622503748Sequence informationTable 1: Amino acid sequencesSEQ Sequence SequenceID NO: name1 PPI1 MALWMRLLPLLALLALWG2 PPI2 LLPLLALLALWGPDPAAA3 PPI3 LLALWGPDPAAAFVNQHL4 PPI4 PDPAAAFVNQHLCGSHLV5 PPI5 FVNQHLCGSHLVEALYLV6 PPI6 CGSHLVEALYLVCGERGF7 PPI7 EALYLVCGERGFFYTPKT8 PPI8 CGERGFFYTPKTRREAED9 PPI9 FYTPKTRREAEDLQVGQV10 PPI10 RREAEDLQVGQVELGGGP11 PPI11 LQVGQVELGGGPGAGSLQ12 PPI12 ELGGGPGAGSLQPLALEG13 PPI13 GAGSLQPLALEGSLQKRG14 PPI14 PLALEGSLQKRGIVEQCC15 PPI15 SLQKRGIVEQCCTSICSL16 PPI16 IVEQCCTSICSLYQLEN17 PPI17 TSICSLYQLENYCN18 Signal Peptide MALWMRLLPLLALLALWGPDPAAA(full-length)19 Insulin B-chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT (full-length)20 C-peptide (full- EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ length 31-mer)21 Insulin A-chain GIVEQCCTSICSLYQLENYCN(full-length) - *isoacyl bondfor A-chainshown in italicwith underline.22 4mer#1 QHLC23 4mer #2 EVYI10062250374924 4mer #3 MWGP25 4mer #4 KTFR26 Signal Peptide MALWMRLLP9mer_1 PP11-927 Signal Peptide ALWMRLLPL9mer_2 PPI2- 1028 Signal Peptide LWMRLLPLL9mer_3 PPI3- 1129 Signal Peptide WMRLLPLLA9mer_4 PPI4- 1230 Signal Peptide MRLLPLLAL9mer_5 PPI5- 1331 Signal Peptide RLLPLLALL9mer_6 PPI6- 1432 Signal Peptide LLPLLALLA9mer_7 PPI7- 1533 Signal Peptide LPLLALLAL9mer_8 PPI8- 1634 Signal Peptide PLLALLALW9mer_9 PPI9- 1735 Signal Peptide LLALLALWG9mer_10PPI10-1836 Signal Peptide LALLALWGP9mer_11PPI11-1937 Signal Peptide ALWGPDPAA9mer_12PPI15-2338 SerineFVNQHLSGSHLVEALYLVSGERGFFYTPKT substitutedInsulin B-chain(full-length)100622503750*Substitutedserines in bold39 HIP-7 GQVELGGGFLGEGHH40 HIP-8 GQVELGGGSSPETLI41 HIP-13 SLQPLALTPIESHQ42 CHGA342-355 WSKMDQLAKELTAE43 GAD121-140 YVVKSFDRSTKVIDFHYPNE44 GAD274-286 IAFTSEHSHFSLK45 GAD555-567 NFFRMVISNPAAT46 I-A2318-342 GDRGEKPASPAVQPDAALQRLAAVL47 I-A2752-775 KLKVESSPSRSDYINASPIIEHDP48 X-ID ARQEDTAMVYYFDYW49 Islet amyloid KLQVFLIVLpolypeptideIAPP5-1350 Islet-specific LNIDLLWSVglucose-6- phosphatasecatalyticsubunit-relatedproteinIGRP228-23651 Glutamic acid VMNILLQYVVdecarboxylase65-kilodaltonisoform GAD- 65114-12352 INS B18-27 VCGERGFFYT53 Full length M RSAAVLALLLCAGQVTALPVNSPM N KGDTEVM KCI VEV Chromogranin- ISDTLSKPSPMPVSQECFETLRGDERILSILRHQNLLKEL A (CHGA) QDLALQGAKERAHQQKKHSGFEDELSEVLENQSSQAEL KEAVEEPSSKDVMEKREDSKEAEKSGEATDGARPQALP EPMQESKAEGNNQAPGEEEEEEEEATNTHPPASLPSQK YPGPQAEGDSEGLSQGLVDREKGLSAEPGWQAKREEE EEEEEEAEAGEEAVPEEEGPTVVLNPHPSLGYKEIRKGE SRSEALAVDGAGKPGAEEAQDPEGKGEQEHSQQKEEE EEMAWPQGLFRGGKSGELEQEEERLSKEWEDSKRWS KMDQLAKELTAEKRLEGQEEEEDNRDSSMKLSFRARAY GFRGPGPQLRRGWRPSSREDSLEAGLPLQVRGYPEEK KEEEGSAN RRPEDQELESLSAI EAELEKVAHQLQALRRG54 Full length MASPGSGFWSFGSEDGSGDSENPGTARAWCQVAQKFGlutamic acid TGGIGNKLCALLYGDAEKPAESGGSQPPRAAARKAACA100622503751decarboxylase CDQKPCSCSKVDVNYAFLHATDLLPACDGERPTLAFLQD 65-kilodalton VMNILLQYVVKSFDRSTKVIDFHYPNELLQEYNWELADQ isoform PQNLEEILMHCQTTLKYAIKTGHPRYFNQLSTGLDMVGL (GAD65) AADWLTSTANTNMFTYEIAPVFVLLEYVTLKKMREIIGWP GGSGDGIFSPGGAISNMYAMMIARFKMFPEVKEKGMAA LPRLIAFTSEHSHFSLKKGAAALGIGTDSVILIKCDERGKM I PSDLERRI LEAKQKGFVPFLVSATAGTTVYGAFDPLLAV ADICKKYKIWMHVDAAWGGGLLMSRKHKWKLSGVERA NSVTWNPHKMMGVPLQCSALLVREEGLMQNCNQMHAS YLFQQDKHYDLSYDTGDKALQCGRHVDVFKLWLMWRA KGTTGFEAHVDKCLELAEYLYNIIKNREGYEMVFDGKPQ HTNVCFWYIPPSLRTLEDNEERMSRLSKVAPVIKARMME YGTTMVSYQPLGDKVNFFRMVISNPAATHQDIDFLIEEIE RLGQDL55 Full length MRRPRRPGGLGGSGGLRLLLCLLLLSSRPGGCSAVSAH Insulinoma- GCLFDRRLCSHLEVCIQDGLFGQCQVGVGQARPLLQVT associated SPVLQRLQGVLRQLMSQGLSWHDDLTQYVISQEMERIP protein 2 (I-A2) RLRPPEPRPRDRSGLAPKRPGPAGELLLQDIPTGSAPAA QHRLPQPPVGKGGAGASSSLSPLQAELLPPLLEHLLLPP QPPHPSLSYEPALLQPYLFHQFGSRDGSRVSEGSPGMV SVGPLPKAEAPALFSRTASKGIFGDHPGHSYGDLPGPSP AQLFQDSGLLYLAQELPAPSRARVPRLPEQGSSSRAED SPEGYEKEGLGDRGEKPASPAVQPDAALQRLAAVLAGY GVELRQLTPEQLSTLLTLLQLLPKGAGRNPGGWNVGAD IKKTMEGPVEGRDTAELPARTSPMPGHPTASPTSSEVQ QVPSPVSSEPPKAARPPVTPVLLEKKSPLGQSQPTVAG QPSARPAAEEYGYIVTDQKPLSLAAGVKLLEILAEHVHMS SGSFINISVVGPALTFRIRHNEQNLSLADVTQQAGLVKSE LEAQTGLQILQTGVGQREEAAAVLPQTAHSTSPMRSVLL TLVALAGVAGLLVALAVALCVRQHARQQDKERLAALGPE GAHGDTTFEYQDLCRQHMATKSLFNRAEGPPEPSRVSS VSSQFSDAAQASPSSHSSTPSWCEEPAQANMDISTGHM ILAYMEDHLRNRDRLAKEWQALCAYQAEPNTCATAQGE GNIKKNRHPDFLPYDHARIKLKVESSPSRSDYINASPIIEH DPRMPAYIATQGPLSHTIADFWQMVWESGCTVIVMLTPL VEDGVKQCDRYWPDEGASLYHVYEVNLVSEHIWCEDFL VRSFYLKNVQTQETRTLTQFHFLSWPAEGTPASTRPLLD FRRKVN KCYRGRSCPI I VHCSDGAGRTGTYI LI DMVLN R MAKGVKEIDIAATLEHVRDQRPGLVRSKDQFEFALTAVA EEVNAILKALPQ56 Full length Islet MGI LKLQVFLI VLSVALN HLKATPI ESHQVEKRKCNTATCA amyloid TQRLAN FLVHSSN N FGAI LSSTN VGSNTYGKRNAVEVLK polypeptide REPLNYLPL(IAPP)57 Full length MDFLHRNGVLIIQHLQKDYRAYYTFLNFMSNVGDPRNIFF Islet-specific IYFPLCFQFNQTVGTKMIWVAVIGDWLNLIFKWILFGHRP glucose-6- YWWVQETQIYPNHSSPCLEQFPTTCETGPGSPSGHAMphosphatase GASCVWYVMVTAALSHTVCGMDKFSITLHRLTWSFLWS100622503752catalytic VFWLIQISVCISRVFIATHFPHQVILGVIGGMLVAEAFEHT subunit-related PGIQTASLGTYLKTNLFLFLFAVGFYLLLRVLNIDLLWSVPI protein (IGRP) AKKWCANPDWIHIDTTPFAGLVRNLGVLFGLGFAINSEM FLLSCRGGNNYTLSFRLLCALTSLTILQLYHFLQIPTHEEH LFYVLSFCKSASIPLTVVAFIPYSVHMLMKQSGKKSQ58 Full length MEFLERTYLVNDKAAKMYAFTLESVELQQKPVNKDQCP Zinc RERPEELESGGMYHCHSGSKPTEKGANEYAYAKWKLC transporter 8 SASAICFIFMIAEWGGHIAGSLAVVTDAAHLLIDLTSFLLS (ZnT8) LFSLWLSSKPPSKRLTFGWH RAEI LGALLSI LCI WVVTGV LVYLACERLLYPDYQIQATVMIIVSSCAVAANIVLTVVLHQ RCLGHNHKEVQANASVRAAFVHALGDLFQSISVLISALIIY FKPEYKI ADPICTFI FSI LVLASTITI LKDFSI LLM EGVPKSLN YSGVKELILAVDGVLSVHSLHIWSLTMNQVILSAHVATAA SRDSQVVRREIAKALSKSFTMHSLTIQMESPVDQDPDCL FCEDPCD59 GAD113-132 DVMNILLQYVVKSFDRSTKV60 GAD115- MNILLEYVVKSFD127_Q120E61 GAD265- KGMAALPRcitLIAFTSEHSHFS284_R272Cit62 GAD335-352 TAGTTVYGAFDPLLAVAD63 GAD521-535 ERMSRLSKVAPVIKARMMEYGTT64 I-A2198- SLSYEPALLEPYLFHEFGS216_Q207E, Q213E65 I-A2293-311 VPRLPEQGSSSRAEDSPEG66 I-A2449-488 SPLGQSQPTVAGQPSARPAAEEYGYIVTDQKPLSLAAGV K67 I-A2523- QNLSLADVTEEAGL536_Q532E, Q533E68 I-A2545- TGLEILETGVGEREEAAA562_Q548E, Q551E,Q556E69 I-A2654-674 VSSVSSQFSDAAQASPSSHSS70 I-A2709-736 LAKEWQALCAYQAEPNTCATAQGEGNIK71 I-A2854-872 FYLKNVQTQETRTLTQFHF72 I-A2955-975 SKDQFEFALTAVAEEVNAI LK73 IAPP65-84_R73Cit, VGSNTYGKRcitNAVEVLKRcitEPLR81Cit74 IGRP23-35 YTFLNFMSNVGDP75 IGRP226-238 RVLNIDLLWSVPI76 IGRP247-259 DWIHIDTTPFAGL10062250375377 ZnT8l-27_Y18H MEFLERTYLVNDKAAKMHAFTLESVEL78 ZnT8io6-i32 HLLIDLTSFLLSLFSLWLSSKPPSKRL79 ZnT8120-i46 SLWLSSKPPSKRLTFGWHRAEILGALL80 ZnT82i 1-237 N ASVRAAFVHALG DLFQSI SVLI SALI81 ZnT8244-258 YKIADPICTFIFSIL82 ZnT8266-285 ILKDFSILLMEGVPKSLNYS83 ZnT8267-293 LKDFSI LLM EG VPKSLN YSGVKELI LA84 ZnT8309-335TMNQVILSAHVATAASRDSQVVRREIARcit = citrullineDetailed description of the embodiments

[0156] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0157] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0158] All of the publications referred to herein are incorporated by reference in their entirety.

[0159] The terms "a," "an," or "the" as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a plurality of such polypeptides and reference to "the agent" includes reference to one or more agents known to those skilled in the art, and so forth. For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0160] The inventors have developed simple, sensitive, and robust assays for monitoring and detecting β-cell antigen-specific T cell responses in peripheral blood samples obtained from individuals who are at risk of developing T1D.100622503754

[0161] Existing assays that are commonly used to detect β-cell antigen-specific T cells (e.g. CD4+) in peripheral blood include: (i) a CFSE-based proliferation assay and related dye dilution assays, (ii) ELISpot assays, (iii) tetramer staining and (iv) activation marker upregulation. Each of these assays face significant intrinsic obstacles as screening tools for widespread use, including: extensive laboratory-based preparation, large volumes of blood, the need for highly skilled technicians, and in many cases customized reagents and / or sophisticated bioinformatic analysis.

[0162] In the work described herein, the inventors develop T cell assays for the detection and monitoring of T-cell responses associated with T1D in a clinical setting. A first assay detects and monitors effector T cell (Teff) responses that include responses by CD4+ and CD8+ T cells, and a second assay detects and monitors antigen-specific regulatory T cell (Treg) function (e.g. regulatory CD8+ T cells). The two assays may also be used in combination and / or with other assays (for example, assays that detect islet autoantibodies). Importantly, both assays only require a small blood volume, which makes both assays particularly suitable for use in pediatric screening and longitudinal monitoring of T1D progression. Further, assays described herein allow identification of individuals who will develop the disease before any clinical symptoms are manifest.

[0163] The major advantages of certain aspects and embodiments of the methods and assays of this disclosure include one or more or all of: (i) it requires minimal blood, as low as 1.0 ml (for two 0.5 ml replicates) per antigen treatment, (ii) it is very simple to perform, (iii) it has a short 24 h culture period, (iv) plasma samples can be stored, shipped and analyzed in batches, (v) surplus plasma is generated, which can be easily assayed for additional biomarkers, (vi) it is antigen-specific, (vii) it measures T cell function (by IL-2 production or inhibition of IL-2 production). This assay will be very useful for the monitoring of changes in β-cell antigen-specific memory T-cell function in clinical trials and other clinical research settings. This assay may allow more accurate prediction of the time taken for an individual to progress to Stage 3 T1 D and require insulin therapy.Definitions

[0164] As used herein, ‘detecting’ or ‘monitoring’ is intended to refer to at least the identification of a disease or condition, or identification of increase in severity of a100622503755disease or condition relative to a baseline. In some embodiments, this includes identifying at least one pre-clinical symptom of the disease or condition in an individual at risk, where the individual has yet to experience or display clinical symptoms of the disease. In another embodiment, this includes identification of an increase in severity of the at least one pre-clinical symptom of the disease or condition to a clinical symptom of the disease or condition.Type 1 Diabetes

[0165] The term "type 1 diabetes mellitus (T1D)" refers to an immune-mediated disease in which insulin producing beta-cells (pancreatic islet beta-cells) are completely or near completely destroyed, resulting in life-long dependence on exogenous insulin, in other words resulting in insulin deficiency. It is a chronic and potentially disabling disease that represents a major public health and clinical concern. Symptomatic T1D is diagnosed by hyperglycemia often in combination with symptoms of weight loss, thirst, fatigue, and frequent urination, sometimes with ketoacidosis.

[0166] The clinical onset of symptomatic T1D is preceded by a pre-symptomatic phase. Pre-symptomatic or stage 1 T1D is defined as being normoglycemic but multiple beta cell autoantibody positive. The development of multiple beta cell autoantibodies is defined as being positive for circulating multiple beta cell autoantibodies to beta-cell antigens (GADA, IA-2A, IAA and ZnT8A). First, seroconversion to islet autoantibodies (islet autoantibody positive, IAAb+) occurs in a subject, before said subject may develop multiple islet autoantibodies (multiple islet autoantibody positive).

[0167] Beta-cell autoimmunity is the pre-symptomatic form of T1D that precedes the onset of symptomatic T1 D and comprises the development of islet autoantibodies and the development of multiple islet autoantibodies.

[0168] Further, stage 2 T1D is characterized by having abnormal glucose tolerance and multiple beta cell autoantibodies. Stage 3 is symptomatic T1D with hyperglycemia and clinical signs, and typically requires insulin. Time from stage 1 to stage 3 varies between months and decades.

[0169] In this context, the term "type 1 diabetes (T1D)" comprises both the pre-symptomatic (Stages 0, 1, and 2) and symptomatic phase (Stage 3 or 4) of type 1100622503756diabetes. In particular, the pre-symptomatic form of T1D namely "beta-cell autoimmunity" may also be preferred in the present disclosure. Thus, the present invention may also comprise a method of detecting and monitoring progression of pre-symptomatic T1 D to symptomatic T1 D by measuring T cell responses.

[0170] Progression of T1D may comprise progression within a single stage, or progression to a higher stage, for example, from stage 1 to stage 2, stage 2 to stage 3 or stage 3 to stage 4.

[0171] The present invention may also be used for detecting and monitoring beta cell autoimmunity in individuals who may be pre-symptomatic for type 1 diabetes, have recent-onset diabetes, or be at risk of developing type 1 diabetes. For example, it may be used for monitoring efficacy of therapeutics administered to an individual to prevent or treat pre-clinical type 1 diabetes in a clinical trial.

[0172] The present invention may also be used for detecting and monitoring beta cell autoimmunity in individuals with clinical, symptomatic T1 D. For example, it may be used for monitoring efficacy of therapeutics administered to an individual to treat T1 D.

[0173] Additionally, the present invention may distinguish between individuals with T1D and other forms of diabetes, which may be difficult for clinicians to distinguish using existing methods.

[0174] Individuals who are at increased risk to develop multiple beta-cell autoantibodies (pre-symptomatic T1 D) and symptomatic type 1 diabetes can now be monitored for T 1 D-specific T cell responses. This provides opportunity for introducing early therapies to prevent pre-symptomatic and symptomatic type 1 diabetes.Individual at risk of developing T1D

[0175] Type 1 diabetes has a multifactorial etiology, which is determined by genetic and environmental factors. Certain HLA DR-DQ genotypes confer markedly elevated risk for type 1 diabetes. Notably, infants who have the HLA DR3 / DR4-DQ8 or the DR4-DQ8 / DR4-DQ8 genotype have a risk of around 5%. In some embodiments, family history, genetic markers, and genotyping at T1D susceptibility regions may be used to100622503757identify individuals who are at risk of developing T1 D, and who would benefit from screening and monitoring for T1D.

[0176] As used herein, the term “subject” or “individual” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human. The terms “subject” and “individual” may be used interchangeably.

[0177] The term "adult" refers to a person with an age above 18 years. A “child” refers to a person with an age below 18 years. A “teen” refers to a person of age 13-10 years. A “school-aged child” refers to a person of age 5-12 years. A “preschooler” refers to a person of age 3-4 years. A “toddler” refers to a person of age 1-2 years. An “infant” refers to a person of age 3-11 months. A “newborn” refers to a person of age 0-2 months.

[0178] In some embodiments of the invention, the individual at risk is an adult. In some embodiments, the individual at risk is a child.

[0179] In any aspect or embodiment, the individual is an adult.

[0180] In any aspect or embodiment, the individual is a child.

[0181] In any aspect or embodiment, the individual is male.

[0182] In any aspect or embodiment, the individual is female.

[0183] In any aspect or embodiment, the individual aged from 2 years old to 70 years old, from 2 years old to 60 years old, from 2 years old to 50 years old, from 2 years old to 40 years old, from 2 years old to 30 years old, from 2 years old to 20 years old, from 10 years old to 70 years old, from 10 years old to 60 years old, from 10 years old to 50 years old, from 10 years old to 40 years old, from 10 years old to 30 years old, or from 10 years old to 20 years old. Also contemplated is an individual with an age of, or between any two ages of, 2 years old, 3 years old, 10 years old, 20 years old, 30 years old, 40 years old, 50 years old, 60 years old, 70 years old, or any other age described herein, including in Tables 2 and 3.100622503758

[0184] In any aspect or embodiment, the individual aged from about 2 years old to about 70 years old, from about 2 years old to about 60 years old, from about 2 years old to about 50 years old, from about 2 years old to about 40 years old, from about 2 years old to about 30 years old, from about 2 years old to about 20 years old, from about 10 years old to about 70 years old, from about 10 years old to about 60 years old, from about 10 years old to about 50 years old, from about 10 years old to about 40 years old, from about 10 years old to about 30 years old, or from about 10 years old to about 20 years old. Also contemplated is an individual with an age of, or between any two ages of, about 2 years old, about 3 years old, about 10 years old, about 20 years old, about 30 years old, about 40 years old, about 50 years old, about 60 years old, about 70 years old, or about any other age described herein, including in Tables 2 and 3.

[0185] In any aspect or embodiment, the individual aged from 2 years old to 20 years old, from 2 years old to 17 years old, from 2 years old to 11 years old, from 2 years old to 8 years old, from 2 years old to 6 years old, from 2 years old to 4 years old, from 4 years old to 20 years old, from 6 years old to 20 years old, from 8 years old to 20 years old, from 11 years old to 20 years old, from 17 years old to 20 years old, from 4 years old to 17 years old, from 6 years old to 17 years old, from 8 years old to 17 years old, from 11 years old to 17 years old, or from 3 years old to 11 years old. Also contemplated is an individual with an age of, or between any two ages of, 2 years old, 3 years old, 4 years old, 6 years old, 8 years old, 11 years old, 17 years old, 20 years old, or any other age described herein, including in Table 3.

[0186] In any aspect or embodiment, the individual aged from about 2 years old to about 20 years old, from about 2 years old to about 17 years old, from about 2 years old to about 11 years old, from about 2 years old to about 8 years old, from about 2 years old to about 6 years old, from about 2 years old to about 4 years old, from about 4 years old to about 20 years old, from about 6 years old to about 20 years old, from about 8 years old to about 20 years old, from about 11 years old to about 20 years old, from about 17 years old to about 20 years old, from about 4 years old to about 17 years old, from about 6 years old to about 17 years old, from about 8 years old to about 17 years old, from about 11 years old to about 17 years old, or from about 3 years old to about 11 years old. Also contemplated is an individual with an age of, or between any two ages of, about 2 years old, about 3 years old, about 4 years old, about 6 years old, about 8100622503759years old, about 11 years old, about 17 years old, about 20 years old, or any other age described herein, including in Table 3.

[0187] In any embodiment of the present disclosure, the individuals may have been diagnosed as having or being at risk of having T1D by a physician.

[0188] In any embodiment of the present disclosure, the individuals in need thereof may also have an impaired ability to clear glucose from the blood, and / or be diagnosed with T1D.

[0189] In any embodiment, the individual may be one that has been identified as having a reduced level of circulating insulin. Preferably the level of circulating insulin that is reduced is at a fasting state or is a basal level.

[0190] In any embodiment, the individual may be one that has been identified as having an insulin deficiency.

[0191] In some embodiments, the individuals may have mild insulin deficiency, and not require specific treatment with insulin. In embodiments the individuals may have moderate insulin deficiency. In embodiments the individuals may have severe insulin deficiency. In embodiments the individuals have absolute insulin deficiency. In embodiments the individual may be being administered insulin.

[0192] The methods of the invention described herein are for detecting and monitoring progression T1D in at risk individuals to prevent disruption in normal regulation of blood glucose and symptoms associated therewith. Typically, that means that the levels of T cell responses are monitored longitudinally in an at risk individual and compared relative to a baseline level.

[0193] In any aspect or embodiment, the individual may be, or have been identified as, at risk of developing type 1 diabetes. For example, the individual may have one or more risk factors, including risk factors as described herein. In one embodiment, the individual has increased genetic risk for type 1 diabetes. For example, the individual may be identified as having a genetic risk score indicating that they are at risk of developing type 1 diabetes.100622503760

[0194] In any aspect or embodiment, the individual may have, or have been identified as having, recent-onset type 1 diabetes. For example, the individual may have one or more signs or symptoms of recent-onset type 1 diabetes, including signs or symptoms as described herein.

[0195] In one embodiment, the individual may express the HLA alleles DQ8, DQ2, DR3 and / or DR4. Preferably, the individual expresses the HLA alleles DQ2, DQ8, or DQ2 and DQ8. The DQ8 allele may be HLA-DQA1 *03:01 or HLA-DQB1*03:02. The DQ2 allele may be DQA1*05:01 or DQB1*02:01. The DRB1 allele may be DRB1*04:0X.

[0196] In any aspect or embodiment, the individual may be or has been tested as multi-antibody-positive.

[0197] In any aspect or embodiment, the individual is enrolled in a clinical trial of a therapy intended to prevent / delay on the onset of T1 D.Polypeptides and pools of polypeptides

[0198] In any aspect or embodiment, the signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0199] In any aspect or embodiment, the B-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 19, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0200] In any aspect or embodiment, the C-peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,100622503761at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0201] In any aspect or embodiment, the A-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 21, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0202] In any aspect or embodiment, the fragment of a signal peptide, a B-chain, a C-peptide or an A-chain of proinsulin as described herein may be at least 14, at least 15, at least 16, at least 17 or at least 18 amino acids in length, or may be 14, 15, 16, 17 or 18 amino acids in length.

[0203] In any aspect or embodiment, the fragment of a signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ I D NO: 1, 2 or 3, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0204] In any aspect or embodiment, the fragment of a signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 26, 21, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Preferably, the signal peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 30 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.100622503762

[0205] In any aspect or embodiment, the fragment of a B-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8 or 9, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0206] In any aspect or embodiment, the fragment of a C-peptide comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 9, 10, 11, 12, 13 or 14, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0207] In any aspect or embodiment, the fragment of a A-chain comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 14, 15, 16 or 17, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0208] ln any aspect or embodiment, the X-ID peptide comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 48, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0209] In any aspect or embodiment, HIP-7 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 39, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.100622503763

[0210] I n any aspect or embodiment, HIP-8 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0211] ln any aspect or embodiment, HIP-13 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 41, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0212] In any aspect or embodiment, CHGA comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0213] In any aspect or embodiment, GAD65 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0214] In any aspect or embodiment, I-A2 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 55 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.100622503764

[0215] In any aspect or embodiment, IAPP comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 56 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0216] I n any aspect or embodiment, IGRP comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 57 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0217] In any aspect or embodiment, ZnT8 comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 58 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0218] In any aspect or embodiment, the pool of polypeptides is two or more polypeptides or fragments thereof described herein. For example, the pool of polypeptides may be two or more of any polypeptide that comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NOs: 1 to 21 or 26 to 37. The pool of polypeptides may be polypeptides that comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NOs: 1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; 11 and 12; 13 and 14; 15 and 16, or 18 and 17. The pool of peptides may also be polypeptides that comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NOs: 26 and 27, 28 and 29, 30 and 31, 32 and 33, 34 and 35, or 36 and 37.Driver antigens100622503765

[0219] Driver antigens that may be useful in some aspects of the invention include influenza haemagluttinin (Native Ag company), influenza neuraminidase (Native Ag company), the spike protein of SARS-CoV-2 (Aero Biosystems), Diptheria toxoid (CSL Sequiris, List Labs), tetanus toxoid (CSL Sequiris, List Labs), inactivated polio virus (Sanofi Pasteur, GlaxoSmithKline, Bilthoven Biologicals, and the Staten Serum Institute), hepatitis B antigen (Abeam), or human papilloma virus antigen (SinoBiological).

[0220] The skilled person is aware that a number of different driver antigens may be used, including multivalent vaccines that include multiple vaccine-derived epitopes, and that the examples provided are not limiting.Cytokines

[0221] The methods and assays of the disclosure include determining the presence of, or amount of, a cytokine or fragment thereof in an assay or control sample.

[0222] It is contemplated that detection of a cytokine may be detection of a whole or intact cytokine, or detection of a fragment or part of a cytokine that allows identification of the cytokine. For example, detection of a fragment or part of a cytokine that is unique or characteristic of the whole cytokine is contemplated.

[0223] In any aspect or embodiment, the cytokine may be a cytokine that is known to be associated with T1D pathogenesis and / or that is expressed by a T cell in response to a Beta cell antigen. Cytokines that are associated with T1D pathogenesis and / or that are expressed by a T cell in response to a Beta cell antigen include cytokines that play an anti-inflammatory role such as IL-10, TGF-β, and type 2 cytokines, and cytokines that play a pro-inflammatory role, such as IL-1, IL-6, TNF-α, IFN-α, IL-17, and IL-21.Cytokines that are associated with T1 D pathogenesis and / or that are expressed by a T cell in response to a Beta cell antigen also include cytokines that have pleiotropic effects, such as IL-2, IFN-γ, or IL-15. Cytokines that are associated with T1D pathogenesis and / or that are expressed by a T cell in response to a Beta cell antigen are well known to the skilled person in the art, and are not limited to the examples provided.100622503766

[0224] The presence of, or increase in amount of, a cytokine in the assay sample may be determined by a method for analysing and detecting cytokines. Numerous methods for analysing and detecting cytokines are well known to the skilled person in the art, and include flow cytometry, quantitative real-time PCR (qPCR), enzyme linked immunosorbent assays (ELISA), multiplex arrays, or immunoassays, including electrochemiluminescence (ECL)-based immunoassays. The examples provided are not limiting, and the skilled person knows that many methods that can analyse and detect cytokines in assay samples may be used.

[0225] In a preferred embodiment, the presence of, or increase in amount of, a cytokine in the assay sample is determined by electrochemiluminescence (ECL)-based immunoassay.

[0226] An exemplary method of detecting a cytokine, for example IL-2, is outlined in Example 1. Other potential useful methods for detecting cytokines in plasma may include other immunoassays methods including standard ELISA or high sensitivity ELISA (e.g.: Human IL-2 kit, High Sensitivity, ThermoFisher Cat# BMS221-2HS), which may include amplification of standard immunoassay signal beyond established colorimetric readouts using additional or alternative readouts including chemiluminescence or PCR-based detection systems (e.g.: ProQuantum IL-2 Immunoassay, ThermoFisher, Cat# A35603). Other recently developed platforms include single molecule array and related technologies (e.g.: Simoa Complex Human IL-2 kit for use with SP-X reader, Cat # 100-0487, Quanterix) that would be suitable to detect sub-pg / ml concentration differences of cytokine present in plasma.Methods and assays

[0227] In any method or assay of the disclosure, a whole blood sample may be obtained or provided from an individual as described herein. The whole blood sample may be a peripheral blood sample. A whole blood sample is a sample of blood in blood drawn directly from the body from which none of the components, such as plasma or platelets, has been removed.

[0228] In any embodiment, the whole blood sample is preferably taken from the hand (in particular venous blood is taken from the back of the hand) or the arm, for example by100622503767venipuncture. In infants, older children, or adults, the blood sample is preferably taken from the arm.

[0229] Preferably, the whole blood sample is heparinised. In this case the whole blood sample may be referred to as a heparinised sample. In one embodiment, the only treatment of the whole blood sample after being obtained from an individual and before being contacted with at least one T-cell autoantigen or fragment thereof in a method of the disclosure is heparinization.

[0230] In any aspect or embodiment, the whole blood is untreated or unpurified.

[0231] In any aspect or embodiment, the volume of the whole blood sample contacted with at least one T-cell autoantigen or fragment thereof is greater than 0.2 ml but equal to or less than 60 ml; greater than 0.2 ml but equal to or less than 50 ml; greater than 0.2 ml but equal to or less than 40 ml; greater than 0.2 ml but equal to or less than 30 ml; greater than 0.2 ml but equal to or less than 20 ml; greater than 0.2 ml but equal to or less than 10 ml; greater than 0.2 ml but equal to or less than 5 ml; greater than 0.25 ml but equal to or less than 2 ml; greater than 0.2 ml but equal to or less than 1 ml; equal to or greater than 0.5 ml but less than or equal to 5 ml; equal to or greater than 0.5 ml but less than or equal to 2 ml; or equal to or greater than 0.5 ml but less than or equal to 1 ml.

[0232] In any aspect or embodiment, the volume of the whole blood sample contacted with at least one T-cell autoantigen or fragment thereof, and in certain embodiments a driver antigen, is greater than 0.2 ml but equal to or less than about 60 ml; greater than 0.2 ml but equal to or less than about 50 ml; greater than 0.2 ml but equal to or less than about 40 ml; greater than 0.2 ml but equal to or less than about 30 ml; greater than 0.2 ml but equal to or less than about 20 ml; greater than 0.2 ml but equal to or less than about 10 ml; greater than 0.2 ml but equal to or less than about 5 ml; greater than 0.2 ml but equal to or less than about 2 ml; greater than 0.2 ml but equal to or less than about 1 ml; equal to or greater than about 0.5 ml but less than or equal to about 5 ml; equal to or greater than about 0.5 ml but less than or equal to about 2 ml; or equal to or greater than about 0.5 ml but less than or equal to about 1 ml. The volume of the whole blood sample may be 600pl, 700pl, 800pl and 900pl, or about 600pl, about 700pl, about 800pl and about 900pl.100622503768

[0233] In any aspect or embodiment, the volume of the whole blood sample contacted with at least one T-cell autoantigen or fragment thereof is 0.5 ml or 1 ml.

[0234] In any aspect or embodiment, the cells in the whole blood sample are not labelled, for example with a detectable label.

[0235] In any aspect or embodiment, the cells in the whole blood sample are not labelled with 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE).

[0236] The whole blood sample, preferably after heparinization, is contacted with at least one T-cell autoantigen or fragment thereof, and in some aspects or embodiments, a driver antigen. The contact with at least one T-cell autoantigen or fragment thereof may be by aliquoting the whole blood sample, preferably after heparinization, to a vessel pre-aliquoted with the at least one T-cell autoantigen or fragment thereof. A whole blood sample may be contacted with a control, for example comprising polypeptides or pool of polypeptides, in the same way. Typically, the vessel is a tube, preferably a sterile, capped, polypropylene tube.

[0237] In any aspect or embodiment, the concentration of the at least one T-cell autoantigen or fragment thereof contacted with the blood sample is greater than or equal to 1 μM, greater than or equal to 2 μM, greater than or equal to 10 μM, greater than or equal to 1.0 μM, greater than or equal to 2.0 μM, greater than or equal to 10.0 μM, greater than or equal to 1 μM but less than or equal to 10 μM, greater than or equal to 1 μM but less than or equal to 2 μM, greater than or equal to 1.0 μM but less than or equal to 10.0 μM, or greater than or equal to 1.0 μM but less than or equal to 2.0 μM.

[0238] In some aspects or embodiments, the concentration of the driver antigen contacted with the blood sample is 0.01 - 300 pg / ml. Preferably, the concentration of the tetanus toxoid is 0.33 Lfu / ml (72 pg / ml).

[0239] In any aspect or embodiment, the whole blood sample (preferably after heparinization) and at least one T-cell autoantigen or fragment thereof are incubated for a period of time sufficient for stimulation to produce a cytokine. The time period is sufficient to allow production of cytokines. Typically, the period of time is 8 hours; about 8 hours; greater than or equal to 8 hours but less than 7 days; 24 hours; about 24 hours; greater than or equal to 24 hours but less than 7 days.100622503769

[0240] A control sample may be a whole blood sample not contacted with a polypeptide nor pool of polypeptides. Preferably, the whole blood sample from which the control sample is formed is derived from the same individual or from the same sample from the same individual to the whole blood sample which is contacted with a polypeptide nor pool of polypeptides. A control sample may be a whole blood sample contacted with a solvent or one or more control or irrelevant peptides (e.g. not derived from preproinsulin).

[0241] After a period of incubation, plasma may be harvested, for example by centrifugation, and then the presence of, or amount of, a cytokine as described herein is determined in the plasma. This plasma may be referred to herein as a plasma sample. In an embodiment, the assay sample may be a plasma sample.

[0242] In any aspect or embodiment of the invention where the whole blood sample is contacted with at least one T-cell autoantigen or fragment thereof and not a driver antigen, the change (e.g. increase or decrease) or lack thereof in amount of a cytokine may be measured in change in concentration and / or fold change in comparison to a reference value. Preferably, the reference value is derived from a whole blood sample not contacted with at least one T-cell autoantigen or fragment thereof from the same individual or from the same sample from the same individual to the whole blood sample which is contacted with at least one T-cell autoantigen or fragment thereof. Preferably, this reference value is derived from whole blood samples contacted with at least one T-cell autoantigen or fragment thereof from a population of individuals not known to have type 1 diabetes or not at risk of developing type 1 diabetes. Alternatively, a reference value is derived from whole blood samples contacted with at least one T-cell autoantigen or fragment thereof from a population of individuals known to have type 1 diabetes or at risk of developing type 1 diabetes.

[0243] In any aspect or embodiment of the invention where the whole blood sample is contacted with at least one T-cell autoantigen or fragment thereof and a driver antigen, the change (e.g. increase or decrease) or lack thereof in amount of a cytokine may be measured in change in concentration and / or fold change in comparison a reference value. Preferably, this reference value is derived from a whole blood sample not contacted with at least one T-cell autoantigen or fragment thereof but contacted with the driver antigen and is derived from the same individual or from the same sample from the100622503770same individual to the whole blood sample which is contacted with at least one T-cell autoantigen or fragment thereof. Preferably, this reference value is derived from whole blood samples contacted with a driver antigen and at least one T-cell autoantigen or fragment thereof from a population of individuals not known to have type 1 diabetes or not at risk of developing type 1 diabetes. Alternatively, a reference value is derived from whole blood samples contacted with at least one T-cell autoantigen or fragment thereof from a population of individuals known to have type 1 diabetes or at risk of developing type 1 diabetes.

[0244] In any aspect or embodiment, the change in amount of a cytokine may be a change relative to the amount of a cytokine present to a reference value. Therefore, in any method of the disclosure, the method further comprises a step of comparing the determined amount of the cytokine in the whole blood with the reference value.ExamplesExample 1: Materials and methodsStudy Design

[0245] This study was conducted in two phases. For the initial phase, patients with T1 D were recruited during regular consultations at The Royal Children’s Hospital (Victoria, Australia) endocrinology clinic or at baseline sampling prior to entry into the BANDIT trial (Waibel, M. et al (2023) N Engl J Med, 389(23): 2140-2150). Non-diabetic control samples were recruited through the SVI Living Biobank. For the validation phase, additional samples were obtained from research participants attending Royal Melbourne Hospital. Ethical approval was given by St Vincent’s Hospital HREC (Approval Numbers: HREC-A 161.15 and HREC-A 135 / 08) and Southern Health (Royal Children’s Hospital, approval number: 12185B). Further approval (number: 2009.026) was given by Melbourne Health. All participants provided written informed consent. Participants are without T1D or diagnosed according to American Diabetes Association criteria. HLA typing of donor samples was provided by Victorian Transplantation and Immunogenetics Services (Victoria, Australia). Blood donors were classed based on the presence of HLA-DR3-DQ2 (HLA-DQB1*02:01, HLA-DQA1*05:01) or HLA-DR4-DQ8 (HLA-DQB1*03:02, HLA-DQA1*03:01) haplotypes with high risk for T1D. Donors100622503771possessing the HLA-DQA1*03:03 variant were also included within the HLA-DQ8 haplotype (as established in Enczmann, J. etal (2021) Genes (Basel) 12(12): 1879).

[0246] The demographics and HLA of all blood donors are summarised in Table 2. Table 2: Demographics and HLA of non-T1D and T1D blood donorsNon-T1D T1D No. individual participants 44 137 Age (years) 37.23 ± 16.59 (SD) 13.87 ± 4.25 (SD) range: 8-66 range: 3 - 28 Sex (male | female) 19 | 25 86 | 47 Disease duration (years) N / A 4.71 ± 4.47 (SD)Range: 0 - 15.72 % HLA-DQ2+ 11.36 % 43.07 % % HLA-DQ8+ 29.55 % 38.69 % % HLA-DQ2 / HLA-DQ8+ 2.27 % 18.98 % % Neither HLA-DQ2+ nor HLA- 54.5 % 11.7 % DQ8+

[0247] The demographics and HLA of the pediatric cross-sectional validation cohort are summarised in Table 3.Table 3: Demographics of pediatric participants in cross-sectional validationAt-risk Low-risk New-Onset Established participants pediatric T1D T1D participantsNo. individual 21 10 12 20 participantsAge (years) ± (SD) 6.63 ± 2.3 15.40 ± 3.89 11.42 ± 4.23 13.35 ± 3.31 range: 3- 11 range: 8 - 20 range: 4-17 range: 6 - 17Sex (male | female) 13 | 8 4 | 6 8 | 4 17 | 3 Disease N / A N / A 0.02 ± 0.04 7.45 ± 4.60 duration (years)100622503772% HLA-DQ2+ 38.1 % (8 / 21) 0% (0 / 10) 58.33% 70% (14 / 20)(7 / 12)% HLA-DQ8+ 38.1% (8 / 21) 20% (2 / 10) 41.67% 60% (12 / 20)(5 / 12)% HLA-DQ2 / HLA- 9.5 % (2 / 21) 0% (0 / 10) 16.67% 35% (7 / 20) DQ8+ (2 / 12)% Neither HLA-DQ2+ 33.3 % (7 / 21) 80% (8 / 10) 16.7% (2 / 12) 5% (1 / 20) nor HLA-DQ8+% ZnT8 AAB+ 19 % (4 / 21) N / A N / A % GADA+ 9.5 % (2 / 21) N / A N / A % IAA+ 9.5 % (2 / 21) N / A N / A % IA-2A+ 9.5 % (2 / 21) N / A N / A % Multiple AAB+ 14.3 % (3 / 21) N / A N / APeptides and Stimuli

[0248] Preproinsulin peptides at > 90% purity (supplied by Genscript except for isoacyl A-chain, which was prepared in-house) were dissolved in DMSO, or acetonitrile / water with or without the addition of 0.5% acetic acid, or 100 mM ammonium bicarbonate, to a concentration of 5.0 mM, aliquoted and stored at -80 °C. Pre-dissolution of isoacyl A-chain in acidic conditions prevented base-mediated isomerization. A full list of peptides is shown in Table 4.Table 4: Peptide sequencesPeptide Sequence Pool PPI1 MALWMRLLPLLALLALWG POOL1 PPI2 LLPLLALLALWGPDPAAA POOL1 PPI3 LLALWGPDPAAAFVNQHL POOL2 PPI4 PDPAAAFVNQHLCGSHLV POOL2 PPI5 FVNQHLCGSHLVEALYLV POOL3 PPI6 CGSHLVEALYLVCGERGF POOL3 PPI7 EALYLVCGERGFFYTPKT POOL4 PPI8 CGERGFFYTPKTRREAED POOL4 PPI9 FYTPKTRREAEDLQVGQV POOL5100622503773PPI10 RREAEDLQVGQVELGGGP POOL5 PPI11 LQVGQVELGGGPGAGSLQ POOL6 PPI12 ELGGGPGAGSLQPLALEG POOL6 PPI13 GAGSLQPLALEGSLQKRG POOL7 PPI14 PLALEGSLQKRGIVEQCC POOL7 PPI15 SLQKRGIVEQCCTSICSL POOL8 PPI16 IVEQCCTSICSLYQLEN POOL8 PPI17 TSICSLYQLENYCN POOL8 Signal Peptide MALWMRLLPLLALLALWGPDPAAA PPI pool (full-length)Insulin B-chain FVNQHLCGSHLVEALYLVCGERGFFYTPKT PPI pool (full-length)C-peptide (full- EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ PPI pool length 31-mer)Insulin A-chain GIVEQCCTSICSLYQLENYCN PPI pool(full-length)*isoacyl bond for A-chain shown in italic with underline.

[0249] Initial experiments used commercially available CEFX (JPT Peptide Technologies, Germany), a pool of peptide epitopes from pathogens known to stimulate CD4+ T-cell responses, at 1 pg / ml. Tetanus toxoid was used at 0.33 Lfu / ml and supplied by Statens Serum Institut, Copenhagen, Denmark.β-cell antigen specific T-cell assay - BASTA

[0250] Briefly, heparinized peripheral blood was aliquoted within 3 hours of blood draw into sterile, capped, polypropylene tubes containing pre-aliquoted peptides or control stimuli, or similarly prepared cryovials with external-threaded caps. Blood without any additional stimulation, or the peptide solvent alone was an additional negative control. After incubation for 24 h at 37 °C with 5% CO2, samples were centrifuged, plasma was harvested, aliquoted and frozen. Cytokines in the plasma were measured in duplicate using an electrochemiluminescence (ECL)-based immunoassay platform (Meso Scale Discovery (MSD), Rockville, MD, USA), using lot-validated 96-well plate format MSD V-plex kits measured on a MESO QuickPlex SQ 120 reader. Data was analysed using the Discovery Workbench software. If below lower limit of detection (LLOD), results were100622503774set to the LLOD for each assay run based on the standard curve. Response cut-off was selected as 3-fold over the unstimulated or solvent only control blood cultures.CFSE-Based Proliferation Assay

[0251] The CFSE (5,6-carboxylfluorescein diacetate succinimidyl ester) proliferation assays were performed as described previously (Mannering, S. I.et al (2003) J Immunol Methods, 283(1-2): 173-83). Briefly, PBMC from T1D subjects or non T1D HLA matched donors labelled with 0.1 mM CFSE (Life Technologies, Carlsbad, CA) were cultured either with: no additional stimulus, or C-peptide (10 pM), or tetanus toxoid (0.33 LfU / ml). After 7 days of culture the cells were washed in PBS and stained on ice with anti-human CD4-AlexaFluor-647 (clone OKT4, prepared in house). CD4+ T-cell proliferation was measured by determining the number of CD4+, CFSEdimcells for every 5,000 CD4+ CFSE bright cells. The results are presented as a cell division index (CDI) which is the ratio of the number of CD4+ cells that have proliferated in the presence of antigen: without antigen (Mannering, S.l. et al (2005) J Immunol Methods, 298(1-2): 83-92). HLA-blocking

[0252] As previously described (Mannering, S.l. et al (2009) Clin Exp Immunol, 156(2): 226-31; Mannering, S.l. et al (2005) J Immunol Methods, 298(1-2): 83-92), TCR recognition of peptide HLA complexes on antigen presenting cells was blocked through the addition of monoclonal antibodies specific for either HLA-DP (clone: B7 / 21), HLA-DQ (clone: SPV-L3), HLA-DR (clone: L243), the addition of all 3 pooled antibodies, or pan anti-H LA-class I (clone: W6 / 32, 2 pg / ml final concentration). Each was added in combination to either blood with no additional stimulus or peptide / protein stimuli as indicated.IL-2 capture

[0253] IL-2+ cells were enriched for further analysis using a magnetic bead-based IL-2 capture system (Miltenyi) according to manufacturer’s instructions. Briefly, whole blood was stimulated overnight as above, in 5 ml volumes using 50 ml tubes. After 16 h incubation, erythrocytes were lysed (using Red Cell Lysis Buffer, Miltenyi), and cytokine secreting cells identified through further labelling with capture reagents and incubation. Cells were then stained with PE-labelled anti-IL-2 antibodies, and the following panel of100622503775antibodies against surface markers: CD3-PerCP (clone: LICHT1, Biolegend), CCR7-APC-Cy7 (clone: G043H7, Biolegend), CD45RO-BV650 (clone: UCHL1, BD biosciences), CD4-AF647 (clone: OKT4, prepared in-house), CD8-V500 (clone: RPA-T8, BD biosciences), CD69-FITC (clone: FN50, BD Biosciences), prior to magnetic enrichment using MS columns.Flow cytometry

[0254] Flow-cytometry was performed as previously described (So, M. et al (2018) Proc Natl Acad Sci USA, 115(42): 10732-10737; Mannering, S.l. etal (2003) J Immunol Methods, 283(1-2): 173-83.), cells were acquired using the flow cytometer LSRFortessa (BD Biosciences) or Cytek Aurora (Cytek Biosciences) and analyzed in FlowJo 10 (v10 TreeStar). Live cells were gated on by excluding cells positive for propidium iodide (PI). Whole blood tetanus toxoid-suppression assay

[0255] For the tetanus toxoid (TT)-suppression assay, larger blood volumes (total 1 ml culture volume) were used compared to the effector T cell assay (BASTA), to account for the very low frequency of Ag-responsive suppressive cells. A modified version of the BASTA whole blood assay from autoreactive effector cells is used. 900 pl of whole blood is added to a 5 ml polypropylene sterile capped round bottom tube pre-aliquoted with 100 pl of PBS containing either no stimulus, TT alone (0.33 Lfll / ml), TT with DMSO as a solvent control, or TT plus a pool of irrelevant 4mer peptides at equal relative amino acid content to candidate suppressive islet peptides. Preproinsulin peptides were also added alongside TT, so that their final concentration in the 1 ml culture was 10 pM. Each stimulation was performed in duplicate. Suppressive capacity is interpreted as the IL-2 levels in plasma after culture in the tubes with TT and suppressive peptides calculated as a percent of the mean IL-2 levels in the TT plus irrelevant control (nominally set as 100) from a separate aliquot of blood from the same blood draw from the same individual.

[0256] The peptide sequences that were tested in the whole blood tetanus toxoidsuppression assay are represented in Table 5.Table 5: Peptides used in tetanus toxoid-suppression assay100622503776Peptide Sequence Pool PPI#1 MALWMRLLPLLALLALWG POOL1 PPI#2 LLPLLALLALWGPDPAAA POOL1 PPI#3 LLALWGPDPAAAFVNQHL POOL2 PPI#4 PDPAAAFVNQHLCGSHLV POOL2 PPI#5 FVNQHLCGSHLVEALYLV POOL3 PPI#6 CGSHLVEALYLVCGERGF POOL3 PPI#7 EALYLVCGERGFFYTPKT POOL4 PPI#8 CGERGFFYTPKTRREAED POOL4 PPI#9 FYTPKTRREAEDLQVGQV POOL5 PPI10 RREAEDLQVGQVELGGGP POOL5 PPI11 LQVGQVELGGGPGAGSLQ POOL6 PPI12 ELGGGPGAGSLQPLALEG POOL6 PPI#13 GAGSLQPLALEGSLQKRG POOL7 PPI#14 PLALEGSLQKRGIVEQCC POOL7 PPI#15 SLQKRGIVEQCCTSICSL POOL8 PPI#16 IVEQCCTSICSLYQLEN POOL8 PPI#17 TSICSLYQLENYCN POOL8 PPI Signal Peptide MALWMRLLPLLALLALWGPDPAAA(full-length)4mer #1 QHLC 4mers 4mer #2 EVYI 4mers 4mer #3 MWGP 4mers4mer #4 KTFR 4mersDetection of antigen-specific CD8+ T-cell responses to preproinsulin and influenza derived 9mer peptides

[0257] A series of overlapping 9mer peptides covering almost all of the 24 amino acids of preproinsulin signal peptide were used to stimulate 0.5 mL duplicate cultures of heparinized whole blood taken from four subject with longstanding T1D (> 100-days since diagnosis, in this case donors all had T1 D for >4 years). Negative controls were: (i) no added peptide / antigen and (ii) peptide solvent alone (DMSO). Positive controls were: (i) an HLA-A2 restricted influenza matrix protein (FluMPss-ee) (Townsend et al.,100622503777(1986), Cell, 44(6): 959-68) epitope, (ii) tetanus toxoid, and (iii) a commercially available pool of influenza peptides (InfA Ultra, JPT, Berlin, Germany).

[0258] The peptides used in the whole blood assay for CD8+ T-cell responses are listed in Table 6.Table 6: Peptides used in CD8+ T cell whole blood assayPeptide name Abbreviation Amino acid sequenceSignal Peptide PPI1-24 MALWMRLLPLLALLALWGPDPAAA 9mer_1 PPI1-9 MALWMRLLP9mer_2 PPI2-10 ALWMRLLPL9mer_3 PPI3-11 LWMRLLPLL9mer_4 PPI4-12 WMRLLPLLA9mer_5 PPI5-13 MRLLPLLAL9mer_6 PPI6-14 RLLPLLALL9mer_7 PPI7-15 LLPLLALLA9mer_8 PPI8-16 LPLLALLAL9mer_9 PPI9-17 PLLALLALW9mer_10 PPI10-18 LLALLALWG9mer_11 PPI11-19 LALLALWGP9mer_12 PPI15-23 ALWGPDPAAFlu MP58-66 MP58-66GILGFVFTLStatistical analysis

[0259] Statistical tests were performed using GraphPad Prism 10 for MacOS (GraphPad Software, San Diego California USA). Datasets with a normal distribution were analyzed using paired or unpaired two-tailed Student’s t-test. Non-parametric data was assessed using an unpaired two-tailed Mann- Whitney U-test or Wilcoxon matched-pairs signed rank test, corrected for multiple comparisons where appropriate. P-values < 0.05 were considered statistically significant. Analysis between multiple groups was performed using ANOVA or Kruskal-Wallis test for non-parametric data, with Dunn’s post-hoc test for comparison between individual groups. Area under the receiver operator characteristic curve (AUROC) calculated using inbuilt function in Prism. The Coefficient100622503778of variation (CV) was calculated as the ratio of the standard deviation to the mean, expressed as a percentage (%CV = SD / mean x 100).Example 2: Effector T cell assay

[0260] Peripheral blood is the only tissue that is routinely available to measure human immune responses. Whole blood assays have been used to detect T-cell responses to the vaccine antigens tetanus toxoid and SARS-CoV-2 by measuring cytokines in the plasma after a period of in vitro culture. In fact, stimulation of whole, heparinized blood, has become a standard diagnostic to detect responses to mycobacterial infection. However, these whole blood functional T-cell assays are not sufficiently sensitive to measure autoimmune T-cell responses. Here, the inventors describe the development and validation of a simple whole blood assay capable of detecting p-cell antigen-specific CD4+ T-cell responses well suited to use in a clinical setting called BASTA (p-cell antigen specific T-cell assay).BASTA is superior to the CFSE-based Proliferation Assay

[0261] To evaluate BASTA, the inventors compared CD4+ T-cell responses to full-length C-peptide detected using the CFSE-based proliferation assay to responses detected using BASTA from the same blood samples (Fig. 1A). In the CFSE-based proliferation assay, the number of cells that proliferate, in response to an antigen over 7 days are counted by flow cytometry. Responses are expressed as a ratio (cell division index, or CDI) of the number of cells that have proliferated with and without antigen. For this cohort, while the CDI values were increased in the T1D group (n = 17) compared to the non-T1D samples (n = 8), this was not statistically significant (Mann-Whitney, P = 0.92) (Fig. 1B). In contrast, using BASTA C-peptide specific responses were determined by measuring the concentration of IL-2 present in the plasma after 24-hour of autoantigen stimulation, in the high sensitivity Meso Scale Discovery (MSD) ECL-based immunoassay platform. The inventors chose to measure IL-2 because this cytokine has a very low basal concentration and consequently, gives the best signal-to-noise ratio compared to I FNy, TNFa, IL-6, IL-10, and IL-13 (Figure 2C). The positive control was the vaccine antigen tetanus toxoid (TT), which gave comparable results to a commercially available pool of viral peptides (CEFX) (Fig. 2B-C). IFNy was also higher in C-peptide-stimulated T1D samples compared to non-T1D samples (Figure 2B),100622503779however high background IFNy in unstimulated blood impacted fold-change cytokine increase compared to IL-2 measurements (Figure 2C), which were lower than those observed for IL-2 (Wilcoxon Matched-pairs test, p = 0.03) (Figure 2D). Antigen-stimulated plasma IL-2 concentrations were reported as fold-change over background levels in control, untreated blood samples that were also cultured for 24h. BASTA detected C-peptide responses in >60% (12 of 17) of samples from subjects recently diagnosed with T1D (<100 days), but only in 1 of 8 HLA-matched non-T1D subjects with no family history of T1D (Mann-Whitney, P = 0.0008) (Fig. 1D). This corresponded to an area under the receiver-operator characteristic curve (AUROC) of 0.89 (P = 0.0015) for BASTA and 0.52 (P = 0.8676) for the CFSE-based Proliferation Assay. Furthermore, the CFSE assay showed lower signal per sample (expressed as the cell division index, GDI) than BASTA (Wilcoxon matched-pairs signed rank test, P = 0.04) (Fig. 1F). A higher proportion of samples from non-diabetic subjects had positive responses (CDI>3.0) in the CFSE assay than with BASTA (Fig. 1G). The inventors conclude that BASTA is more sensitive and specific for detecting full-length C-peptide specific responses in T1D than the CFSE-based proliferation assay.Optimizing BASTA for detecting T-cell responses to preproinsulin

[0262] Seventeen overlapping 18mer peptides which spanned the entire length of human preproinsulin (PPI) were tested. These peptides were combined into eight pools of two or three peptides (Fig. 3A). While responses were detected in blood samples from subjects with T1 D from across the entire sequence of preproinsulin, the most T1 D-specific responses in this cohort were detected to pools 1, 3, and 8, corresponding to the signal peptide, B-chain, and A-chain of preproinsulin (T1D n = 18, non-T1D n = 9) (Fig. 3A).

[0263] To determine the effect of peptide length on T-cell responses measured using BASTA, the inventors compared pools of overlapping 18mer peptides to single longer peptides (referred to as ‘full-length’) which covered the sequence of four regions of preproinsulin: signal peptide, B-chain, C-peptide and A-chain (Fig. 3B) (peptide sequences listed in Table 4). Full-length signal peptide stimulated stronger responses in samples from people with T1D (n = 9) than overlapping 18mers, each at the same molar concentration (Wilcoxon multi-test, adjusted P = 0.02). 56% (5 of 9) samples responded to full-length C-peptide, but none responded to the corresponding pooled10062250378018mers (0 of 9). In contrast, responses to insulin B-chain were unaffected by peptide length and responses to full-length A-chain were diminished compared to 18mers (Wilcoxon multi-test, adjusted P = 0.06). A concentration of 10.0 pM C-peptide induced highly disease-specific responses. Titration experiments revealed that 2.0 pM preproinsulin signal peptide and insulin B-chain increased the T1D specificity of responses (Fig. 4A), whereas 10.0 pM was optimal for insulin A-chain.

[0264] The presence of cysteines in the peptides can impact their immunogenicity and stability. Full-length insulin A-chain is very difficult to synthesize and work with due to its insolubility and reactivity, which may have contributed to its relatively poor performance in these assays. To find more stable variant peptides the inventors evaluated the effect of substituting cysteine residues with serine in insulin B-chain and A-chain. Serine substitution for cysteine had no apparent deleterious effect on responses to B-chain, but destroyed recognition of the A-chain peptide (Fig 4B). To address the issues with handling the full-length insulin A-chain peptide, an isoacyl bond was introduced between Thr8 and Ser9. The isoacyl bond increases the solubility of the peptide and suppresses aggregation, but spontaneously isomerizes to native A-chain at physiological pH (Liu, F. et al (2014) Angew Chem Int Ed Engl, 53(15): 3983-7; Karas, J. A. et al (2014) Chemistry, 20(31): 9549-52). Full-length insulin A-chain elicited responses from all three test T1 D samples (Fig. 4B).

[0265] In a pediatric clinical setting, the blood volume required for a test is often limiting. To determine the minimum volume of blood required for the assay responses to preproinsulin peptides from 1.0, 0.5 or 0.25 ml of blood were compared, maintaining relative volume and concentration of peptide stimulus. There was no decrease in IL-2 response to all four preproinsulin peptides using 0.5ml compared to 1.0 ml (Fig. 4C-D) of blood from three individuals. However, reducing the volume to 0.25 ml increased the inter-assay variability (Wilcoxon matched pairs signed rank test, P = 0.0494) (Fig. 4E). Hence, 0.5 ml blood per antigen treatment was chosen for future assays. The inventors also compared preproinsulin induced IL-2 responses after culture in two different vessels: 1.0 ml polypropylene cryovials (Falcon), or 5.0 ml round-bottom polypropylene capped tubes (SPL Life Sciences) (Fig. 4G). Responses were not significantly different between the different tubes and gave similar %CV. 5.0 ml capped tubes was chosen for handling convenience. In summary, the optimized BASTA format is: full-length peptides100622503781at a final concentration of 2.0 pM for the preproinsulin signal peptide and insulin B-chain, or 10.0 pM for C-peptide and insulin A-chain (as isoacyl A-chain), delivered as concentrated peptide stock in minimal solvent to 0.5 ml whole blood per antigen treatment in a capped 5.0 ml polypropylene tube.T 1 D-associated IL-2 responses to proinsulin peptides

[0266] Individual peptides with sequences corresponding to all four regions of preproinsulin (signal, B-chain, C-peptide and A-chain) drove increased IL-2 responses in 33 subjects with T1D compared to 19 non-T1D subjects (Multiple Mann-Whitney tests, Signal peptide: adj. P = 0.03, B-chain: adj. P = 0.00008, C-peptide adj. P = 0.008, A-chain adj. P = 0.008) (Fig. 3C).

[0267] Native insulin B-chain showed reduced utility as a BASTA stimulus in pediatric subjects. A modified form of B-chain with both cysteine residues substituted with serine was tested for reduction of potential non-specific stimulation resulting from potential oxidisation of the cysteines or other difficulties arising from this less stable peptide format. Comparing 6 non-T1D subjects to 6 T1D subjects with established disease, C-to-S substituted B-chain, there were clear responders within the T1D group not observed within the non-T1D group (Multiple Mann- Whitney, adjusted p = 0.06), and no discernible difference between donors based on T1D-status for native B-chain (adjusted p = 0.81) (Fig. 3E). Non-T1D associated responses were more associated with native B-chain, with responses to this form showing a trend to being increased in non-T1D, and decreased in T1 D subjects, relative to responses to the modified form (Wilcoxon signed-rank matched pairs test, p = 0.06 for non-T 1 D, p = 0.09 for T 1 D). Utility of B-chain with C-to-S substitution was confirmed using additional subjects for validation, comparing a total of 13 non-T1D to 17 T1D subjects, with a clear significant T1D-associated BASTA response (Mann-Whitney, p = 0.0062) (Fig. 3G). This gave an AUROC of 0.77 (p = 0.01) (Fig. 3H).

[0268] With the aim of further minimizing volume of blood required, responses against a pool of all four full-length preproinsulin peptides (Signal peptide, B-chain, C-peptide, isoacyl-A-chain) was tested. Responses from 11 adult participants without T1 D, including roughly 40% with T1 D-associated high-risk HLA-DR3-DQ2 and HLA-DR4-DQ8 HLA alleles, were compared to 20 subjects with T1D disease (duration ranging100622503782from the day of diagnosis to 12 years after diagnosis with T1D). IL-2 responses to the preproinsulin peptides were clearly elevated in samples from T1D donors (Mann-Whitney, P <0.0001). Responses to preproinsulin pool were very effective at distinguishing subjects with T1D from those without T1D, giving an AUROC of 0.93 (P < 0.0001) (Fig. 5A). A majority of T1D subjects in this cohort had longstanding T1D, which contributed to the low proportion of C-peptide responders (Fig. 6A).

[0269] The inter- and intra-assay variability of BASTA was then evaluated using individual preproinsulin peptides and a pool of all four preproinsulin peptides. The intra-assay variability was low (Fig. 7A). In samples from four subjects with established T1D, duplicate stimulations of blood from the same draw using the preproinsulin pool in separate stimulation tubes had a mean CV of 13.7%, and duplicate wells of plasma in the IL-2 assay had a mean CV of 3.7%. While this provides evidence of strong reproducibility, it indicates individual blood aliquots and their stimulation as a greater source of experimental variation compared to IL-2 measurement in the MSD assay (Wilcoxon signed-rank test, P = 0.0076) (Fig. 7A). The inter-assay variability was determined by testing samples from four individuals with longstanding T1D (T1D#1-4) on four occasions 1-3 weeks apart. Triplicate whole-blood cultures were set up for each individual preproinsulin antigen, preproinsulin pool, or tetanus toxoid. After collecting the triplicate plasma samples, a single IL-2 measurement was made from each. All individuals consistently exhibited responses to the preproinsulin pool (Fig. 7B). Interassay variability for responses to the preproinsulin pool had a mean CV of 31.7-54.7% (Fig. 8B). For individual antigens, the inter-assay variability was lowest for responses to tetanus toxoid (mean CV: 11.72%) and highest for responses to preproinsulin signal peptide (mean CV: 53.9%) (Fig. 8C).Whole Blood IL-2 release assay measures antigen-specific CD4+ memory T cell responses

[0270] The inventors then sought to determine the cellular source of the IL-2 detected in the whole blood assay in responses to antigenic stimulation. First, the requirement for HLA class II was investigated by adding HLA-class II blocking mAbs to the whole blood culture. IL-2 release was modestly inhibited in the presence of blocking mAb specific for HLA-DR, -DP and -DQ, but almost completely inhibited in the presence of all three mAbs (One-way ANOVA with Holm-Sidak multiple-comparisons test, adj. P = 0.0003)100622503783(Fig. 9A). Similar trends were observed from tetanus toxoid stimulations (P = 0.0775) (Fig. 10B). In contrast, an anti-HLA class-l mAb (W6 / 32), of the same isotype (murine lgG2a) did not attenuate the IL-2 responses (Fig. 10A). Next, the inventors directly identified the IL-2 producing cells using IL-2 surface capture and flow cytometry. In response to a pool of preproinsulin peptides, or tetanus toxoid, a distinct population of IL-2+ CD4+ T-cells was seen (Fig. 9B). The number of CD4+, IL-2+ T-cells recovered using the surface IL-2 capture assay was strongly correlated with IL-2 concentrations from a whole blood assay run in parallel (Pearson r² = 0.93, P < 0.0001) (Fig. 9C). Phenotypic analysis of the IL-2 producing cells (Fig. 10C) revealed that after preproinsulin peptide stimulation, the CD4+, IL-2+ T cell subset contained significantly fewer naive cells (CD45RO-, CCR7+) than the CD4+, IL-2- subset (multiple paired T-test, adj. P = 0.03) (Fig. 9D). Conversely, the CD4+, IL-2+ T cell subset shows a trend towards being enriched in CD45RO+ memory cells, particularly CD45RO+, CCR7-effector / effector memory T cells (adj. P = 0.09). Furthermore, CD69 was found exclusively expressed on CD4+, IL-2+ T cells (Mann-Whitney, P = 0.008) (Fig. 9E). Hence, the inventors conclude that BASTA measures memory / effector CD4+ T-cell responses to p-cell antigens and tetanus toxoid.BASTA measures T1D-specific CD4+ T-cell responses in children

[0271] Up to this point, the inventors had largely used HLA-matched adult donors for the non-T1D control samples. To determine if the age of the subject impacted CD4+ T-cell responses to preproinsulin, the assay was validated by measuring responses to preproinsulin peptides in a cross-sectional cohort of children and adolescents, ages 3-to 20-years-old (as per scheme Fig. 12A, see also Table 3 for participant details). Test subjects included participants without T1D (“low-risk”), recruited from the community (n = 10) as well as children at-risk for developing T1 D because they have a first-degree relative (parent or sibling) with T1 D. Samples for the whole blood assays were taken at a single time point in subjects greater than 3 years of age. These at-risk participants were further stratified into two groups: (i) negative for islet autoantibodies (“AAB-”) (n = 14), or (ii) positive for one or more islet autoantibody (“AAB+”) (n = 8). Subjects with a clinical T1D diagnosis were also recruited and divided into two groups: (i) pediatric participants within 100 days of diagnosis with T1D (“New-OnsetT1D”) (n = 12), or (ii) those with established T1D, > 100 days post diagnosis (“Established T1D”) (n = 20). In100622503784contrast to the test cohort using adult non-diabetic samples (in Fig. 5A), whole blood from young non-T1D pediatric subjects in the lAAb- and IAAb+ groups gave IL-2 responses to the pool of four preproinsulin peptides similar to those from T1 D participants (Fig. 11 B). As a result, the preproinsulin pool did not distinguish samples based on T1D status (AUROC = 0.55, P = 0.5182) (Fig. 11C), with no increase in AUROC by comparing non-T1D samples with only New-Onset T1 D (Fig. 11 D). In marked contrast, responses to full-length C-peptide were clearly associated with T1D (Fig. 11D). These largely distinguished non-T1D from T1D samples, although lower prevalence of C-peptide responders in established T1D participants lowered the overall AUROC (AUROC = 0.77, P = 0.0002) (Fig. 11E). CD4+ T-cell responses to C-peptide detected by BASTA were markedly different between new-onset T1 D and subjects without T1 D (AUROC = 0.86, P = 0.0003) (Fig. 11 F). When CD4+ T-cell responses to individual preproinsulin peptides were evaluated, many pediatric non-diabetic subjects had detectable responses to the preproinsulin signal peptide and insulin B-chain. There was no difference between autoantibody positive (IAAb+) and negative (lAAb-) non-diabetic children with a family history of T1D (Fig. 12). In contrast, non-T1D subjects, with no family history of T1D, showed significantly lower responses to insulin B-chain compared to the other groups, notably the AAB- non-T 1 D children with a first-degree relative with T1D (Kruskal-Wallis using Dunn’s post-hoc test, adj. P=0.04) (Fig. 12B). A similar trend is reflected in the differential responses of these groups to the preproinsulin pool (P = 0.06) (Fig. 12A). As in the initial test cohort (Fig. 1D), responses to C-peptide above 3-fold over background are highly specific for individuals with T1 D and most prevalent in New-Onset T1 D (Kruskal-Wallis using Dunn’s post-hoc test comparing non-T1D to New-Onset T1D, adj. P = 0.01) (Fig. 12D). Responses to the isoacyl insulin A-chain were low in all groups (Fig. 12E). IL-10 secretion was minimal in response to all stimuli for all subject groups (Fig. 13A). When stratifying for H LA-type, individuals with T1D who are heterozygous for high-risk HLA-DQ2 and -DQ8 alleles showed the strongest IL-2 responses to full-length C-peptide (Kruskal-Wallis using Dunn’s post-hoc test comparing non-T 1 D X / X to T1 D DQ2 / DQ8 [adj. P = 0.0026]) (Fig.13B). Upon further stratification, responses to C-peptide were also indeed higher in samples from at-risk pediatric donors if positive for multiple islet autoantibodies, compared to those positive for a single autoantibody (Kruskal-Wallis using Dunn’s post-hoc test, adj. P = 0.02) (Fig. 13C). This was specific for C-peptide, as increased IL-2100622503785from multi-antibody-positive individuals was not observed in response to any other preproinsulin peptide (Fig. 13C). The inventors concluded that full-length C-peptide specifically stimulates T1 D associated CD4+ T-cell responses in young people which can be measured with the whole blood assay.

[0272] BASTA is a simple whole blood assay that is suitable for the routine clinical measurement of p-cell antigen-specific CD4+ memory T-cell responses. This assay overcomes many of the obstacles inherent in assays for CD4+ T-cell autoimmunity against p-cell antigens. The major advantages of this assay include: (i) it requires minimal blood, as low as 1.0 ml (for two 0.5 ml replicates) per antigen treatment, (ii) it is very simple to perform, (iii) it has a short 24 h culture period, (iv) plasma samples can be stored, shipped and analyzed in batches, (v) the platform can be easily adapted to different antigens and peptide epitope combinations, (vi) surplus plasma is generated, which can be easily assayed for additional biomarkers. The inventors anticipate that this assay will be very useful for the monitoring of changes in p-cell antigen-specific memory CD4+ T-cell function in clinical trials and other clinical research settings. This assay may allow more accurate prediction of the time taken for an individual to progress to Stage 3 T1D and require insulin therapy.

[0273] The C-peptide and, to a lesser degree, insulin A-chain showed specificity for T1D (Fig. 3C, Fig. 11). Pooling of preproinsulin peptides did show advantages in greatly enhancing the proportion of responders (as in Fig. 8A) and the absolute IL-2 concentrations, but at the cost of reduced T1 D specificity in younger donors (Fig. 11 B, C). This suggests that pooling peptides will be a useful strategy for increasing the breadth of T-cell responses measured, but that the composition of this peptide pool needs to be carefully evaluated.

[0274] In response to preproinsulin peptides, CD4+ T-cell effector cells are the predominant source of IL-2. The inventors found little evidence for IL-2 production by CD8+ T-cells, or other cell types. This is not surprising since relatively long peptides were used, which would be expected to favour HLA class II restricted, CD4+ T-cell responses and a relatively short culture period, and favours activation of memory T cells. An increase in IL-10 secretion, in response to preproinsulin and other islet autoantigens has been reported in non-diabetic siblings of children with T1D. IL-10 was100622503786interpreted to be a surrogate for regulatory T cells. However, the inventors did not detect any evidence that IL- 10 is useful for measuring regulatory T-cell responses. Although IFNy is commonly measured in immunoassays, the high background of IFNy in cultured whole blood makes this cytokine unsuitable for this application. IL-2 signals were reproducible across assays, and showed comparable variability (%CV) to previously described T-cell assays for p-cell antigens using tetramers (mean CV 14-23%) or the CFSE-based Proliferation Assay (mean CV 30-53%).

[0275] The preliminary assay validation and optimization used samples from adults without T1 D (>20 years of age) as negative controls, compared to T1 D samples recruited from a pediatric (<18 years of age) endocrinology clinic. However, preproinsulin signal peptide and insulin B-chain later showed less T1D specificity when used to analyze samples from T1D children compared to age-matched negative controls. Most strikingly, while responses to the preproinsulin pool from non-T1D adults were low or absent (Fig. 5A), responses to this pool from non-T1D children were similar to those of age-matched children with T1 D (Fig. 11 B). This could indicate dynamics of beta cell antigen-specific T-cell reactivity that change with age, or more simply general changes to reactivity of T-cells. The test cohort included children with and without islet autoantibodies. While the overall presence or absence of islet autoantibodies did not give any clear association between CD4+ T-cell responses to preproinsulin peptides (Fig. 12A-E), further subset analysis revealed that multi-antibody positive children had stronger C-peptide responses compared to single antibody positive children, in accordance with the heightened T1 D risk in this subgroup. This further indicates a potential role for full-length C-peptide-directed CD4+ T-cell responses as a biomarker of stage 1 or 2 T1 D.

[0276] In conclusion, T1D specific CD4+ T-cell responses to full-length C-peptide can be readily measured using a simple whole blood-based assay. Due to its simplicity and robustness, BASTA will be a useful and powerful tool for both dissecting autoimmune CD4+ T-cell responses and monitoring changes in clinical trials and community settings. This work paves the way for the development of a long-sought after T-cell assay that is suitable for routine clinical monitoring of CD4+ T-cell responses in people with, or at risk of T1D.100622503787Example 3: Regulatory T cell (Treg) assay

[0277] Beta cell antigen specific regulatory T cell responses to beta cell peptides were evaluated through a modified BASTA designed to measure bystander suppression, the hallmark of regulatory T cells, induced by preproinsulin peptides added to whole blood and co-incubated with driver vaccine antigen tetanus toxoid (TT) (as per scheme in Fig.14A). Overlapping 18mer peptides which spanned the entire length of human preproinsulin (PPI) were tested for their capacity to suppress TT-induced IL-2 release. These peptides were combined into eight pools of two or three peptides (Fig. 14B). Negative controls were TT added to whole blood alone with peptide solvent control (DMSO), or TT-stimulated whole blood with additional short irrelevant peptides (4mers), generally considered too short to serve as typical T cell epitopes. Strikingly, IL-2 appeared reduced after the addition of PPI pool 1 + TT in samples from non-T1D subjects, on average giving only 40-50% of IL-2 signal observed forTT plus irrelevant 4mer peptides (Fig. 14B), while no similar change was observed in the T1D samples (non-T 1D n = 7, T1D n = 5). A second validation cohort of blood samples from non-T 1 D (n=12) and T1D ( n=15) confirmed a clear, significant decrease in typical TT-induced IL-2 signal when PPI pool 1 is added alongside TT to non-T1D samples, with samples from T1D subjects showing no such reduction (Mann- Whitney U test, p < 0.0001).Corresponding ROC analysis gave an AUROC of 0.94 ( p <0.0001) (Fig. 14C). PPI pool 1 consists of sequences from the preproinsulin signal peptide. Initial investigation into the mechanisms behind this suppression compared full-length 24mer peptide corresponding to the entire sequence of PPI signal peptide to PPI pool 1, or either of the single 18mer peptides derived from signal peptide that comprise PPI pool 1. While samples from non-T1D subjects reproduced an average 50% reduction in IL-2 with the additional of PPI pool 1 to TT compared to TT with negative controls alone, no such reduction was induced by full-length signal peptide (Fig. 14D). A partial reduction was observed in non-T1D samples with the addition of each 18merfrom signal peptide alone with TT, most clearly PPI#1, corresponding to the first 18 amino acids of the signal peptide. This highlights a significant difference between reduced reactivity of blood samples to driver antigen through the addition of beta cell peptides with clear association to T1 D status. This points to a potential role for antigen-specific, PPI signal peptide-reactive suppressor cells in mediating this effect, and a novel approach for measuring functional regulatory responses in a simple assay.100622503788

[0278] Figure 15 shows that CD8+ T-cell responses to some 9mer peptides that mimic the sequence of preproinsulin can be readily detected. A full-length preproinsulin leader sequence peptide (Signal Peptide 24mer) did not stimulate an IL-2 response suggesting that the responses detected were mediated by HLA-Class I restricted CD8+ T cells. All three HLA-A2+ donors showed responses to FluMP58-66. Two were above the 3-fold cutoff used in the CD4+ whole blood assays. As expected, the HLA-A2⁻ᵛᵉ subject did not respond to Flu MP₅₈₋₆₆. Importantly, all four long-standing T1D subjects had some response to the PPI5-14 (MRLLPLLAL).Example 4: Characterisation of regulatory T cell response in T1D

[0279] The inventors will define the immune cell population that mediates the suppression of the responses to tetanus toxoid. To identify and isolate the antigen activated population of T cells, the inventorswill identify the regions, or epitopes, from within the preproinsulin (PPI) leader sequence peptide that are recognized by the putative CD4+ regulatory T cells. There may be overlapping sequences that are recognised by regulatory T cells, or specific sequences that are recognized by the Treg cells.

[0280] The inventors will also determine the mechanism of by which the putative regulatory T cells suppress the tetanus toxoid specific responses in the Treg assay. Many mechanisms have been described by which regulatory T cells suppress bystander T-cell responses in other diseases and models. The inventors will identify which, if any of these are used by the regulatory T cells of the invention.

[0281] To characterise the Tregs associated with this effect, the inventors will enrich rare antigen-specific effector and regulatory T cells, as described for commensal microbes, infectious agents and allergens in Bacher et al (2016) Cell,167(4):1067-1078.e16. This involves staining for effector markers (for example, CD154, CD69, 0X40) that are characteristic of recent antigen-specific reactivation of circulatory memory CD4+T cells present in peripheral blood samples. In tandem, the inventors will stain for CD137, a similar marker that can identify regulatory T cell upon stimulation with their cognate antigen.100622503789

[0282] Preliminary analysis using control stimuli for these cells show a high proportion of CD127+, CD25lo, FoxP3_CTLA-4' T effector cells within the cells identified as CD154+CD137 antigen-reactive conventional effector T cells. Similarly, high proportions (~90%+) of CD137+CD154_antigen-reactive CD4+ T cells are CD25hiCD127lo cells, indicating a T regulatory phenotype, further confirmed by high proportions of FoxP3+CTLA-4+ positivity. The inventors will analyse the T cells responsive to signal peptide (including the peptides showing suppressive capacity in the TT-suppression assay) in terms of different proportions between people with and without T1D, and compare the phenotype of these cells between people based on disease state.

[0283] The inventors will further evaluate whether any differences in the proportion and phenotype of these cells between groups based on T1 D status are associated with responses in the whole blood TT-suppression assay, as evidence for the association of these cells with the effect. For example, a higher proportion of CD137+, CD154- cells may be associated with protection from T1 D. As a proof of principle, the set-up of the whole blood assay will be adapted to a less clinically useful PBMC-based system, which will allow mechanistic exploration, including depleting Tregs or blocking their functions to further demonstrate a role for Tregs in the mechanism of suppression during the suppression of TT-induced IL-2. After enriching sufficient numbers of rare signal peptide-specific Tregs, the inventors will further demonstrate their role in the bystander suppression of IL-2 responses to antigen, by comparing the suppressive function of these cells in T1D and non-T1D individuals using in vitro suppression assays for bystander T cell responses.

[0284] After validating the biological relevance of the bystander suppression capacity of the core beta cell peptides from preproinsulin signal peptide, and the association of this effect with T1 D disease status, the inventors will screen a panel of up to 34 recently custom synthesised peptides with sequences from beta cell proteins previously identified to stimulate T 1 D-associated effect responses. The bystander TT-suppression assay will be used to evaluate the potential of these additional candidate peptides to reveal Treg responses that diverge from normal ranges in T1D.

[0285] The initial experiments show very low induction of IL-10 (the prototypical regulatory cytokine) in the whole blood assay. The inventors will expand their analysis100622503790to include other regulatory cytokines, including IL-35 and TGF-β, to measure alongside IL-2.

[0286] Similar HLA-A2+subjects without T1D will be tested to determine if the responses detected are T1D specific. Several other beta-cell antigen peptides that have been identified as being the targets of human islet-infiltrating CD8+T cells will also be tested.

[0287] Collectively, the whole blood assay can measure CD4+effector, CD4+regulatory and CD8+effector T-cell responses against beta-cell antigens to provide a complete picture of the adaptive immune response against beta cells in human T1D.Example 5: Testing of combined T1D assay in patient cohort

[0288] A combined whole blood assay includes testing effector responses to the candidate beta cell epitope peptides and TT-suppression capacity in tandem.

[0289] The inventors will use the combined assay (i.e. method / assays of the present disclosure which involve where an assay sample is formed by contacting a whole blood sample with a stimulant, and methods / assays of the present disclosure which involve where an assay sample is formed by contacting a whole blood sample with a stimulant and a driver antigen) to evaluate the benefits of a combined metric, compared to either assay alone to stratify T1D patients, non-diabetic controls, and those with signs of early disease (multi-antibody-positive individuals).Example 6: Testing of assay in a young cohort

[0290] The inventors will confirm that young (~8-18years of age) individuals without T1D have a regulatory T cell response to PPI leader sequence peptides or fragments thereof, similar to the adults tested. A small number of these individuals (n=~3) have been tested and this number will be increased to about 10.

[0291] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.100622503791Example 7: CD4+Teff responses with non-insulin p cell antigens (NIBA) and pooled peptide antigens

[0292] Peptides were screened in two steps. First, the inventors identified 30 non-insulin cell antigen (NIBA) peptides for screening. The 30 NIBA peptides are listed in Table 7 and are included in Table 1.Table 7: NIBA peptides for screeningProtein Peptide Amino acid sequencenameCHGA CHGA342- WSKMDQLAKELTAE355GAD65 GAD113- DVMNILLQYVVKSFDRSTKV132GAD65 GAD115- MNILLEYVVKSFD127_Q120EGAD65 GAD121- YVVKSFDRSTKVIDFHYPNE140GAD65 GAD265- KGMAALPRcitLIAFTSEHSHFS284_R272CitGAD65 GAD274- IAFTSEHSHFSLK286GAD65 GAD335- TAGTTVYGAFDPLLAVAD352GAD65 GAD521- ERMSRLSKVAPVIKARMMEYGTT535GAD65 GAD555- NFFRMVISNPAAT567I-A2 I-A2198- SLSYEPALLEPYLFHEFGS216_Q207E,Q213EI-A2 I-A2293- VPRLPEQGSSSRAEDSPEG311I-A2 I-A2318- GDRGEKPASPAVQPDAALQRLAAVL342I-A2 I-A2449- SPLGQSQPTVAGQPSARPAAEEYGYIVTDQKPLSLAAGVK488100622503792I-A2 I-A2523- QNLSLADVTEEAGL536_Q532E,Q533EI-A2 I-A2545- TGLEILETGVGEREEAAA562_Q548E,Q551E,Q556EI-A2 I-A2654- VSSVSSQFSDAAQASPSSHSS674I-A2 I-A2709- LAKEWQALCAYQAEPNTCATAQGEGNIK 736I-A2 I-A2752- KLKVESSPSRSDYINASPIIEHDP775I-A2 I-A2854- FYLKNVQTQETRTLTQFHF872I-A2 I-A2955- SKDQFEFALTAVAEEVNAI LK975IAPP IAPP65- VGSNTYGKRcitNAVEVLKRcitEPL 84_R73Cit,R81CitIGRP IGRP23-35 YTFLNFMSNVGDPIGRP IGRP226- RVLNIDLLWSVPI238IGRP IGRP247- DWIHIDTTPFAGL259ZnT8 ZnT81- MEFLERTYLVNDKAAKMHAFTLESVEL 27_Y18HZnT8 ZnT8106- HLLIDLTSFLLSLFSLWLSSKPPSKRL 132ZnT8 ZnT8120- SLWLSSKPPSKRLTFGWHRAEILGALL 146ZnT8 ZnT8211- NASVRAAFVHALGDLFQSISVLISALI 237ZnT8 ZnT8244- YKIADPICTFIFSIL258ZnT8 ZnT8266- ILKDFSILLMEGVPKSLNYS285ZnT8 ZnT8267- LKDFSI LLM EG VPKSLN YSG VKELI LA 293ZnT8 ZnT8309- TMNQVILSAHVATAASRDSQVVRREIA335100622503793Rcit=citrulline

[0293] E, in bold text = glutamic acid ( E) in place of the naturally occurring glutamine (Q).

[0294] The inventors then screened the 30 N I BA with whole blood samples from individuals without T1D (non-T1D n = 6) samples. Then the inventors selected 11 NIBAS (CHGA342-355, GAD65l21-140, GAD65274-286, GAD65555-567, I-A2l98-216 Q-> E, I-A2293-311, I-A2318-342, I-A2545-562 Q-> E, I-A2654-674, I-A2709-736 and l-A2752-77s) that did not stimulate any responses in non-T1Ds. These 11 non-insulin beta-cell antigens (NIBA) peptides were then tested using BASTA with whole blood samples from 12 T1D individuals. Based on area under the receiver operator curve (AUROC) ranking, the inventors selected 6 NIBAs that yielded AUROC greater than 0.55 (CHGA342-355, GAD65121-140, GAD65274-286, GAD65555-567, I-A2318-342 and I-A2752-775).

[0295] The inventors also tested a peptide known as “X-ID” peptide using the BASTA assay. Briefly, heparinized peripheral blood was aliquoted within 3 hours of blood draw into sterile, capped, polypropylene tubes containing pre-aliquoted X-ID peptide or peptide solvent control (40% acetonitrile, 0.5% acetic acid, water) stimuli. Blood without any additional stimulation, or the peptide solvent alone was an additional negative control. After incubation for 24 h at 37 °C with 5% CO2, samples were centrifuged, plasma was harvested, aliquoted and frozen. Cytokines in the plasma were measured in duplicate using an electrochemiluminescence (ECL)-based immunoassay platform (Meso Scale Discovery (MSD), Rockville, MD, USA), using lot-validated 96-well plate format MSD V-plex kits measured on a MESO QuickPlex SQ 120 reader. Data was analysed using the Discovery Workbench software. If below lower limit of detection (LLOD), results were set to the LLOD for each assay run based on the standard curve. Response cut-off was selected as 3-fold over the unstimulated or solvent only control blood cultures. Non-parametric data was assessed using an unpaired two-tailed Mann-Whitney U-test corrected for multiple comparisons where appropriate. P-values < 0.05 were considered statistically significant. These data indicate that the X-ID peptide stimulates CD4+T-cell responses that are detectable using the BASTA assay.

[0296] Fig. 16 shows the T cell response, measured by IL-2 in the plasma of a heparinized whole blood sample, when contacted with X-ID. Responses to X-ID peptide100622503794were detected in one of seven samples tested from individuals without T1 D (non-T 1 D), whereas 10 of 14 individuals with T1D had responses that were detectable using the BASAT assay platform. This indicates that the X-ID peptide is able to stimulate detectable responses predominantly in individuals with T1D, but rarely in those without T1D.

[0297] X-ID was then pooled with the 6 NIBA peptides identified in the screen and used to stimulate T cell responses using the BASTA assay.

[0298] Briefly, heparinized peripheral blood was aliquoted within 3 hours of blood draw into sterile, capped, polypropylene tubes and were contacted with the pool of polypeptides in Table 8.Table 8: Peptides for the poolPeptide name Amino acid sequenceProinsulin C-peptide EAEDLQVGQVELGGGPGAGSLQPLALEGS (SEQ ID NO: 20) LQInsulin C to S INS-B FVNQHLSGSHLVEALYLVSGERGFFYTPKT (SEQ ID NO: 38)Hybrid insulin HIP-7 GQVELGGGFLGEGHHpeptidesHIP-8 GQVELGGGSSPETLI(SEQ ID NOs:39-41) HIP-13 SLQPLALTPIESHQCHGA342-355 WSKMDQLAKELTAENon-insulin beta GAD121-140 YVVKSFDRSTKVIDFHYPNEcell antigens GAD274-286 IAFTSEHSHFSLK(SEQ ID NOs: GAD555-567 NFFRMVISNPAAT42-47) I-A2318-342 GDRGEKPASPAVQPDAALQRLAAVLI-A2752-775 KLKVESSPSRSDYI NASPI I EH DPOtherX-ID ARQEDTAMVYYFDYW(SEQ ID NO: 48)

[0299] Blood without any additional stimulation, or the equivalent volume of peptide solvent (40% alone was an additional negative control. After incubation for 24 h at 37 °C with 5% CO2, samples were centrifuged, plasma was harvested, aliquoted and frozen. IL-2 in the plasma were measured in duplicate using an electrochemiluminescence100622503795(ECL)-based immunoassay platform (Meso Scale Discovery (MSD), Rockville, MD, USA), using lot-validated 96-well plate format MSD V-plex kits measured on a MESO QuickPlex SQ 120 reader. Data was analysed using the Discovery Workbench software. If below lower limit of detection (LLOD), results were set to the LLOD for each assay run based on the standard curve. Response cut-off was selected as 3-fold over the unstimulated or solvent only control blood cultures. Non-parametric data was assessed using an unpaired two-tailed Mann-Whitney U-test corrected for multiple comparisons where appropriate. P-values < 0.05 were considered statistically significant.

[0300] Fig. 17 shows the responses of blood samples from individuals without (non-T1D), or with type 1 diabetes were tested, using the BASTA assay, to a pool (Table 8) of p-cell antigen derived peptides. Responses to the peptides were detected by measuring the IL-2 in the plasma. The data indicate that responses were rarely detectable in blood from individuals without T1 D, but commonly detected in blood from individuals with T1D. The difference in responses was statistically significant (Mann-Whitney U test) significantly (p<0.05) greater in individuals with T1D. This indicates that the BASTA assay with this pool of peptides can detect CD4+T cell responses associated with type 1 diabetes.Example 8: CD8+Teff responses with peptides

[0301] The inventors also used the BASTA assay to detect CD8+T cell responses associated with type 1 diabetes. Heparinized peripheral blood was aliquoted within 3 hours of blood draw into sterile, capped, polypropylene tubes a single polypeptide peptide stimulant was added (see Table 9) to a final concentration of 10 pM. Blood without any additional stimulation, or the equivalent volume of peptide solvent (DMSO) alone was an additional negative control. After incubation for 24 h at 37 °C with 5% CO2, samples were centrifuged, plasma was harvested, aliquoted and frozen. IL-2 in the plasma were measured in duplicate using an electrochemiluminescence (ECL)-based immunoassay platform (Meso Scale Discovery (MSD), Rockville, MD, USA), using lot-validated 96-well plate format MSD V-plex kits measured on a MESO QuickPlex SQ 120 reader. Data was analysed using the Discovery Workbench software. If below lower limit of detection (LLOD), results were set to the LLOD for each assay run based on the standard curve. Response cut-off was selected as 3-fold over100622503796the unstimulated or solvent only control blood cultures. Non-parametric data was assessed using an unpaired two-tailed Mann-Whitney U-test corrected for multiple comparisons where appropriate. P-values < 0.05 were considered statistically significant. 7 peptides (Table 9) stimulated CD8+T cell responses associated with type 1 diabetes.Table 9: Peptides that stimulate CD8+T-cell responses in whole bloodAntigen Amino acid Amino acid number sequence Preproinsulin leader sequence (PPI) 4-12 WMRLLPLLA(SEQ ID NO:29)10-18 LLALLALWG(SEQ ID NO:35)11-19 LALLALWGP(SEQ ID NO:36)Insulin B-chain 18-27 VCGERGFFYT (SEQ ID NO:52)Islet amyloid polypeptide (IAPP) 5-13 KLQVFLIVL(SEQ ID NO:49)Islet-specific glucose-6-phosphatase 228-236 LNIDLLWSV catalytic subunit-related protein (IGRP) (SEQ ID NO:50)Glutamic acid decarboxylase 65-kilodalton 114-123 VMNILLQYVV isoform (GAD-65) (SEQ ID NO:51)

[0302] The response to each of the seven peptides that stimulate CD8+T cell responses are shown separately in Fig. 18. The data show that all peptides stimulate a response in some of the donors with T1 D, but rarely in those donors without T1 D. This indicates that these peptides stimulate a T1D specific CD8+T cell response in individuals with T1D, but not in those without T1D. Some peptides (eg PPI10-18, PPI11-19, IGRP228-236 and IAPP5-13) stimulated responses in one or both of the individuals tested who had islet100622503797autoantibodies, but had not yet developed clinical, (stage 3) T1D. This indicates the whole blood assay as described here can detect CD8+T cell responses to beta cell antigens. Furthermore, this data shows that responses to some peptides can be detected in individuals before they develop insulin-requiring, or stage 3, T1D.

[0303] The inventors then also tested a pool comprising the 7 peptides from Table 9 in the BASTA assay to determine if the pool could stimulate CD8+T cell responses. Heparinized peripheral blood was aliquoted within 3 hours of blood draw into sterile, capped, polypropylene tubes and were contacted with a pool of the polypeptides in Table 9 to a final concentration of 5 or 1 pM for each peptide within the pool. Blood without any additional stimulation, or the equivalent volume of peptide solvent (DMSO) alone was an additional negative control. After incubation for 24 h at 37 °C with 5% CO2, samples were centrifuged, plasma was harvested, aliquoted and frozen. The concentration of IL-2 in the plasma was measured in duplicate using an electrochemiluminescence (ECL)-based immunoassay platform (Meso Scale Discovery (MSD), Rockville, MD, USA), using lot-validated 96-well plate format MSD V-plex kits measured on a MESO QuickPlex SQ 120 reader. Data was analysed using the Discovery Workbench software. If below lower limit of detection (LLOD), results were set to the LLOD for each assay run based on the standard curve. Response cut-off was selected as 3-fold over the unstimulated or solvent only control blood cultures. Nonparametric data was assessed using an unpaired two-tailed Mann-Whitney U-test corrected for multiple comparisons where appropriate. P-values < 0.05 were considered statistically significant.

[0304] Fig. 19 shows that the whole blood assay, using a pool of seven p-cell antigen derived peptides, is able to detect CD8+T cell responses in all individuals with islet autoantibodies and the individual with established T1D. In contrast, no responses were detected in the blood samples from individuals who did not have T1D. This was seen at both concentrations of peptide tested. As such, the whole blood assay using these peptides can identify T cell response in individuals who have not yet developed insulinrequiring, or stage 3, T1 D.

Claims

100622503798CLAIMS1. A method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample;wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof indicates the individual has type 1 diabetes.

2. A method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample;wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof indicates the individual is displaying early signs or symptoms of type 1 diabetes.

3. A method for detecting and / or monitoring effector T cell (Teff) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample;wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof detects and / or monitors effector T cell (Teff) responses in the individual.1006225037994. A method for detecting and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual at a first time point with at least one T-cell autoantigen or fragment thereof to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with at least one T-cell autoantigen or fragment thereof to form a second assay sample;wherein the second time point is a period of time after the first time point;- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, a cytokine in the second assay sample compared to the first assay sample indicates progression of type 1 diabetes in the individual.

5. A method of determining whether an individual has or is at risk of type 1 prediabetes, the method comprising:- contacting a whole blood sample from an individual with at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample;wherein presence of, or increase in amount of, the cytokine in the assay sample following contact with the at least one T-cell autoantigen or fragment thereof indicates the individual has or is at risk of type 1 pre-diabetes.

6. A method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;1006225037100- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual has type 1 diabetes.

7. A method of determining whether an individual has type 1 diabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual does not have type 1 diabetes.

8. A method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual is displaying early signs or symptoms of type 1 diabetes.

9. A method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising:1006225037101- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual is not displaying early signs or symptoms of type 1 diabetes.

10. A method for detecting and / or monitoring regulatory T cell (Treg) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof detects and / or monitors regulatory T cell (Treg) responses in the individual.

11. A method for detecting and / or monitoring regulatory T cell (Treg) responses in an individual, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof detects and / or monitors regulatory T cell (Treg) responses in the individual.100622503710212. A method for detecting and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes, the method comprising:- contacting a whole blood sample from the individual at a first time point with at least one T-cell autoantigen or fragment thereof to form a first assay sample;- contacting a whole blood sample from the individual at a second time point with at least one T-cell autoantigen or fragment thereof to form a second assay sample;wherein the second time point is a period of time after the first time point;- determining the presence of, or amount of, a cytokine in the first and second assay samples;wherein presence of, or increase in amount of, a cytokine in the second assay sample compared to the first assay sample indicates progression of type 1 diabetes in the individual.

13. A method of determining whether an individual has or is at risk of type 1 prediabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;- determining the presence of, or amount of, a cytokine in the assay sample;wherein presence of, or increase in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual has or is at risk of type 1 pre-diabetes.

14. A method of determining whether an individual has or is at risk of type 1 prediabetes, the method comprising:- contacting a whole blood sample from an individual with a driver antigen and at least one T-cell autoantigen or fragment thereof to form an assay sample;1006225037103- determining the presence of, or amount of, a cytokine in the assay sample; wherein the absence of, or decrease in amount of, a cytokine in the assay sample following contact with the driver antigen and at least one T-cell autoantigen or fragment thereof indicates the individual does not have or is not at risk of type 1 pre-diabetes.

15. The method of any one of claims 1-14, wherein the at least one T-cell autoantigen or fragment thereof comprises a polypeptide or pool of polypeptides which comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NOs: 1-84, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

16. The method of any one of claims 1 to 5, wherein the at least one T-cell autoantigen or fragment thereof comprises a polypeptide or a pool of polypeptides comprising:(a) a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, and / or(b) a hybrid insulin peptide, non-insulin cell antigen, or other T-cell autoantigen, or a fragment thereof.

17. The method of claim 16, wherein the polypeptide or pool of polypeptides comprises a C-peptide or B-chain of preproinsulin or a fragment thereof.

18. The method of claim 17, wherein the C-peptide or B-chain of preproinsulin or a fragment thereof comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 3-14, 20, or 52, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.100622503710419. The method of any one of claims 16-18, wherein the polypeptide or pool of polypeptides comprises the X-ID peptide.

20. The method of any one of claims 16-19, wherein the polypeptide or pool of polypeptides comprises a hybrid insulin peptides (HIP) 7 (HIP-7), HIP-8, HIP-13, Chromogranin-A (CHGA), Glutamic acid decarboxylase 65-kilodalton isoform (GAD65), Insulinoma-associated protein 2 (I-A2), Islet amyloid polypeptide (IAPP), Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), Zinc transporter 8 (ZnT8), or a fragment thereof.

21. The method of claim 19 or 20, wherein the X-ID, hybrid insulin peptides (HIP) 7 (HIP-7), HIP-8, HIP-13, Chromogranin-A (CHGA), Glutamic acid decarboxylase 65-kilodalton isoform (GAD65), Insulinoma-associated protein 2 (I-A2), Islet amyloid polypeptide (IAPP), Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), Zinc transporter 8 (ZnT8), or a fragment thereof comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NOs: 39-84, or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

22. The method of any one of claims 6 to 14, wherein the at least one T-cell autoantigen or fragment thereof comprises a polypeptide or a pool of polypeptides comprising:(a) a signal peptide, a B-chain, a C-peptide or an A-chain of preproinsulin, or a fragment thereof, and / or(b) a hybrid insulin peptide, non-insulin cell antigen, or other T-cell autoantigen, or a fragment thereof.

23. The method of claim 22, wherein the polypeptide or pool of polypeptides comprises a signal peptide of preproinsulin or a fragment thereof.100622503710524. The method of claim 23, wherein the signal peptide of preproinsulin or a fragment thereof comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 1-3, 18, or 26-37 or an amino acid sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

25. The method of any one of claims 6 to 14 or 22 to 24, wherein the driver antigen includes an antigen that is used in vaccination.

26. The method of claim 25, wherein the driver antigen includes influenza haemagglutinin, influenza neuraminidase, the spike protein of SARS-CoV-2, Diptheria toxoid, tetanus toxoid, inactivated polio virus, hepatitis B antigen, or human papilloma virus antigen.

27. The method of claim 25 or 26, wherein the driver antigen includes tetanus toxoid.

28. The method of any one of claims 25 to 27, wherein concentration of the driver antigen contacted with the blood sample is 0.01 - 300 μg / ml, preferably 72 μg / ml.

29. A method of determining whether an individual has type 1 diabetes, the method comprising performing the method of any one of claims 1 or 16 to 21 and performing a method of any one of claims 6, 7 or 22 to 28, wherein:an individual is determined to have type 1 diabetes when a method of any one of claims 1 or 16 to 21 and / or a method of any one of claims 6, 7 or 22 to 28 indicate the individual has type 1 diabetes; oran individual is determined not to have type 1 diabetes when both a method of any one of claims 1 or 16 to 21 and a method of any one of claims 6, 7 or 22 to 28 indicate the individual does not have type 1 diabetes.

30. A method of determining whether an individual at risk of developing type 1 diabetes is displaying early signs or symptoms of the disease, the method comprising performing the method of any one of claims 2 or 16 to 21 and performing a method of any one of claims 8, 9 or 22 to 28, wherein:1006225037106an individual is determined to be displaying early signs or symptoms of the disease when a method of any one of claims 2 or 16 to 21 and / or a method of any one of claims 8, 9 or 22 to 28 indicate the individual is displaying early signs or symptoms; oran individual is determined not to be displaying early signs or symptoms of the disease when both a method of any one of claims 2 or 16 to 21, and a method of any one of claims 8, 9 or 22 to 28 indicate the individual is not displaying early signs or symptoms.

31. A method of detecting and / or monitoring effector T cell (Teff) responses and / or regulatory T cell (Treg) responses in an individual, the method comprising performing the method of any one of claims 3 or 16 to 21 and performing a method of any one of claims 10, 11 or 22 to 28.

32. A method of detecting and / or monitoring progression of type 1 diabetes in an individual at risk of developing type 1 diabetes or in an individual diagnosed with type 1 diabetes, the method comprising performing the method of any one of claims 4 or 16 to 21 and performing a method of any one of claims 12 or 22 to 28.

33. A method of determining whether an individual has or is at risk of type 1 prediabetes, the method comprising performing the method of any one of claims 5 or 16 to 21 and performing a method of any one of claims 13, 14 or 22 to 28, wherein:an individual is determined to have or be at risk of type 1 pre-diabetes when a method of any one of claims 5 or 16 to 21 and / or a method of any one of claims 13, 14 or 22 to 28 indicate the individual has or is at risk of type 1 pre-diabetes; oran individual is determined not to have or be at risk of type 1 pre-diabetes when both a method of any one of claims 5 or 16 to 21, and a method of any one of claims 13, 14 or 22 to 28 indicate the individual does not have or is not at risk of type 1 pre-diabetes.100622503710734. The method of any one of claims 1 to 33, wherein the cytokine is IL-2, IL-10, TGF, a type 2 cytokine, IL-1, IL-6, IFN, IL-15, IL-17, GM-CSF, or IL-21, ora fragment thereof.

35. The method of claim 34, wherein the cytokine is IL-2 or a fragment thereof.

36. The method of any one of claims 1 to 35, wherein the volume of the whole blood sample contacted with a polypeptide or pool of polypeptides is greater than 0.2 ml but equal to or less than 60 ml; greater than 0.2 ml but equal to or less than 50 ml; greater than 0.2 ml but equal to or less than 40 ml; greater than 0.2 ml but equal to or less than 30 ml; greater than 0.2 ml but equal to or less than 20 ml; greater than 0.2 ml but equal to or less than 10 ml; greater than 0.2 ml but equal to or less than 5 ml; greater than 0.2 ml but equal to or less than 2 ml; greater than 0.2 ml but equal to or less than 1 ml; equal to or greater than 0.5 ml but less than or equal to 5 ml; equal to or greater than 0.5 ml but less than or equal to 2 ml; or equal to or greater than 0.5 ml but less than or equal to 1 ml.

37. The method of claim 36, wherein the volume of the whole blood sample contacted with a polypeptide or pool of polypeptides is 0.5 ml or 1 ml for each polypeptide or pool of polypeptides.

38. The method of any one of claims 1 to 37, wherein the cells in the whole blood sample are not labelled, for example with a detectable label.

39. The method of claim 38, wherein the cells in the whole blood sample are not labelled with 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE).

40. The method of any one of claims 1 to 39, wherein the whole blood sample is contacted with the polypeptide or pool of polypeptides and / or the driver antigen for 8 hours; about 8 hours; greater than or equal to 8 hours but less than 7 days; 24 hours; about 24 hours; greater than or equal to 24 hours but less than 7 days.

41. The method of any one of claims 1 to 40, wherein the individual is a child.

42. The method of any one of claims 1 to 40, wherein the individual is an adult.100622503710843. The method of any one of claims 1 to 42, wherein the individual may be, or have been identified as, at risk of developing type 1 diabetes.

44. The method of any one of claims 1 to 43, wherein the individual has not previously been diagnosed with type 1 diabetes.

45. The method of any one of claims 1 to 43, wherein the individual has been identified as having recent-onset type 1 diabetes.

46. The method of any one of claims 1 to 43, wherein the individual has previously been diagnosed with type 1 diabetes.

47. The method of any one of claims 1 to 47, wherein the individual expresses the HLA alleles DQ8, DQ2, DR3 and / or DR4.

48. A polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence of SEQ ID NO: 38.

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