immunoinformatics in diabetes

8/20/2019 Immunoinformatics in Diabetes

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[Draft 1] Diabetes — Immunology

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Type 1 Diabetes Mellitus (T1DM) is a T cell mediated autoimmune disease in which the

insulin-producing pancreatic islet beta-cells in the pancreas of genetically susceptible are

selectively eliminated. Such destruction within the pancreas leads to insuling deficiency, and

eventually hyperglycemia—an excess of glucose in the body (Scott et al. 2010). Studies inmice and men in recent studies show that autoreactive CD8 T cells in humans are involved in

both the initiation of the disease and the destruction of beta-cells (Unger et al. 2011). Although

CD4 and CD8 cells are requried for disease development, the latter plays an important role in

the initiation of tissue damage and promotion of diabetes progression by directly targeting

beta-cells. They also target, aside from insulin, different autoantigens in the ß-cells such as

islet specific glucose-6-phosphatase catalytic subunit related protein (IGRP), dystrophia

myotonica kinase (DMK), and glutamic acid decarboxylase (GAD). Among these, insulin is the

autoantigens detected in mice, leading to the suggestion that removing or regulating insuling-

reactive CD8 T cells may protect against diabetes (Scott et al. 2010). T cells that react to islet

beta cells can contribute to the autoimmune response in diabetic patients and also play a part

in self-tolerance in healthy individuals. The rarity of these cells and inadequate technology

has impaired the examination of this paradigm; thus, the mechanism for the development of

T1DM are not fully understood.

This gap in our knowledge is a result of many constraints on such studies. Most

importantly, islet-infiltrating T cells from patients with diabetes are very rarely available for

expansion and cloning to examine their CTL potential, while the search for such cells

trafficking in the peripheral blood requires prior knowledge of the autoantigens targeted and

the epitopes and HLA-presenting molecules involved. To address these requirements, a study

was conducted by Skowera et al. (2008) created surrogate ! cells expressing a single

autoantigen and HLA class I molecule and investigated their naturally displayed peptide

repertoire. This uncovered the signal peptide (SP) as a source of preproinsulin (PPI) epitopes


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that constitute major targets of CD8+ T cell responses in HLA-A2+ patients with type 1

diabetes. (Skowera et al. 2008).

Epitopes have through the process referred to as “reverse” immunology requiring the

immunization of HLA-A2 transgenic mice. Although, this strategy may introduce false-positiveresults as the majority of epitopes identified display at least one amino acid difference with the

endogenous murine sequence, rendering them xeno-antigens to the HLA-A2 transgenic mice.

Nonetheless, several of these epitopes are recognized by CD8 T-cells of type 1 diabetic

patients. Alternatively, islet-specific autoreactive CD8 T-cell reactivity has been detected

against an insulin epitope identified by high HLA-binding affinity using HLA-A2 tetramers and

cytokine secretion assays. An epitope that lies in the signal peptide (i.e., PPI15-24) was

identified as a major target of CD8 T-cell responses in HLA-A2" T1D patients (Unger 2011).

To examine the functional properties of PPI SP–reactive CD8+ T cells, short-term

CD8+ T cell lines from HLA-A2+ patients with type 1 diabetes who showed appropriate

ELISPOT reactivity were established; focusing on the PPI15–24

epitope because of its

relative immunodominance. From these lines, cells staining with PPI15-24-loaded HLA-A2

tetramers (PPI15–24 –Tmr) were sorted by flow cytometry and expanded, yielding 5 PPI15–

24 –Tmr +CD8+ T cell clones from a single patient. Clones showed HLA-A2–restricted

responses to PPI15–24, either when PPI15–24 peptide was presented after pulsing of HLA-

A2-autologous antigen-presenting cells or when it was presented naturally by K562-PPI-A2

cells. These results indicate that circulating autoreactive CD8+ T cells in patients with type 1

diabetes recognize ! cell–specific targets and have cytotoxic capability (Skowera et al. 2008).


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In recent years, it has become apparent that in some patients, there is a considerable

overlap between type 1 and type 2 diabetes. A small subset of patients with type 2 diabetes

(10%) in the course of disease development produce autoantibodies characteristic of type 1

diabetes: antibodies to insulin, GAD, and IA-2 (McDevitt 2005). Autoantibodies are created by the immune system when it fails to distinguish between

“self” and “non-self”. As mentioned above, A small subset of patients with type 2 diabetes

(10%) in the course of disease development produce autoantibodies characteristic of type 1

diabetes: antibodies to insulin, GAD, and IA-2.

It has been reported that approximately 50% of the genetic risk for T1DM can be

attributed to the HLA region. The highest risk HLA-DR3/4 DQ8 genotype has been shown to

be highly associated with beta-cell autoimmunity. The first antibodies described in association

with the development of T1DM were islet cell autoantibodies (ICA). Subsequently, antibodies

to insulin (IAA), glutamic acid decarboxylase (GAA or GAD) and protein tyrosine phosphatase

(IA2 or ICA512) have all been defined. The number of antibodies, rather than the individual

antibody, is thought to be most predictive of progression to overt diabetes (Taplin & Barker,

2008).

Antibodies to Insulin

The fact that 10% of patients with type 2 diabetes are found to have autoantibodies

characteristic of type 1 diabetes and to have lower C-peptide levels suggests that this is not a

case of a simple coincidence of two relatively common diseases. This result is because of the

known incidence of type 1 diabetes in the general population. In the general population, 20%

lack aspartic acid at DQ 57 on both chromosomes. Thus, at HLA, 20% of the population could

be said to be potentially susceptible to type 1 diabetes. Despite this, only 0.5–0.8% of the

population develop type 1 diabetes. Assuming that there is no association between HLA and


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type 2 diabetes (which has been shown in several early studies), the existence in 10% of type

2 diabetic patients of autoantibody characteristics of type 1 diabetes suggests that the

additional stress produced on islet cells by the necessity to produce more than usual amounts

of insulin can, in individuals who are susceptible at the HLA region, lead to the developmentof autoantibodies, insulitis, and exhaustion of the insulin-producing cells, with a corresponding

decrease in C-peptide and a distinctly different phenotype (McDevitt 2005).

When IgM and IgG antibodies against insulin, the body reacts as if the insulin is

foreign. This makes insulin less effective, or not effective at all. This process results to

hyperglycemia, eventually progressing to T1DM (Eisenbarth 2011).

Regardless of purity and origin, therapeutic insulins continue to be immunogenic in

humans. However, severe immunological complications occur rarely, and less severe events

affect a small minority of patients. Insulin autoantibodies (IAAs) may be detectable in insulin-

naive individuals who have a high likelihood of developing type 1 diabetes or in patients who

have had viral disorders, have been treated with various drugs, or have autoimmune

disorders or paraneoplastic syndromes. This suggests that under certain circumstances,

immune tolerance to insulin can be overcome. Factors that can lead to more or less

susceptibility to humoral responses to exogenous insulin include the recipient’s immune

response genes, age, the presence of sufficient circulating autologous insulin, and the site of

insulin delivery. Little proof exists, however, that the development of insulin antibodies (IAs) to

exogenous insulin therapy affects integrated glucose control, insulin dose requirements, and

incidence of hypoglycemia, or contributes to !-cell failure or to long-term complications of

diabetes. (Fineberg et al. 2013).

Past epitope analysis of IAA have utilized naturally occurring isoforms of insulin. In two

studies, human, bovine and porcine insulin were used to reveal differences in the binding

characteristics of IAA and anti-bodies to exogenous administered insulin (IA), locating the


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major binding site of IA to the A-chain, while type 1 diabetes-associated IAA recognize a

conformational epitope requiring both the A- and B-chain. Differences in epitope specificities

of IAA and IA were also detected using random peptide phage display. Monoclonal antibodies

and their Fab are being used successfully in the epitope analysis of other autoantibodies intype 1 diabetes. This method is particularly useful in the study of conformational epitopes as

the structure of the antigen remains intact (Padoa et al. 2005).

IA responses consisting of virtually all Ig classes and IgG subclasses have been

reported. Insulin-specific antibodies are primarily composed of IgG1–4 antibodies, but IgM,

IgA, and IgE have been reported. Anti-insulin IgM has been detected during early insulin

treatment. Also reported are the presence of that class in patients with immunological insulin

resistance. IgA have been detected in patients and were associated IgA with allergic reactions

in patients with diabetes (Fineberg et al. 2013)

These autoantibodies to insulin and their corresponding epitopes have not been well

characterized until a study by Padoa et al. (2005) cloned and characterized the recombinant

Fab of the insulin-specific monoclonal anti-body CG7C7. In an effort to identify the epitope

recognized by CG7C7, competition assays with insulin-specific monoclonal anti-bodies mAb 1

and mAb 125 were performed. Both antibodies have well-defined epitopes, binding of mAb 1

is strictly dependent on amino acid residue B30, binding of mAb 125 is directed predominantly

to the A-chain loop.

Antibodies to Protein Tyrosine Phosphatase (IA2 or ICA512)

Islet antigen-2 (IA-2), previously known also as ICA-512, is a major target of islet cell

autoantibodies. The protein is found in neural tissue and cells of the pancreatic islets, and its

gene has been localized to chromosome 2q35. The cDNA encodes a 979 amino acid


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transmembrane protein which is enzymatically inactive, and a related PTP-like molecule

termed IA-2! or phogrin is also a major islet autoantigen whose location and intracellular

domain are 74% identical to IA-2. Autoantibodies to IA-2 are present in up to 80% of children

and adolescents at diagnosis of type 1 diabetes.

IA-2 could be a primary target of the immune process which is believed to destroy the

insulin-secreting islet cells. Alternatively, IA-2A might develop as a consequence of this

destructive process, releasing the sequestered antigen IA-2 and thus inducing the immune

response (Decochez 2014).

Antibodies to GAD

Antibodies to the 64,000 M r , proteins are the earliest and most reliable predictive

marker of IDDM in humans and are also present in the two animal models for IDDM, the non-

obese diabetic (NOD) mouse and the Bio-breeding rat (Kaufman et al. 1992).

Baekkeskov et al. (1990) reported that the 64,000 M r islet cell auto-antigen is a form of

glutamate decarboxylase (GAD; E.C.4.1.1.15), the enzyme responsible for the synthesis of y-

aminobutyric acid (GABA) in brain, peripheral neurons, pancreas, and other organs. It is

shown that the brain contains two forms of GAD, which are encoded by two separate genes.

The two GADs (GAD65 and GAD67) differ in molecular size (with M r s = 65,000 and 67,000)

and amino acid sequence (with ~30% sequence divergence), as well as in their intracellular

distributions and interactions with the GAD cofactor pyridoxal phosphate (Erlander et al.

1991).

Both GAD67 and GAD65 are targets of autoantibodies in people who later develop type

1 diabetes mellitus or latent autoimmune diabetes. Lethagen et al. (2001) screened 441


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nondiabetic patients with autoimmune thyroiditis (AT) for GADab to study whether antibodies

to glutamic acid decarboxylase (GADab) are associated with subclinical !-cell damage and

impaired insulin secretion.

Islet Cell Antibodies

The pancreatic islets or islets of Langerhans are the regions of the pancreas that

contain its endocrine (i.e., hormone-producing) cells. The beta cells of the pancreatic islets

secrete insulin, and so play a significant role in diabetes. It is thought that they are destroyed

by immune assaults. However, there are also indications that beta cells have not been

destroyed but have only become non-functional.

In an experiement conducted by Bottazzo et al. (1974), Antibodies to pancreatic islet

cells were found by immunofluorescence in the sera of 13 patients with multiendocrine

deficiencies associated with organ-specific autoimmunity. 10 of these patients were diabetic.

The antibodies were complement fixing and of IgG class; titres varied from 1 to 160 and were

independent of insulin treatment. The presence of organ-specific pancreatic antibodies

supports the hypothesis of an autoimmune form of diabetes mellitus put forward to explain the

histological " insulitis " found in selected cases of this disease. This new marker allows the

segregation of a homogeneous group of insulin-dependent diabetics who may well prove to

have a different metabolic pattern from that in other forms of inherited diabetes mellitus.


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References

Baekkesko S, Aanstoot S, Christgau A,Reetz M, Solimena M.Cascalho F, Foli H ,Richter-Olesen and DeCamilli P. (1990).Identification of the 64K autoantigen ininsulin-dependent diabetes as theGABA-

synthesizing enzyme glutamic aciddecarboxylase. Nature(London.).347:151-156.

Bottazzo GF, Florin-Christensen A,Doniach D 1974 Islet-cell antibodies indiabetes mellitus with autoimmunepolyendocrine deficiencies. Lancet 2:1279–

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Decochez, Katelijn. IA-2 antibodies[internet]. 2014 Aug 13; Diapedia21042821251 rev. no. 14. Availablefrom:http://dx.doi.org/10.14496/dia.21042821251.14

Eisenbarth GS, Buse JB. (2011). Type 1diabetes mellitus. In: Melmed S,Polonsky KS, Larsen PR, Kronenberg HM,eds. Williams Textbook of Endocrinology.12th ed. Philadelphia, PA: SaundersElsevier; chap 32.

Taplin C & Barker J. 2008. Autoantibodies in type1 diabetes. Autoimmunity. Vol. 4 1 ( 1 ) . p p11-18 DOI:10.1080/08916930701619169

Erlander MG, Tillakaratne S, Feldblum N,Patel and Tobin AJ. (1991)Two genesencode distinct glutamate decarboxylaseswith different responses to pyridoxalphosphate. Neuron. 7:91-100.

Fineberg, E., Kawabata, T., Finco-Kent, D.,Fountaine, R., Finch, G., & Krasner, A.(2007). Immunological responses toexogenous insulin. Endocrine SocietyJournals and Publications, 28(6).

Kaufman DL, Erlander MG, Clare-Salzler M, Atkinson MA, Maclaren NK, Tobin AJ

(1992). "Autoimmunity to two forms ofglutamate decarboxylase in insulin-dependent diabetes mellitus". J. Clin.Invest. 89 (1): 283–92.doi:10.1172/JCI115573. PMC 442846.PMID 1370298.

Lethagen, Å., Erricson, U., Hallengren, B.,Groop, L., & Tuomi, T. (2001).Glumatic acid decarboxylaseantibody positivity is associated with animpaired insulin response to glucose and

arginine in nondiabetic patients withautoimmune thyroditis. Journal of ClinicalEndrocrinology and Metabolism, 87(3).

McDevitt, H. (2006). Characteristics ofautoimmunity in type 1 diabetes and type1.5 overlap with type 2 diabetes.Diabetes, 54(2), 4-10.

Pihoker, C., Gilliam, L., Hampe, C., & Lernmark, Å.(2005). Antibodies in diabetes. Diabetes,54(2), 52-61.

Scott, G., Fishman, S., Khai Siew, L.,

Margalit, A., Chapman, S., Chervonsky, A.,# Wong, S. (2010). Immunotargeting ofinsulin reactive CD8 T cells to preventDiabetes. Journal of Autoimmunity, 35,390-397.

Skowera, A., Ellis, R., Varela-Calviño, R., Arif, S.,Huang, G., Van Krinks, C., #Peakman, M.(2008). CTLs are targeted to kill beta-cells inpatients with type 1 diabetes throughrecognition of a glucose-regulatedpreproinsulin epitope. The Journal ofClinical Investigation, 118(10), 3390-3401.

Unger, W., Velthuis, J., Abreu, J., Sandra, S.,Quinten, E., Kester, M., #Roep, B.(2011).Discovery of low-affinity preproinsulinepitopes and detection of autoreactive CD8T-cells using combinatorial MHC multimers.Journal of Autoimmunity, 37, 151-159.
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