Lecture 15 - Organ-specific autoimmunity

Question 1

What is T1D pathogenesis from the thymus to beta cell destruction?

Answer 1

Thymus
>Developing T cells undergo selection
>positive selection to progress further and enter the periphery, or negative selection because they react to self-antigens and should be deleted (central tolerance)
>Those at risk of developing T1D have T cells that are self-reactive to beta cell antigens (failure of negative selection)

> organ specific autoimmune disease
represents a failure of self from non-self discrimination (basically a fundamental task of the immune system)

> Pancreatic lymph node
In the periphery, these naive t cells become activated, presumably in the draining lymph node or in the pancreatic islets themselves
due to the high conc of beta cell antigens present in both these places presented by the APCs that picked them up in the beta cells and moved back to the draining lymph nodes
activate the naive t cells that have escaped the thymus and these self-reactive t cells go on to infiltrate the islets and mediate the destruction of beta cells, resulting in dysregulation of glucose homeostasis

Question 2

What is the role of the human leukocyte antigen (HLA) locus on chromosome 6?

Answer 2

HLA Class II molecule:
>(alpha chain + beta chain)
>present peptides to CD4+ T cells

HLA Class I molecule:
>(alpha chain + beta-2-microglobulin chain (chr15))
>present peptides to CD8+ T cells

• Alleles for genes encoding MHC molecules confer the highest risk of T1D

Question 3

What are the functions of T cells in T1D?

Answer 3

CD8+ T cells recognise peptides presented by HLA Class I molecules
>cytotoxic function
>directly kill the cells presenting that peptide on the HLA class I molecule

CD4+ T cells recognise peptides presented by HLA class II molecules
>usually from a professional APC
>will activate the APC to go on to do other functions e.g might be a B cell which goes on to produce (in the case of T1D) autoantibodies
>helper function

Question 4

What is the DQ locus?

Answer 4

DQ locus consists of 2 genes
>gene that encodes the beta chain
>gene that encodes the alpha chain
>DQ2 and DQ8 is shorthand nomenclature to tell you what those alleles are for those 2 genes

Question 5

What is the odds ratio for the HLA/MHC class II molecules?

Answer 5

HLA class II alleles confer the highest risk for type 1 diabetes

HLA/MHC class II Odds Ratio
DQ2; DQ8 >16.6
DQ8; other >11.4
DQ2; other >3.6

DQ2; DQ8 heterozygosity confers the highest risk of T1D

Question 6

How do high risk alleles present beta-cell antigen-derived peptides to T cells?

Answer 6

• HLA class II molecules shape the T-cell repertoire during T cell development
• HLA class II molecules also activate the CD4+ T cells in the periphery

Question 7

Why is the HLA class II locus associated so strongly with T1D (and other autoimmune diseases)?

Answer 7

• Beta cell antigens may not be presented efficiently in the thymus by particular HLA molecules
• This enables the escape of low affinity T cells from the thymus and subsequent activation by beta cell antigens that are present at high concentration in the pancreas and draining lymph nodes

Question 8

How are genetic polymorphisms for insulin associated with thymic expression and T1D risk?

Answer 8

Key concept = Dysregulation of negative selection generates a peripheral pool of anti-self T cells displaying increased avidity/affinity and likely an enhanced pathogenic potential
A lot of self antigens are expressed in the thymus and presented by various cells in the medullary, so when the T cells interact with them, it sends out a signal to them to be deleted before they can get out into the periphery

Insulin (INS) = a key autoantigen for human T1D

> VNTR upstream element of INS (variable number of tandem repeats)
»>Class I (26-63 repeats) = predisposing allele
»>Class III (140-210 repeats) = protective allele

o Genetic polymorphisms that affect thymic insulin expression are associated with T1D
 Decreased thymic expression = increased T1D risk
 Increased thymic expression = decreased T1D risk

Question 9

What is the effect of reduced thymic insulin expression?

Answer 9

 Reduce negative selection of insulin-specific single positive thymocytes (precursor to the developed T cell that leaves and enters periphery)
 Limit thymic development of beta cell-specific regulatory T cells (FOXP3+ CD4+ T cells)

Question 10

What is PTPN22?

Answer 10

PTPN22 is a negative regulator of T cell receptor (TCR) signalling

Is expressed in developing thymosites that become t cells in the thymus)
>genetic variant (R620W, 1858C>T mutation associated with increased phosphatase activity and increased T1D risk

Question 11

What is elevated phosphatase activity (PTPN22) predicted to do?

Answer 11

Reduce TCR signalling and diminish apoptosis induction in beta cell-specific thymocytes
>autoreactive T cells escape to the periphery

> similar to insulin, limits thymic development of beta cell-specific regulatory T cells (FOXP3+ CD4+ T cells)

o Alter BCR signalling that results in defective B cell tolerance and allows autoreactive B cells to escape into the periphery

Question 12

What is the association between PTPN22 and INS in terms of defective negative selection and T1D risk?

Answer 12

During negative selection
>strong signal to developing thymocytes
>cause them to undergo apoptosis and die

> when you have PTPN22 allele, get increased phosphatase activity
reduce TCR signalling
reduces apoptosis induction in thymocytes that recognise B cell antigen and escape into periphery

> When you have reduced insulin expression in thymus
thymocytes who have low affintty for insulin can escape
once they get to pancreas lymph nodes, high amount of insulin makes up for that low affinity
cause immune response

Question 13

What are the beta-cell antigen-specific autoantibodies?

Answer 13

Autoreactive B and T cells in the periphery that have escaped negative selection
Islet cell auto-antibodies (ICAs)

Question 14

What are some islet self-antigens and what is their significance?

Answer 14

Present in serum of people with T1D

 Insulin: hormone produced by pancreatic beta cells
 GAD65: glutamic acid decarboxylase; expression not exclusive to pancreatic beta cells
 IA-2: tyrosine phosphatase-like protein islet antigen 2; not exclusive to pancreatic beta cells
 ZnT8: zinc transporter 8; zinc plays a role in storage and secretion of insulin; highly expressed in the endocrine, but absent in exocrine pancreas, also detected in extra-pancreatic sites

These are antigens expressed by the beta cells

Question 15

What are the beta cell antigens?

Answer 15

Insulin granule
>insulin
>IA-2 (islet antigen 2)
>ZnT8 (zinc transporter 8)

Not in insulin granule
>GAD65 (glutamate decarboxylase)

Question 16

What is the significance for islet auto-antibodies and clinical presentation?

Answer 16

Allows for the prediction and diagnosis + distinguishment of T1D from T2D as there are now biomarkers

• Positive seroconversion to islet cell autoantibodies (ICA) in at-risk individuals
• Not all islet cell autoantibodies are the same:
  o Insulin autoantibodies (IAAs) are typically the first to be detected
  o Glutamic acid decarboxylase autoantibodies (GADAs) and IAAs are the most frequent in childhood
  o High affinity IAAs and GADAs associated with progression to multiple islet autoantibodies and diabetes
  o Other islet autoantibodies typically appear later and indicate further progression of disease

Question 17

How are islet-specific autoantibodies detected?

Answer 17

Radiobinding assay
>to detect islet-specific autoantibodies (ICAs)

1) Collect serum from patient with suspected T1D
>serum containing autoantibodies
+
>radio-labelled insulin (or other beta-cell antigen)

2) Incubate and allow antigen/antibody complexes to precipitate

3) Measure radioactivity in the precipitate
>the amount of insulin specific antibody is proportional to the amount of radioactivity in the precipitate
>antibodies will start to complex and form these lattices which will precipitate in solution

Note:
Serum will contain a large collection of antibodies, with different specificities, only some will be ICAs

Question 18

Are beta-cell antigen-specific autoantibodies directly pathogenic?

Answer 18

• Islet autoantibodies do not have a direct cytotoxic effect on human islets in vitro
• Transfer of maternal autoantibodies to the foetus does not increase the offspring’s risk of developing T1D
• BCR (membrane bound versions of the secreted antibodies) capture beta cell autoantigens
  o Process and present peptides by HLA class II molecules to CD4+ T cells
  o CD4+ T cells are activated and secrete cytokines that stimulate B cells to secrete antibodies

Question 19

Where are beta cell specific T cells found in T1D patients?

Answer 19

In the periphery of T1D patients

Question 20

What is the phenotype of beta-cell specific T cells in T1D patients vs healthy controls?

Answer 20

• Beta cell specific T cells are relatively rare, but detectable in the peripheral blood of T1D patients. Sometimes possible to detect in healthy individuals too.
• The phenotype of circulating beta-cell specific T cells is distinct in T1D patients
  o T cells from patients = effector/memory phenotype
  o T cells from healthy controls = naïve phenotype
  o T cells from patients = proinflammatory cytokines (E.G., IFN-gamma)
  o T cells from healthy controls = regulatory responses (e.g. IL-10)

Question 21

How can one detect beta cell specific T cells?

Answer 21

Isolate peripheral blood mononuclear cells
1. T-cell proliferation asay + beta cell antigens –> measure cell proliferation through FACS analysis

2. MHC Tetramer Detection Assay + MHC tetramer loaded with peptides from beta cell proteins –> measure tetramer binding through FACS analysis

Question 22

What are T-cell antigen targets associated with T1D?

Note: some overlap between B-cell autoreactive responses and T cell autoreactive responses

Answer 22

GAD65
IA-2
ZnT8
Proinsulin
IGRP
IAAP
Chromogramin A

Question 23

Where is insulin C-peptide found?

Answer 23

Insulin C-peptide specific CD4+ T cells are detectable in peripheral blood

Question 24

What occurs inside of the islets during T1D?

Answer 24

• Insulitis is the pathologic hallmark of T1D: Immune cell infiltration of the islets associated with beta cell loss
• Insulitis is often variable in pancreatic tissue from T1D
• Insulitis is typically observed as peri-insulitis or intra-insulitis in insulin-positive islets
• Infiltration is not homogenous
• Some part of the pancreas have more infiltration than others (i.e., insulitis is lobular)
• There are pseudo-atrophic islets devoid of beta cells (still other pancreatic cells present)

Question 25

What is the relationship between the immune cells and islets?

Answer 25

Immune cells can be within and around the islets
* An islet showing early stages of destruction:
o Insulin expression still present
o Insulitis present at the top of this islet
o B cells and T cells detected
o CD4 (helper) and CD8 (killer) T cells detected
* Multiple CD8+ T cell autoreactivities against islet autoantigens (i.e. beta-cell proteins) were detected within islets of T1D patients, even up to 8 years after clinical diagnosis
Both CD4+ and CD8+ T cells are detected in the islets that recognise beta-cell derived antigens

Question 26

What is the relationship between infection and T1D?

Answer 26

Triggers such as viral infection and innate immune activation lead to type I interferon production
* Increased IFN-alpha is detected in the pancreas of type 1 diabetic subjects
* Interferon-stimulated genes are over-expressed in patients recently diagnosed with T1D

Question 27

What is the relationship between autoreactive CD8+ T cells and beta cells?

Answer 27

Autoreactive CD8+ T cells interact with beta cells through T-cell receptor binding to peptide/HLA class I complex

Activated CD8+ T cells secrete perforin and granzymes which directly kill beta cells by inducing cell death

Question 28

What is the expression like of HLA class I and II molecules in the islets T1D patients?

Answer 28

• Hyperexpression of HLA class I molecules are observed in tissues from patients with recently diagnosed type 1 diabetes
• HLA class I and class II molecules can be upregulated in vitro by culturing human islets in vitro with IFN-gamma
• Increased expression of HLA molecules and presentation of beta cell autoantigens make beta cells more susceptible to destruction by infiltrating autoreactive T cells
  Upregulation of HLA class I correlated with number of CD8+ T cells

Question 29

What is the suspected path of the autoreactive T cell?

Answer 29

1. Naive beta-cell autoreactive T cells escape the thymus
2. Activation and expansion of beta-cell autoreactive T cells + upregulation of activation markers + dendritic cells pick up antigens in the islets and bring the back to the pancreatic lymph node
3. Beta-cell autoreactive T cells gain effort function and expand. Produces cytokines and cytotoxic molecules. Activated T cells may also leave the islets and re-circulate in the blood