An Unfinished Journey: Molecular Pathogenesis to Prevention

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An Unfinished JourneyMolecular Pathogenesis to
Prevention
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An Unfinished JourneyMolecular Pathogenesis to
Prevention
Stephan, Stephanie, and Frieda, plus Tracy,
Oscar, Karl, Austin
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An Unfinished JourneyMolecular Pathogenesis to
Prevention
Harold Lebovitz
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An Unfinished JourneyMolecular Pathogenesis to
Prevention
NIDDK NIAID ADA JDRF CDF ITN Brehm
Coalition
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An Unfinished JourneyMolecular Pathogenesis to
Prevention
Joslin
Barbara Davis Center
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Immunology of Diabetes

• Human Type 1 diabetes
• NOD mouse model

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Take Home Message

• Type 1A diabetes is hard-wired in
• the genome
• Insulin is the primary autoantigen
• Prediction is now possible and
• prevention will be achievable

Immunology of Diabetes WWW.BARBARADAVISCENTER.ORG
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Stages in Development of Type 1A Diabetes
(?Precipitating Event)
Genetic Predisposition
Overt immunologic abnormalities
Progressive loss insulin release
Normal insulin release
Overt diabetes
Beta cell mass
Glucose normal
C-peptide present
No C-peptide
Eisenbarth NEJM 1986
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Stages Type IA Diabetes

• I Genetic Susceptibility
• II Triggering-Environment
• III Active Autoimmunity
• IV Progressive Metabolic Abnormalities
• V Overt Diabetes
• VI Insulin Dependence

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Immune Mediated Diabetes

• Monogenic
• IPEX syndrome
• Autoimmune Polyendocrine Type 1
• Polygenic
• Autoimmune Polyendocrine Type 2
• Type 1A diabetes

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Child with IPEX Syndrome
9 Months of Age
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IPEX Immune Dysfunction, Polyendocrinopathy,
Enteropathy, X-linked

• Mutation of FoxP3 gene- Controls regulatory T
  cells
• Approximately 80 of children with syndrome
  develop diabetes
• Bone marrow transplant can reverse

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APS-I Syndrome

• Loss of thymic tolerance to peripheral antigens
  
• Results from mutations AIRE gene
• Multiple autoimmune disorders, Addisons disease,
  hypoparathyrodism, mucocutaneous candidiasis
• 18 Type 1 diabetes

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Eisenbarth GS, Gottlieb PA. NEJM 3502068-79 2004
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(No Transcript)
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(No Transcript)
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Progression to Diabetes in Monozygotic Twins
N 83
Redondo, Jeffrey, Fain, Eisenbarth, Orban NEJM
3592849 2008
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Genome-wide Associations in Type 1 Diabetes
6.50
2.25
2.00
1.75
1.50
1.25
1.00
HLA
Locus
ß cell apoptosis protection
Insulin production metabolism
Immunity
Unknown function
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The Major Histocompatibility Complex
Class II
Class III
Class I
Human
DP DQ DR B C
A
Chromosome 6
Allele
DRB10401
Haplotype
Genotype
Type 1 Diabetes Molecular, Cellular and Clinical
immunology http//www.uchsc.edu/misc/diabetes/eis
enbook.html
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Antigen
Endocytosis
CD4 T cell
APC
T Cell Receptor
Trimolecular Complex
MHC II
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Daisy Study Population
NEwborn Cohort (NEC) General population cohort
(SOC) Sibling/Offspring Cohort

• screened 33,096

offspring
siblings
Enrolled 391 high risk (2.4 ) 456 moderate
risk 384 average-low risk
51 111 509
49 65 254
368
671
1,231
Total enrolled 2,270
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Extreme Risk for DiabeticAutoimmunity in DR3/4
Siblings
Autoantibody Positive
Siblings at high risk (Share 2)
100
90
Siblings at low risk (Share 0 or 1)
80
N 29
70
60
50
40
30
20
N 19
10
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
Age (y)
Aly et al. PNAS 10314074-9 2006
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Progression to Diabetic Autoimmunity in DR3/4
General Population Children
Autoantibody Positive ()
Baschal et al. Diabetes 562405-9 2007
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Barbara Davis Center Autoimmune Risk Testing

• Transglutaminase autoantibodies (5 celiac
  disease)
• 21-Hydroxylase autoantibodies
• (1 positive predictive Addisons disease)
• Sensitive TSH assay (hypothyroidism very common)

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Stages Type IA Diabetes

• I Genetic Susceptibility
• II Triggering-Environment
• III Active Autoimmunity
• IV Progressive Metabolic Abnormalities
• V Overt Diabetes
• VI Insulin Dependence

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Environment

• Type 1 Diabetes incidence
• doubling in developed societies every 20 years
• especially for youngest onset
• Kilham rat virus model
• Susceptible HLA molecules
• Induction innate immunity

• hygiene hypothesis

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PENDING ZIPRIS
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Stages Type IA Diabetes

• I Genetic Susceptibility
• II Triggering-Environment
• III Active Autoimmunity
• IV Progressive Metabolic Abnormalities
• V Overt Diabetes
• VI Insulin Dependence

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Major Islet Autoantigens
ZnT8
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15
10
with islet autoantibodies
5
0
0
2
4
6
8
10
12
14
16
Age (years)
Bonifacio, Ziegler BABYDIAB
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Prediabetic Child 00379-0 3X
DM
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DPT-1 Time to Diabetes by Number of Antibodies
N 24151
1.0
0.9
N 1718
0.8
0.7
0.6
Survival Distribution
0.5
0.4
N 405
N 378
P- Valuelt 0.001 (Log Rank Test)
0.3

0.2
N 147
0.1
n 26799
0.0
0
1
2
3
4
5
6
7
8
Years Followed
Autoantibodies
1
2
0
3
4
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Stages Type IA Diabetes

• I Genetic Susceptibility
• II Triggering-Environment
• III Active Autoimmunity
• IV Progressive Metabolic Abnormalities
• V Overt Diabetes
• VI Insulin Dependence

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Rising HbA1c Precedes Diabetes
HbA1c ()
Onset of Diabetes
Age (years)
Stene et al DAISY Study
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Barker et al, Diabetes Care 271399 2004
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Stages Type IA Diabetes

• I Genetic Susceptibility
• II Triggering-Environment
• III Active Autoimmunity
• IV Progressive Metabolic Abnormalities
• V Overt Diabetes
• VI Insulin Dependence

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Stages Type IA Diabetes

• I Genetic Susceptibility
• II Triggering-Environment
• III Active Autoimmunity
• IV Progressive Metabolic Abnormalities
• V Overt Diabetes
• VI Insulin Dependence

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PENDING GIANANI
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What Underlines Slow Development of Type 1
Diabetes?
VITILIGO
ALL ISLETS NO BETA CELLS
ALL ISLETS WITH BETA CELLS
nPOD (Atkinson) Pancreas 6038 www.jdrfnpod.org
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Surviving Beta Cells Express Anti-apoptotic
Molecule Survivin
nPOD Gianani
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How Close to the Tipping Point?

• Immune modulation / suppression
• Antigen specific therapies
• Combined therapies

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Positive Trials
ANTIGEN SPECIFIC
IMMUNE MODULATION

• Anti-CD3 Herold Chatenoud JDRF and ITN
• Anti-CD20 Peskovitz

• Oral Insulin Skyler
• TrialNet
• GAD Vaccine Ludviggson DIAMYD

TrialNet
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DPT-1 Oral Study Time to Diabetes by Treatment
1.0
0.9
0.8
Treated
0.7
0.6
Survival Distribution Function
N 186
0.5
P- Value 0.176 (Log Rank Test)
N 186
Control
0.4
0.3
0.2
0.1
STRATA
Oral Placebo
Oral Insulin
0.0
0
1
2
3
4
5
6
7
Years Followed
Diabetes Care 281068-76 2005
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Insulin Effect Most Evident in Subjects with
Baseline IAA 300
Oral Insulin
Placebo
N63 (Ins.) and 69 (Plac.)
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GAD-Alum Vaccine
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Updated Data from Phase I/II Trial ofAnti-CD3 in
New Onset T1DM
150



100
AUC (pmol/ml/240min)
50
0
0
6
12
18
24
plt0.02
Month
Herold et al. Diabetes 541763-9 2005
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PENDING PESCOVITZ
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PENDING PESCOVITZ
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Where Are We Today?

• We can now predict Type 1 diabetes.
• Insulin may be a primary autoantigen in man.
• In man immunomodulatory/ immunosuppressive Rx can
  dramatically delay loss of beta cells, but does
  not halt.
• In man antigen specific Rx may delay loss beta
  cells.
• Multiple trials, including phase IIIs, underway.
• A LOT WE DONT KNOW

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What We Do Not Know?

• Multiple or a single primary target antigen?
• Sequences of T cell receptors?
• Preventive therapy directed at the root cause?
• Animal models as testing ground NOD mouse.

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Root Cause NOD Mouse

• Genome Sequences of Trimolecular Complex
• Insulin peptide sequence
• Susceptible MHC sequence
• T cell receptor Valpha sequence

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The Trimolecular Complex
TCR
PEPTIDE INS B9-23
MHC
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Isolation of Islet Infiltrate Cells
Digest to Islets

(collagenase)
Digest to Single Cells
(trypsin)
Islet Cells
Infiltrating Cells
NOD Spleen
Cell Suspension
Daniel and Wegmann
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NOD Mouse Islet T Cells React with Insulin
90 react with B 9-23 peptide
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Pathogenic Peptide Insulin B9-23
13
A
23
L
A
E
Y
G
V
16
L
R
L
V
E
H
C
G
S
9
Barbara Davis Center
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Creating a Mouse with Only a Mutated Insulin
B9-23 Sequence
X
X
B16A Insulin- transgenic NOD Insulin 1
() Insulin 2 () B16A Insulin 2 ()
Insulin 1-KO NOD Insulin 1 (-) Insulin 2 ()
SHLVEALYLVCGERG (Insulin 2 - B9-23)
Insulin 2-KO NOD Insulin 1 () Insulin 2 (-)
PHLVEALYLVCGERG (Insulin 1 B9-23)
NOD mice lacking both native insulin 1 and
2 Insulin 1 (-) Insulin 2 (-) B16A Insulin 2
() SHLVEALALVCGERG (B16A Insulin 2 B9-23)
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B16A Insulin
Nakayama et al. Nature 435220-3 2005
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Eight Mice Were Enough
The NOD Mouse Recessive Diabetogenic Gene in the
Major Histocompatibility Complex
DIABETIC MICE
All I-Ag7
I-Ag7
I-Ak
Hattori et al Science 1986 231733-735
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Peptide Presentation Model
AA Peptide Side Chains
Peptide
I-Ag7
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Crystal Structure of I-Ag7
ARG(22) GLU GLY CYS VAL LEU TYR LEU ALA(14)
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Pending Stadinsky
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T Cell Receptors
TCR
PEPTIDE INS B9-23
MHC
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T Cell Receptor Gene Segments
Antigen Presenting Cell
HLA Molecule
Peptide
V? J?
V? D? J?
Chr. 14
Chr. 6
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V-ALPHA 5D-404
 GEQVEQLPSILRVQEGSSASINCSYE
CDR1 DSASNY FPWYKQEPGENPKLIID CDR2
IRSNME
RKQTQGLIVLLDKKAKRFSLHITDTQPGDSAMYFC AAS
No Conservation TCR Beta Chain
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The Generation of Retrogenic Mice
Insertion TCR alpha only or alpha_P2A_beta
SCID
Phoenix cells (293T cells transfected with
pCL-Eco)
CMV-env (Mo-MuLV) RSV-gag-pol (Mo-MuLV)
SCID or Ca-KO BM cells
pMIG
pMIG
pMIG
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Retrogenic 5D-4 Alpha Chains are Pathogenic
Insulin Autoantibodies
Nakayama unpublished
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Next Generation Sequencing T Cell Receptors
454 Machine
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A Therapeutic Target?
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Targeting Trimolecular Complex

• May be possible to prevent diabetes with
  Antibodies to pathogenic trimolecular complex
  (e.g. a vaccine)
• In silico predicted therapeutics or potentially
  environmental Small Molecules can alter the
  pathogenic trimolecular complex

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Sites Selected for Molecular Docking
I-Ag7 a
Arg76 a
Ser57 ß
ins9-23
I-Ag7 ß
Ostrov, Gainesville
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Sites selected for molecular docking
I-Ag7 a
Arg76 a
Ser57 ß
I-Ag7 ß
ins9-23
Ostrov Gainesville
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Small Molecule Glyphosine Stimulates T Cell
Receptor Signaling
PeptideGlyphosine
Glyphosine Alone
B9-23
Glyphosine
TT
10nM
500nM
10nM
250nM
500nM
100nM
100nM
250nM
Glyphosine
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Thoughts from the NOD

• Insulin primary target
• Insulin B9-23 is recognized in Register 3 by
  specific TCR Valpha segment sequence
• There are large numbers of TCRs with this TCR
  alpha segment sequence produced in the thymus
• Trimolecular complex can be altered (both for
  good or bad) by small molecules

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Where Next?

• Discover ubiquitous environmental factors
  responsible for increase in type 1 diabetes.
• Isolate T cells from human islets and define
  human trimolecular complex (nPOD).
• Build on current successful trials
• (combination Rx?) to extend, and with luck,
  permanently arrest beta cell destruction.
• For the long-term utilize knowledge of the
  trimolecular complex to develop more specific
  therapies (remove need for luck).

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What is Needed?

• This audience to pioneer the prevention of Type 1
  diabetes.
• Most new onset patients and their relatives
  should have available the option of participating
  in preventive trials.

TRIALNET 1-800-HALT DM1
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2002
1991
2008
DAISY STUDY
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I-Ag7 a
Arg76 a
Ser57 ß
I-Ag7 ß
ins9-23
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I-Ag7 a
Arg76 a
Ser57 ß
I-Ag7 ß
ins9-23
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BETA CELL AREA DECREASED PATTERN A AND B
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BETA CELL AREA DECREASED PATTERN A AND B
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Type 1 diabetes is T cell mediated

• Infiltrating CD4, CD8 T cells
• T cells drive B lymphocytes
• to make Ab

CD4 T-cell
CD8 T-cell
TCR
CD4 Treg
Trimolecular complex
Epitope
HLA II
HLA I
APC
Peakman
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? -chain
Leu56?
?-chain
Asp57?
BDC
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Stages Type IA Diabetes

• I Genetic Susceptibility
• II Triggering-Environment
• III Active Autoimmunity
• IV Progressive Metabolic Abnormalities
• V Overt Diabetes
• VI Insulin Dependence