Peakman

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Hafler DRB10401 A1-15
Peakman DRB10401 C19-A3
Peakman A2 CD8 PPI15-24
Gottlieb B9-23 DQ8
Durinovic-Bello DRB10401 CD4 73-90
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NOD Mouse anti-islet T Cell Clones/transgenics/ret
rogenics
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CTLs are targeted to kill ß cells in patientswith
type 1 diabetes through recognition ofa
glucose-regulated preproinsulin epitope Ania
Skowera,. and Mark Peakman JCI 2008118
3268-3271.
Interferon gamma HLA-A2 ELISPOT peripheral
blood Patients (SIgt3) DM vs Controls
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Natural T Regulatory Cells
Antigen Specific T Reg
T Cell Receptor
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MAN
NOD MOUSE
TRAV5D-04TRAJ53 Restriction?
?
Insulin B9-23 SHLVEALYLVCGERG?
IAg7
DR3/DR4
Homann 2006,JCI
Natural T Regulatory Cells
Antigen Specific T Reg
IPEX Syndrome MAN foxP3 mutant DM in days of
birth!
NOD anti-B9-23insulin TCR foxP3 mutant DM
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ENVIRONMENTAL FACTORS
Incidence type 1 DM Increasing 3/year gt
30 years!
Protective Factor Decreasing -Hygiene Hypothesis
Bach NEJM 347911, 2002 Triggering
Factors -Congenital Rubella-Rubenstein Diabetes
311088, 1982 -Kilham Rat Virus (BB-DR
rat)-Zipris J. Immunol 174131, 2005 -Poly-IC
Induction Interferon Alpha-Devendra Diabetes 54
2005 -Dietary Factors-Scott Ann Rev Nutr 26175,
2006
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Lack of progression to diabetes of NOD mice
lacking both insulin native genes.
25 25
10 14
21 23
1 1
2 4
Ins1-, ins2- n Ins1, ins2- n
Life table update 5/19/05
Nakayama Nature 435220,2005
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Insulin is the primary antigen and precedes IGRP
in hierarchy of autoantigens
Krishnamurthy et al JCI1163258, 2006
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Is there a primary antigen or immune response to
multiple antigens required for autoimmunity?
T cells specific for multiple antigens
T cells specific for one antigen
Insulitis
Insulitis
OR
Epitope and antigen spreading, expansion
Expansion of T cells
Diabetes
Diabetes
Krishnamurthy et al JCI1163258, 2006
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The insulin A-chain epitope recognized by human T
cells is posttranslationally modifiedMannering
et al, JEM 2021191-1197, 2005

• CD4 T cells cloned with CFSE method from
  peripheral blood of one patient with diabetes and
  one child with insulin autoantibodies
• Subset of the clones reacted with insulin A-chain
  1-13 epitope
• T Cells DR restricted and only reacted with
  peptide with vicinal disulfide bond between
  adjacent cysteines A6 and A7
• No reactivity with this peptide of clones from
  two normal controls with DR4
• Oral report by Sally Kent that their A1-15
  peptide reactive T cells from pancreatic lymph
  node do not require vicinal disulfide cross-link

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Antigens for Islet-infiltrating CD8 T Cells in
NOD Mice
DMK, dystrophia myotonica kinase GAD, glutamic
acid decarboxylase IGRP, islet-specific
glucose-6-phosphatase catalytic subunit-related
protein
DiLorenzo
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Antigens for CD8 T Cells in Type 1 Diabetes
Patients
DiLorenzo
GAD, glutamic acid decarboxylase IAPP, islet
amyloid polypeptide
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Natural peptides selected by diabetogenic DQ8 and
murine I-Ag7 molecules show common sequence
homology Suri et al JCI 1152268, 2005Structure
of Human insulin peptide DQ8, Lee et al Nature
Immunology 6501, 2001
Crystal DQ8B9-23 S H L V E A L Y L V
C G E R G
Wiley Nat Immunol
P1
P4
P9
P6
Preferred AA in Bound Peptides
I-Ag7 v,e,q
I,L A,s D,E
12 20 30,11 45
DQ8 E,d
A,S A,V,s E,D
27,17 19
20 60,25
amino acid at position
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Typical Insulitis of man differs from
peri-insulitis NOD mouse Bottazzo
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Expanded T cells from pancreatic lymph nodes of
type 1 diabetic subjects recognize an insulin
epitopeKent et al, Nature 435224, 2005

• Pancreatic LN Single cell cloning - PHA
• 2/3 Patients Clonal expansion duration diabetes
  29 and 15 years
• Vbeta29-103J2-3(50)/Valpha8-302
  J4401(25)Vbeta5-101 J2-3(52)/Valpha3901
  J3301(26)
• DRB10401 RestrictedInsulin A1-15
• Caveat High concentration to stimulate 100uM

BDC
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Amino Acid Sequence of Mouse 1 and 2 and Human
Insulin
Leader 1 MAL LVHFLPLLALLALWEPKPTQA Leader 2
MALWMRFLPLLALLFLWESHPTQA Human
MALWMRLLPLLALLALWGPDPAAA
10
20 B
Chain 1 FVKQHLCGPHLVEALYLVCGERGFFYTPKS B Chain
2 FVKQHLCGSHLVEALYLVCGERGFFYTPMS Human
FVNQHLCGSHLVEALYLVCGERGFFYTPKT 25
30 40
50 C-Peptide 1
RREVEDPQVEQLELGGSPG..DLQTLALEVARQ C-Peptide 2
RREVEDPQVAQLELGGGPGAGDLQTLALEVAQQ Human
RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ
55 60 70
80 A Chain 1 KR
GIVDQCCTSICSLYQLENYCN A Chain 2 KR
GIVDQCCTSICSLYQLENYCN Human KR
GIVEQCCTSICSLYQLENYCN 88
100
B9-23
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Toma et al. Recognition of a subregion of human
proinsulin by class I-restricted T cells in type
1 diabetic patients PNAS 10210581, 2005

• Selected 8-11mer peptides of proinsulin
• PBMCs of 29/32 (90) recentlong term diabetics
  responded IFNgamma in ELISPOT assay versus
  minimal response of normals
• Significant Peptides often overlapped insulin B
  chain peptide B9-23(Proins 33-47) Proins
  34-42(B10-18) Proins 41-50(B17-26)Proins
  42-51Prins44-51 but also B chain-C-peptide
  (49-57)
• Multiple class I alleles, including A1 and B8

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Antigen Recognition by CD8 T Cells
T. DiLorenzo
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Screening of Peptide Fractions by 51Cr Release
Cytotoxicity Assay Method Used to Discover IGRP
APC
T. DiLorenzo
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A MODEL
b-cells
APC
ISLET
Ag
T cells
Pancreatic Lymph Node
Mathis/Benoist
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Checkpoints in the development of autoimmune
diabetes
Checkpoint 1
Insulitis -Starts at weaning immunological
changes related to food uptake and changes in
the intestinal flora -Increased homing of T cells
expression of addressins MadCam and PNAd on
pancreatic blood vessel epithelium
Checkpoint 2
Beta cell loss and diabetes - T cells gain more
aggressive effector mechanisms Th1/Th2 balance,
cytokines, Expression of Fas Ligand on CTLs -
Loss of protective mechanisms Protective
cytokines, Regulatory cells - Amplification
Epitope spreading
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What are we missing?

• Assay for Pathogenic T cells.
• ? TETRAMER
• ? ELISPOT

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Santamria NRP Class I Peptide

• CD8 T cell from NOD islets (e.g. clone 8.3)
• Conserved Valpha/ nDn/ J alpha (Va17,Ja42)Vbeta
  not conserved but contributes
• Accelerated diabetes TCR Transgenic
• Mimotope Defined-Kd Tetramer ProducedNRPKYNKANWF
  L NRP-V7 KYNKANVFL
• Higher Avidity Peptide/Tetramer V7
• 30 Intra-islet post 9 weeksgt.5 in Blood
  Predicts Diabetes NOD Mice

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Tan et al., JCI 1/2003
Female NOD Mice Peripheral Blood
Kd
NRP-V7 Peptide (KYNKANVFL)
Tetramer Analysis
Avidin
Kd
Kd
Kd
Diabetes
No Diabetes