Ψ
IRES
pMIG
Insertion:
TCR alpha only or
alpha_P2A_beta
SCID
Phoenix cells
(293T cells transfected
with pCL-Eco)
MIG
CMV-env (Mo-MuLV)
RSV-gag-pol (Mo-MuLV)
pMIG
pMIG
pMIG
SCID or
Ca-KO
BM cells
Anti-GAD TCR + or – B Lymphocytes
TCR Retrogenic Induction High
Levels GAD65 Autoantibodies
T Cell Islet Accumulation in Type 1 Diabetes is a
Tightly Regulated, Cell-Autonomous Event Lennon et
al Immunity 31, 643-653, 2009
Unique autoreactive T cells recognize insulin
peptides generated within the islets of Langerhans in
autoimmune diabetes James F Mohan,…& Emil R
Unanue Nature Immunology March 2010
Insulinoma
Only islets +
Rag-/- islets
Type A
type B
Type B
CD11c
islets
Insulin
granules
Conserved T Cell Receptor Alpha Chain Induces
Insulin Autoantibodies
Kobayashi et al PNAS 105:10090-94 2008
Anti-B:9-23 TCR alpha Transgene
No Transgene
Nat Immunol. 2010 Mar;11(3):225-31. Epub 2010 Feb 7.
Chromogranin A is an autoantigen in type 1 diabetes.
Stadinski, …Kappler, Haskins
Peptide WE14 bound to the NOD
mouse major histocompatibility
complex class II molecule I-A(g7) in
an atypical manner, occupying only
the carboxy-terminal half of the IA(g7) peptide-binding groove.
Antigen Presenting Cell
Peptide
Va
V
D
Ja
Chr. 14
J
Chr. 6
Antigen Recognition by CD8+ T Cells
T
cell
T cell
receptor
V
Va
class I
MHC
2m
 cell
T. DiLorenzo
Kuby Immunology
AA Peptide Side Chains
Peptide
g7
I-A
GROOVE
“TEETH”
TEETH
T Cell Receptor Analogy
Child with IPEX syndrome
Awaiting Bone Marrow Transplant
9 Months of Age
IPEX: Immune Dysfunction,
Polyendocrinopathy, Enteropathy, Xlinked
• Scurfin gene (Foxp3/JM2)
- Controls Regulatory T Cells!
• Approximately 80% of children with
syndrome develop diabetes!
• Bone marrow transplant can reverse
“Typical” Insulitis of man differs from peri-insulitis NOD mouse:
A MODEL
-cells
APC
ISLET
t
Ag
t
t
T cells
Pancreatic
Lymph
Node
Mathis/Benoist
MAN
?
NOD MOUSE
TRAV5D-04+TRAJ53 Restriction?
Insulin B:9-23: SHLVEALYLVCGERG?
IAg7
DR3/DR4
Homann 2006,JCI
Natural T Regulatory Cells
Antigen Specific T Reg
IPEX Syndrome MAN:
NOD anti-B9-23insulin TCR:
foxP3 mutant DM in days of birth!
foxP3 mutant DM
ENVIRONMENTAL FACTORS
Incidence type 1 DM Increasing 3%/year > 30 years!
Protective Factor Decreasing
-Hygiene Hypothesis – Bach NEJM 347:911, 2002
Triggering Factors
-Congenital Rubella-Rubenstein Diabetes 31:1088, 1982
-Kilham Rat Virus (BB-DR rat)-Zipris J. Immunol 174:131, 2005
-Poly-IC Induction Interferon Alpha-Devendra Diabetes 54 2005
-Dietary Factors-Scott Ann Rev Nutr 26:175, 2006
Natural T Regulatory Cells
T Cell Receptor
Antigen Specific T Reg
Amino Acid Sequence of Mouse 1 and 2 and Human Insulin
Leader 1: MAL LVHFLPLLALLALWEPKPTQA
Leader 2: MALWMRFLPLLALLFLWESHPTQA
Human : MALWMRLLPLLALLALWGPDPAAA
20
10
B Chain 1: FVKQHLCGPHLVEALYLVCGERGFFYTPKS
B Chain 2: FVKQHLCGSHLVEALYLVCGERGFFYTPMS
Human : FVNQHLCGSHLVEALYLVCGERGFFYTPKT
B:9-23
25
30
40
50
C-Peptide 1: RREVEDPQVEQLELGGSPG…..DLQTLALEVARQ
C-Peptide 2: RREVEDPQVAQLELGGGPGAGDLQTLALEVAQQ
Human :
RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ
55
60
70
A Chain 1: KR GIVDQCCTSICSLYQLENYCN
A Chain 2: KR GIVDQCCTSICSLYQLENYCN
Human : KR GIVEQCCTSICSLYQLENYCN
88
100
80
Lack of progression to diabetes of NOD mice lacking
both insulin native genes.
% Diabetes Free
100
ins1-, ins2-, Tg+ (n=25, P<0.01)
80
ins1+, ins2-, Tg+ (n=25)
60
40
20
0
0
10
20
30
40
50
60
Weeks of age
Ins1-, ins2-: n= 25
Ins1+, ins2-: n= 25
21
23
10
14
2
4
1
1
Life table update 5/19/05
Nakayama Nature 435:220,2005
Insulin is the primary antigen and precedes IGRP in
hierarchy of autoantigens
Insulin specific T cells
Insultis
Epitope and antigen spreading,
expansion
Diabetes
Krishnamurthy et al JCI:116:3258, 2006
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
Diabetes
Expansion
of T cells
Diabetes
Krishnamurthy et al JCI:116:3258, 2006
Natural peptides selected by diabetogenic DQ8 and
murine I-Ag7 molecules show common sequence
homology Suri et al JCI 115:2268, 2005
Structure of Human insulin peptide DQ8, Lee et al
Nature Immunology 6:501, 2001
Crystal DQ8;B:9-23: S H L V E A L Y L V C G E R G
Wiley Nat Immunol
P1
Preferred AA in Bound Peptides
I-Ag7
v,e,q
12%
DQ8
% amino acid at position
E,d
P4
I,L
P6
P9
A,s
D,E
20% 30,11% 45%
A,S
A,V,s
27,17% 19% 20%
E,D
60,25%
NOD Mouse anti-islet T Cell
Clones/transgenics/retrogenics
Clone
BDC2.5
BDC10.1
BDC6.9
NY4.1
CD4/CD8
CD4
CD4
CD4
CD4
Source
Spleen
Islet
Islet
Islet
Antigen
TCRα/β
7D-6/2
17/20
5D-4*01/2
5D-4*04/
Tetramer TransgenicRetrogenicComment
yes
yes
yes
10.1 same mimotope
yes
yes
DM+++ retrogenic
yes
yes
Strain specific, chr 6
yes
yes
yes
DM transgenic
Phogrin-13 CD4
Phogrin-18 CD4
10.23
CD4
Immun LN IA2β 640-659
Immun LN IA2β 755-777
IA2 676-688
13-2/3
10/16
13-4/4
2H6
CD4
BDC12-4.1 CD4
BDC12-4.4 CD4
BDC 8-.1.1 CD4
BDC12-1.19 CD4
BDC12-2.35 CD4
BDC6-4.3 CD4
BDC12-2.4 CD4
6C5
CD4
Panc LN
Islet
Islet
Immun LN
Islet
Islet
Islet
Islet
islet
21/31
5D-4*04/1
5D-4*04/5
5D-4*04/15
13-1/19
7-4/13-1
5D-4*04/1
5A
CD4
B16.3
CD4
BW5147
CD4
PA15.14B CD4
IA4
CD4
PA17.9G7 CD4
PA18.10F10 CD4
PA18.9H7 CD4
530.45.19 CD4
Immun Spl GAD
spleen
GAD65 pp286–300
GAD65 pp206–220
spleen
GAD pp206–220
spleen
GAD pp217–236
GAD pp284–300
GAD pp510–524
GAD pp524–538
GAD pp530–543
G9C8
CD8
Islet
insulin B:15-23
yes
8.3
CD8
Islet
IGRP 206-214
yes
insulin B:12-25
insulin B:12-22
insulin B:12-22
insulin B:12-22
insulin B:9-23
insulin B:9-18
insulin B:9-23
insulin B:9-16
yes
yes
yes
yes
yes
yes
yes
yes
9D-4
6-2/5
3/13-2
6D-6/13-3
6-6/2
6-2/4
7-4/4
21/15
(5D-4/2)
yes
yes
yes
yes
yes
yes
yes
Author
Haskins
Haskins
Haskins
Santamaria
insulitis retrogenic
insulitis retrogenic
Hutton
Hutton
Hutton
TGFβ protection
DM retrogenic
DM retrogenic
DM retrogenic
DM retrogenic
DM retrogenic
young NOD
Zekzer
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Fathman
suppress DM
suppress DM
no effect retrogenic
no effect retrogenic
no effect retrogenic
no effect retrogenic
no effect retrogenic
no effect retrogenic
Zekzer
McDevitt
McDevitt
Vignalli
Tisch
Vignalli
Vignalli
Vignalli
Sercarz
Wong
yes
Perforin independent, Fas
Santamaria
Hafler
Peakman
DRB1*0401 A1-15
DRB1*0401 C19-A3
Peakman
Gottlieb
B:9-23 DQ8
Durinovic-Bello
DRB1*0401 CD4 73-90
A2 CD8 PPI:15-24
Expanded T cells from pancreatic
lymph nodes of type 1 diabetic subjects
recognize an insulin epitope
Kent et al, Nature 435:224, 2005
• Pancreatic LN: Single cell cloning - PHA
• 2/3 Patients Clonal expansion
duration diabetes 29 and 15 years
• Vbeta29-1*03J2-3(50%)/Valpha8-3*02 J44*01(25%)
Vbeta5-1*01 J2-3(52%)/Valpha39*01 J33*01(26%)
• DRB1*0401 Restricted
Insulin A1-15
•
Caveat: High concentration to stimulate 100uM
BDC
The insulin A-chain epitope recognized by
human T cells is posttranslationally modified
Mannering et al, JEM 202:1191-1197, 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
Ins2 deficiency augments spontaneous HLAA*0201-restricted T cell responses to Insulin
Jarchum and DiLorenzo J Immunol 2010 vol 184
Humanized HLA-A2 Mice lacking Insulin 2!
Ins B:10-18
CD8
Elispot
Thymus-specific deletion of insulin induces
autoimmune diabetes.
Fan et al EMBO Journal 28:2812-2824 2009
• Ins1-/- mice (only) with cre mediated
thymic mTEC deletion of ins2 develop
autoimmune diabetes by age 3 weeks.
• Diabetes develops in H-2b mice!
• ELISPOT Responses to Insulin B:9-23
though mice do not have I-Ag7.
• MYSTERIES: Need for Ins1-/-; Lack
protection H-2b; Presentation B:9-23 by H2b.
The frequency and immunodominance of Isletspecific CD8+ T-cell responses change after Type 1
Diabetes Diagnosis and Treatment
Martinuzzi et al Diabetes 57:1312-1320, 2008
7-16 months post onset most ELSIPOT (HLA-A2) responses decrease
PREPROINSULIN
PROINSULIN
New
Responses
“Santamria” NRP(IGRP206-214): Class
I (Kd)Recognized 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 Produced
NRP=KYNKANWFL; NRP-V7: KYNKANVFL
• Higher Avidity Peptide/Tetramer V7
• 30% Intra-islet post 9 weeks
>.5% in Blood Predicts Diabetes NOD Mice
Tan et al., JCI 1/2003
Female NOD Mice Peripheral Blood
Kd
Tetramer
Analysis
NRP-V7 Peptide (KYNKANVFL)
Kd
Kd
Avidin
1.2
1
0.8
0.6
0.4
0.2
0
5
10
14
18
21
Age (weeks)
Diabetes
24
27
30
% NRP-V7 tetramer+ CD8+ cells
% NRP-V7 tetramer+ CD8+ cells
Kd
1.2
1
0.8
0.6
0.4
0.2
0
5
9
12
15
18
21
24
Age (weeks)
No Diabetes
27
30
CTLs are targeted to kill β cells in patientswith type 1 diabetes through
recognition ofa glucose-regulated preproinsulin epitope Ania Skowera,…. and
Mark Peakman JCI 2008:118 3268-3271.
Interferon gamma
HLA-A2 ELISPOT
peripheral blood
Patients+ (SI>3)
DM vs Controls
Screening of Peptide Fractions by
51Cr Release Cytotoxicity Assay
Method Used to Discover IGRP
CD8+
CTL
APC
CTL
T. DiLorenzo
Antigens for CD8+ T Cells in Type 1 Diabetes Patients
Antigen
Position
MHC
Why examined
GAD65
114-123
A2
MHC binding
IAPP
5-13
A2
MHC binding
Insulin
B10-18
A2
Proteasome cleavage
B14-22
A3, A11
Proteasome cleavage
B15-23
A24
MHC binding
B15-24
A24
Proteasome cleavage
B17-26
A1, A3, A11
Proteasome cleavage
B18-27
A1, A2, B8, B18
Proteasome cleavage
B20-27
A1, B8
Proteasome cleavage
B21-29
A3
Proteasome cleavage
B25-C1
B8
Proteasome cleavage
B27-C5
B8
Proteasome cleavage
GAD, glutamic acid decarboxylase; IAPP, islet amyloid polypeptide
DiLorenzo
Antigens for Islet-infiltrating CD8+ T Cells
in NOD Mice
Antigen
Position
MHC
T cells
How identified
DMK
138-146
H-2Db
AI4; islet
Positional scanning
libraries
206-214
H-2Kd
8.3; islet
MHC purification and
mass spectrometry
21-29
H-2Kd
Islet
MHC binding
225-233
H-2Db
Islet
MHC binding
241-249
H-2Db
Islet
MHC binding
324-332
H-2Kd
Islet
MHC binding
B15-23
H-2Kd
G9C8; islet
Expression cloning
IGRP
Insulin 1/2
DMK, dystrophia myotonica kinase
GAD, glutamic acid decarboxylase
DiLorenzo
IGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein
Toma et al. Recognition of a subregion of human
proinsulin by class I-restricted T cells in type 1
diabetic patients PNAS 102:10581, 2005
• Selected 8-11mer peptides of proinsulin
• PBMCs of 29/32 (90%) recent+long term
diabetics responded IFNgamma in ELISPOT
assay versus minimal response of normals
• Significant Peptides often overlapped insulin B
chain peptide B:9-23(=Proins 33-47); Proins 3442(B10-18); Proins 41-50(B17-26);Proins 4251;Prins44-51; but also B chain-C-peptide (49-57)
• Multiple class I alleles, including A1 and B8
T cell epitopes on the diabetes autoantigen Phogrin (IA-2beta) are conserved
among different species
C terminus
TM
PTP
N terminus
Peptide 2: Amino acids 643-658
GADPSADATEAYQEL (rat)
GADPSADATEAYQEL (mouse)
GGDPGADATAAYQEL (human)
Peptide 7: Amino acids 762-777
KNRSLAVLTYDHSRI (rat)
KNRSLAVLTYDHSRI (mouse)
KNRSLAVLTYDHSRV (human)