Chapter 4
The Role of T Cells in Beta Cell
Damage in NOD Mice and
Humans
Identifying a Swarm of T cells Targeting B:9-23 Insulin Peptide?
Trimolecular Recognition Complex
Fundamental Structure Diabetes
Susceptibility
TCR
MHC
PEPTIDE
Pathogenesis of NOD diabetes is initiated by reactivity to the insulin
B chain 9-23 epitope and involves functional epitope spreading
Prasad et al J. Autoimmunity June 2012 on line
LATE PREVENTION ONLY
ECDI-INSULIN
Multiple Antigens are Targeted in
Type 1 Diabetes
Synaptic-like
Microvesicles
Mitochondria
Secretory
Granules
Endoplasmic
Reticulum
Golgi
Network
Nucleus
Cytosol
Figure modified from Tissue Antigens 2003, 62:359
Information from Expert Rev Clin Immunol 2010, 6:939
HUMAN MOUSE
H
M
Ab T Ab T
CD38
Chg A
CPE
DMK
DNA TopII
GAD65
GAD67
GD3
Glima38
GLUT2
GM2-1
GT3
HIP/PAP
Hsp10
Hsp60
Hsp70
Hsp90
IA-2
IA-2
IAPP
ICA69
IGRP
Imogen38
Importin 
Insulin
Jun-B
PDX1
Peripherin
Reg
RegII
S100
SOX13
Sulphatide
ZnT8
x
x
x x x x
x
x
x x x x
x x
x
x
x
x x
x
x
x
x
x
x x x x
x x
x
x x
x
x x
x
x x
x x
x
x
x
x
x
x x x x
x x
x
x x
x x
x
x
x
x
x
x
x
Reported
Antibody
or T Cell
Dilorenzo
A MODEL
-cells
APC
ISLET
t
Ag
t
t
T cells
Pancreatic
Lymph
Node
Mathis/Benoist
AA Peptide Side Chains
Peptide
MHC
V(D)J Rearrangement
Kuby Immunology
GROOVE
“TEETH”
TEETH
T Cell Receptor Analogy
Antigen Presenting Cell
Peptide
V
V
D
J
Chr. 14
J
Chr. 6
Antigen Recognition by CD8+ T Cells
T
cell
T cell
receptor
V
V
class I
MHC
2m
 cell
T. DiLorenzo
“Typical” Insulitis of man differs from peri-insulitis NOD mouse:
Meta-Analysis of Insulitis – T1D Patients Stratified
to Age at Onset and Duration of Disease
In’t Veld and Atkinson, Diabetes (Submitted)
Duration of disease
≤1 week
>1 week ≤1 year
>1 year
total
Onset childhood (014 yrs)
22/23
37/42
3/32
62/97
Onset young adult
(15-39 yrs)
8/14
17/26
1/23
26/63
Pipeleers and Ling, Diabetes/Metabolism Reviews 8:209, 1992.
Gepts et al, 1965, 1978; ,Doniach et al, 1973; Klöppel et al, 1984; Bottazzo et al, 1985; Foulis et al, 1986; Hänninen et
al, 1992; Somoza et al, 1994; Lernmark et al, 1995; Shimada et al, 1999; Dotta et al, 2007; Uno et al, 2007; Butler et al,
2007, Gianani and Atkinson, 2010.
Atkinson
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
Preproinsulin/Proinsulin/Insulin
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
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
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
Insulin gene VNTR genotype associates with frequency and phenotype of the
autoimmune response to proinsulin. I Durinovic-Belló,1* R P Wu,1 V H Gersuk,1 S
Sanda,1 H G Shilling,1 and G T Nepom1
Genes Immun. 2010 March; 11(2): 188–193.
TETRAMERS DRB1*0401
Proinsulin 176-90
p176-90 P9S
High Avidity Peptide
GAD555-567
Control
A306-318
Control
Structural basis for the killing of human beta cells
by CD8(+) T cells in type 1 diabetes. Nat Immunol.
2012 Jan 15. 2012. Bulek……Peakman…Sewell
CD8 Preproinsulin Leader
sequence A2 presented
peptide ALWGPDPAAA:
TCR low affinity, normal
orientation.
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
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
INSULIN
B:9-23
NOD Female
Digest to Islets
(collagenase)
Digest to Single Cells
(trypsin)
Blood Clot
Islet Cells
Infiltrating Cells
Histologic Section
of Pancreas
NOD Spleen
Cell Suspension
Daniel and Wegmann
13
E
V
L
A
L
A
23
Y
G
16
L
R
V
H
E
C
G
S
9
Barbara Davis Center
B16:A Insulin
% of Diabetes Free
100
80
ins1+, ins2- (n=24)
ins1-, ins2- (n=31, P<0.0001)
60
NO EFFECT KO
GAD, IGRP,
IA-2, IA-2β
40
20
0
0
10
20
30
40
50
60
Weeks
Nakayama et al. Nature 435:220-3 2005
NOD ANTI-B:9-23 ALPHA CHAIN T CELL RECEPTORS:
DOMINANT
USAGE TRAV5
Valpha
Junction
Clone ID
12-4.1
12-4.4
4-7.2
6-4.3
6-10.14
8-1.1
8-1.3
8-1.9
8-1.15
12-3.20
6-6.4
12-1.19
12-2.35
12–2.40
6–11.6
I-29
IIT-3
IIT-37
4E4-62
4E7
AS91
AS150
2H6
TRAV
Vα
CDR1
5D-4*04
5D-4*04
5D-4*04
5D-4*04
5D-4*04
5D-4*04
5D-4*01
5D-4*04
5D-4*04
5D-4*04
12
13-1
7D-4*02
5D-4*04
5D-4*04
10*01
5D-4
10/10D
5D-4
5D-4
5-1*01
Vα13.3
Vα13.3
Vα13.3
Vα13.3
Vα13.3
Vα13.3
Vα13.1
Vα13.3
Vα13.3
Vα13.3
Vα8
Vα10
Vα1
Vα13.3
Vα13.3
Vα15
Vα13
Vα15
Vα13
Vα13
-
DSASNY
DSASNY
DSASNY
DSASNY
DSASNY
DSASNY
NSASNY
DSASNY
DSASNY
DSASNY
Not Available
21*01
Vα6
TISGNEY
TTLNS
DRNFQY
DSASNY
DSASNY
DTASSY
DSASNY
DTASSY
DSASNY
DSASNY
DSASVY
CDR2
V
MYFCAAS
IRSNME
MYFCAAS
IRSNME
MYFCASS
IRSNME
MYFCASS
IRSNME
MYFCASS
IRSNME
MYFCASS
IRSNME
MYFCASS
IRSNME
MYFCASS
IRSNME
MYFCASS
IRSNME
MYFCASS
IRSNME
LYYCA
SSTDNKR
TYLCAME
NPSG
LYFCAAI
IFSDG
MYFCAAS
IRSNME
MYFCAAS
IRSNME
IYFCAAS
IRSNVD
MYFCAAS
IRSNME
IYFCAAS
IRSNVD
MYFCAAS
IRSNME
MYFCA
IRSNME
IYFCS
IRSNME
Not Available
GLQQ
VYHCILRV
N
J
Jalpha
TRAJ
GAN
A
AN
ASG
SR
K
ARG
RP
A
KI
PNQ
RS
Q
GAN
S
PS
R
IP
AIG
G
RGN
SGGSNYKLTF
SGGSNYKLTF
GGSNYKLTF
SGGSNYKLTF
GGSNYKLTF
TGGNNKLTF
SGGSNAKLTF
GGSNAKLTF
NSGTYQRF
GGSNAKLTF
GGSAKLIF
SGYNKLTF
NYNQGKLIF
SGGSNYKLTF
SGGSNYKLTF
NSGGSNYKLTF
NYAQGLTF
NSGGSNYKLTF
TGNYKYVF
TGNYKYVF
NNRIFF
53*01
53*01
53*01
53*01
53*01
56*01
42*01
42*01
13*01
42*01
57*01
11*01
23*01
53*01
53*01
53*01
26*01
53*01
40*01
40*01
31*02
D
SGGSNYKLTF
53*01
NOD ANTI-B:9-23 T CELL RECEPTORS: NO DOMINANT USAGE TRBV
Vbeta
Clone ID
TRBV
12-4.1
1*01
12-4.4
5*01
4-7.2
5*01
6-4.3
1*01
6-10.14
8-1.1
15*01
8-1.3
19*01
8-1.9
2*01
8-1.15
12-3.20 31*01
6-6.4
4*01
12-1.19 19*01
12-2.35 13-1*02
12–2.40 13-1*02
6–11.6
5*01
1*01
I-29
16*01
IIT-3
16*01
IIT-37
4E4-62
1*01
4E7
AS91
AS150
2H6
31*01
Junction
Vβ
CDR1
CDR2
Vβ2
Vβ1
Vβ1
Vβ2
NSQYPW
LGHNA
LGHNA
NSQYPW
Vβ12
Vβ6
Vβ4
SGHND
FNHDT
LGHNA
Vβ14
Vβ10
Vβ6
Vβ8.3
Vβ8.3
Vβ1
Vβ2
Vβ11
Vβ11
GKSSPN
LGHDT
FNHDT
NSHNY
NSHNY
LGHNA
NSQYPW
SGHSA
SGHSA
Vβ2
NSQYPW
Vβ14
GKSSPN
LRSPGD
YNLKQL
YNLKQL
LRSPGD
Not
FRSKSL
SITEND
YSYQKL
Not
SITVG
YNNKQL
SITEND
SYGAGN
SYGAGN
YNLKQL
LRSPGD
FRNQAP
FRNQAP
Not
LRSPGD
Not
Not
SITVG
V
LYCTCS
YFCASSQ
YFCASSQ
LYCTCSA
Available
YLCASS
FLCAS
YFCASS
Available
YLCAWS
YLCASS
FLCASS
YFCASS
YFCASS
YFCASS
LYCTCS
YLCAAS
YLCAAS
Available
LYCTCSA
Available
Available
YLCAWS
Jbeta
nDn
J
TRBJ
PGLGN
DT
SRT
AGGG
EQYF
NTGQLYF
GNTLYF
TEVFF
2-7*01
2-2*01
1-3*01
1-1*01
LGWGD
TSGTGQG
PDNA
EQYF
SPLYF
NTEVFF
2-7*01
1-6*01
1-1*01
RLGG
WGQGG
ILGQ
PSGR
PSGR
LTGGAL
AGLGY
AVPGH
PRLGA
NQDTQYF
DTQYF
NTEVFF
NSPLYF
NSPLYF
QYF
EQYF
QDTQYF
SAETLYF
2-5*01
2-5*01
1-1*01
1-6*01
1-6*01
2-7*01
2-7*01
2-5*01
2-3*01
DQ
NQAPLF
1-5*01
PRQNFP
YF
2-3*01
Relevance to Human?
Human Valpha 13-1 (Closest TRAV5D-4) Causes DM
Nakayama
Diabetes 2012
Non-Expanded CD4 POSITIVE CELLS ISOLATED FROM
8 WEEK OLD NOD ISLETS (Kappler PNAS 2011)
CD44
Anti-”B:9-23”
Control Tet
Anti-B:9-23 Tetramer
7/26/2016
Anti-B:9-23 Tetramer
Kappler PNAS 2011
Ostrov
Model
Insulin
Peptide
B:9-23 in
Register 3
of I-Ag7
Ostrov
Model
Insulin
Peptide
B:9-23
in
Register
2 of IAg7
ARG(22)
GLU
GLY
CYS
VAL
LEU
TYR
LEU
INSULIN PEPTIDE B:9-23 IN TWO REGISTERS (arrow 3)
ALA(14)
INSULIN PEPTIDE B:9-23 binding REGISTERS
Insulin B:9-23: S H L V E A L Y L V C G E R G
S H LV EALYLVA E RG
Register 1 R E A L Y L V C E
p1
p4 p6
p9
S H LVE ALYLVAG
RG
R E AY Y V V C E
Register 2
p1
p4 p6
p9
S H LV EALYLVA GE
G
R E AL Y A V
E
Register 3
p1
p4 p6
p9
S H LV EALYLVCG ER G
p1
p4 p6
p9
P1,P4,P6, AND P9
Pockets of I-Ag7 where side chains of peptide bind.
Michels et al
Structure-Based Selection of Small Molecules To Alter
Allele-Specific MHC Class II Antigen Presentation
J Immunol 2011;187;5921-5930
Ostrov
David Ostrov Model Glyphosine in Pocket 9
(amino acids in yellow form p9)
I-Ag7 α
Arg76 α
Ser57 β
I-Ag7 β
Glyphosine enhances T cell stimulation
pe
pt
id
e
no
10
pe
0
pt
nM
id
e
no
pe
10
pt
nM
id
e
no
pe
pt
id
e
Glyphosine
nM
25
0
50
0
nM
no
10
nM
nM
10
0
nM
25
0
nM
50
0
B
:9
-2
3
20
18
16
14
12
10
8
6
4
2
0
TT
SI
8-1.1 alpha1 with Insulin B:9-23
Peptide/[Glyphosine]
Glyphosine only enhances stimulation of the T-cell hybridoma 8-1.1α1
when insulin B:9-23 peptide is present.
Michels
Early Prevention Study with Glyphosine
% Without Diabetes
100
Control, n=12
Glyphosine, n=17
75
P<0.001
50
25
0
0
5
10
15
20
25
30
Weeks of Age
END
START
Michels et al J. Immunol 2011
Multiple Approaches to Therapeutically Target Critical Diabetogenic
Trimolecular Recognition Complexes (MHC+Peptide+TCR)
TCR
Vbeta 13 TCR Targeting unknown beta
cell antigen Liu et al Diabetes 2012
Peptide
MHC
Anti-Rat Vbeta 13
MHC:I-Ag7
Small
Molecules
p6:4
p1:6
p6:17
p9:12
p1:17
p6:18
Insulin
Peptide
p1
Anti-MHC+Insulin Peptide
Zhang et al Diabetes
Metab Res Rev. 2011
p4
p6
p9
MHC
Michels et al J Immunol 2011
Prevention of type 1 diabetes in mice by tolerogenic vaccination with a
strong agonist insulin mimetope
Carolin Daniel,1,4 Benno Weigmann,3 Roderick Bronson,4
and Harald von Boehmer1,2
JEM 2011
Register 3 mimotope
3-4 wk
vaccination
12-14 wk
vaccination
Stadinski et al
Diabetogenic T
cells recognize
insulin bound
to IAg7 in
an unexpected,
weakly binding
register
PNAS
107:10978,
2010
All anti-B:923 only
reacted with
peptide in
Register 3
with arginine
in pocket 3
By mutating the MHC binding groove of NOD’s I-Ag7 insulin peptide B:9-23
is fixed in specific register 3, and this is low affinity register diabetogenic
T cell receptors “see” the peptide.
FIXING IN REGISTER 3 WITH DISULFIDE BONDS
Stadinski et al PNAS 2010
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
IGRP
Islet-specific glucose-6phosphatase catalytic subunit
related protein
“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
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
IAPP
Islet Amyloid Polypeptide
Islet Amyloid Polypeptide Is a Target Antigen for
Diabetogenic CD4+ T Cells
Thomas Delong, …Kathryn Haskins Diabetes 2011
GAD
Glutamic Acid Decarboxylase
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
C-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
Chromagranin A
Peptide WE-14
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.
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
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
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%
Clone
BDC2.5
BDC10.1
BDC5.10.3
BDC5.2.9
BDC6.9
NY4.1
CD4/CD8
CD4
CD4
CD4
CD4
CD4
CD4
Source
Spleen
Islet
Islet
Islet
Islet
Antigen
Chromagranin
Chromagranin
Chromagranin
Amyloid(IAPP 1-20)
? IAPP
TCRα/β
Tetramer Transgenic
7D-6/2
yes
yes
17/20
yes
/2
2/19
5D-4*01/2
yes
5D-4*04/16
yes
Retrogenic Comment
yes
10.1 same mimotope
yes
DM+++ retrogenic
Author
Haskins
Haskins
yes
yes
Strain specific, chr 6
DM transgenic
Haskins
Santamaria
IA2β 640-659
IA2β 755-777
IA2 676-688
13-2/3
10/16
13-4/4
yes
yes
yes
insulitis retrogenic
insulitis retrogenic
Hutton
Hutton
Hutton
TGFβ protection
DM retrogenic
DM retrogenic
DM retrogenic
DM retrogenic
DM retrogenic
Zekzer
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Nakayama
Unanue
Unanue
Unanue
Phogrin-13 CD4
Phogrin-18 CD4
10.23
CD4
Immun LN
Immun LN
Immun LN
2H6
12-4.1
12-4.4
8-1.1
12-1.19
12-2.35
6-4.3
12-2.4
PCR1-10
AS91
AS150
I.29
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
Panc LN
insulin
Islet
insulin
Islet
insulin
Immun LN
insulin
Islet
insulin
Islet
insulin
Islet
insulin
Islet
insulin
anti 8-1.1 isletinsulin
Islet
insulin
Islet
insulin
Islet
insulin
6C5
CD4
islet
5A
B16.3
BW5147
PA15.14B
IA4
CD4
CD4
CD4
CD4
CD4
Immun Spl
spleen
spleen
spleen
PA18.10F10CD4
PA18.9H7 CD4
530.45.19 CD4
PA17.9G7 CD4
4B5
CD4
PA19.9G11 CD4
B:12-25
B:12-22
B:12-22
B:12-22
B:9-23
B:9-18
B:9-23
B:9-16
B:9-23
B12-20
B13-21
21/31
5D-4*04/1
5D-4*04/5
5D-4*04/15 yes
13-1/19
7-4/13-1
5D-4*04/1
yes
yes
yes
yes
yes
5D-4*04/16 yes
5-1/
yes
10/1
Same alpha 8-1.1
yes
9D-4/19
GAD
GAD65 pp286–300
GAD65 pp206–220
GAD pp206–220
GAD pp217–236
6-2/5
3/13-2
6D-6/13-3
6-6/2
Immun
GAD
GAD
GAD
GAD
GAD
GAD
7-4/4
21/15
(5D-4/2)
4.5/10
2.4/6
17.1/5.2
Immun
yes
yes
pp510–524
pp524–538
pp530–543
pp284–300
pp217-236:290-309
pp221-237:284-300
G9C8
CD8
Islet
insulin B:15-23
8.3
AI4
CD8(Kd)
CD8(D6)
Islet
IGRP 206-214
DMK 138-146
yes
yes
yes
yes
yes
yes
yes
yes
17/
yes
young NOD
Fathman
suppress DM
suppress DM
no DM retrogenic
no DM retrogenic
Zekzer
McDevitt
McDevitt
Vignalli
Tisch
no
no
no
no
no
no
Vignalli
Vignalli
Sercarz
Vignalli
Tisch
Vignalli
DM retrogenic
DM retrogenic
DM retrogenic
DM, encephalitis
DM, encephalitis
DM, encephalitis
Wong
yes
Perforin independent, Fas Santamaria
DMK Widespread
DiLorenzo
NOD T CELL CLONES
Clone
BDC2.5
BDC10.1
BDC5.10.3
BDC5.2.9
BDC6.9
NY4.1
CD4/CD8
CD4
CD4
CD4
CD4
CD4
CD4
Source
Spleen
Islet
Islet
Islet
Islet
Antigen
Chromagranin
Chromagranin
Chromagranin
Amyloid(IAPP 1-20)
? IAPP
TCRα/β
Tetramer Transgenic
7D-6/2
yes
yes
17/20
yes
/2
2/19
5D-4*01/2
yes
5D-4*04/16
yes
Retrogenic Comment
yes
10.1 same mimotope
yes
DM+++ retrogenic
yes
yes
Strain specific, chr 6
DM transgenic
IA2β 640-659
IA2β 755-777
IA2 676-688
13-2/3
10/16
13-4/4
yes
yes
yes
insulitis retrogenic
insulitis retrogenic
Hutton
Hutton
Hutton
TGFβ protection
DM retrogenic
DM retrogenic
DM retrogenic
DM retrogenic
DM retrogenic
Zekzer
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Wegmann
Nakayama
Unanue
Unanue
Unanue
Phogrin-13 CD4
Phogrin-18 CD4
10.23
CD4
Immun LN
Immun LN
Immun LN
2H6
12-4.1
12-4.4
8-1.1
12-1.19
12-2.35
6-4.3
12-2.4
PCR1-10
AS91
AS150
I.29
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
CD4
Panc LN
insulin
Islet
insulin
Islet
insulin
Immun LN
insulin
Islet
insulin
Islet
insulin
Islet
insulin
Islet
insulin
anti 8-1.1 isletinsulin
Islet
insulin
Islet
insulin
Islet
insulin
6C5
CD4
islet
5A
B16.3
BW5147
PA15.14B
IA4
CD4
CD4
CD4
CD4
CD4
Immun Spl
spleen
spleen
spleen
PA18.10F10CD4
PA18.9H7 CD4
530.45.19
CD4
PA17.9G7 CD4
4B5
CD4
PA19.9G11 CD4
CD8
8.3
CD8(K d)
6
21/31
5D-4*04/1
5D-4*04/5
5D-4*04/15 yes
13-1/19
7-4/13-1
5D-4*04/1
yes
yes
yes
yes
yes
yes
yes
5D-4*04/16 yes
5-1/
yes
10/1
Same alpha 8-1.1
yes
9D-4/19
GAD
GAD65 pp286–300
GAD65 pp206–220
GAD pp206–220
GAD pp217–236
6-2/5
3/13-2
6D-6/13-3
6-6/2
Immun
GAD
GAD
GAD
GAD
GAD
GAD
7-4/4
21/15
(5D-4/2)
4.5/10
2.4/6
17.1/5.2
Islet
insulin B:15-23
Islet
IGRP 206-214
Immun
G9C8
B:12-25
B:12-22
B:12-22
B:12-22
B:9-23
B:9-18
B:9-23
B:9-16
B:9-23
B12-20
B13-21
pp510–524
pp524–538
pp530–543
pp284–300
pp217-236:290-309
pp221-237:284-300
yes
yes
yes
yes
yes
yes
yes
yes
17/
yes
Author
Haskins
Haskins
Haskins
Haskins
Haskins
Santamaria
young NOD
Fathman
suppress DM
suppress DM
no DM retrogenic
no DM retrogenic
Zekzer
McDevitt
McDevitt
Vignalli
Tisch
no
no
no
no
no
no
Vignalli
Vignalli
Sercarz
Vignalli
Tisch
Vignalli
DM retrogenic
DM retrogenic
DM retrogenic
DM, encephalitis
DM, encephalitis
DM, encephalitis
Wong
yes
Perforin independent, Fas Santamaria
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
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.
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
PreproInsulin
Leader
17-24
A2
Mass Spec MHC elution
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
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
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)