animal model of immune dysregulation
TRANSCRIPT
Type 1 insulin-dependent Type 1 insulin-dependent autoimmune diabetes. autoimmune diabetes.
Ciriaco A. PiccirilloCanada Research Chair
Department of Microbiology & Immunology
McGill University Health Center
Diabetes Classification
• Type 1– Immune Mediated– Insulin deficient, autoantibodies
• Type 2– No Autoantibodies and treated without insulin
• Other Specific forms of Diabetes– Gestational Diabetes
• Monogenic:Monogenic: Single gene defect.Single gene defect. APS-I: AIRE autosomal recessive APS-I: AIRE autosomal recessive IPEX: Scurfy Gene X-linked IPEX: Scurfy Gene X-linked
• Polygenic:Polygenic: Summation of small effects of multiple genes creating diabetes susceptibility Summation of small effects of multiple genes creating diabetes susceptibility – e.g. NOD mousee.g. NOD mouse
The Non-Obese Diabetic (NOD) mouse.Model of spontaneous Type 1 insulin-dependent diabetes (T1D).
-islet Insulin
Normal
Type 1Autoimmune
diabetes
pLN
pancreas
Abnormal peripheral T cell tolerance:
Self-reactive T cell
Checkpoints1. Peri-insulitis (Th2)
2.Insulitis (Th1)
Cellularity heterogeneous
Salivary glandsThyroidNuclear antigensTestesOvaries
Multi-organ Autoimmunity
Cumulative incidence of T1D:
80% in females,
30% in males
(at 30 weeks)
Activated TH1 CD4+ T Cell
CD4+ Cell(TH2 )
CD4+ Cell
(TH0 )
DR3, DR4,,DQ8/insulin peptide
CD2
Macrophage/dendritic cell
Fc R
IFN-
IL-12 CD40L
CD40
, TCR
IL-1, TNF, LT, NO, PGE-2
B Cell?anti-insulin, GAD ab anti-Mog
IL-4
Immunopathophysiology of Diabetes
?Antibody mediated injury
Dendritic cell/APC
CD40L
IL-4CD40L
CD8+ CTL
FasLperforin
cell death islet cells
LOSS OF FIRST PHASE LOSS OF FIRST PHASE INSULIN RESPONSE INSULIN RESPONSE
TIMETIME
Stages in Development of Type 1 DiabetesStages in Development of Type 1 Diabetes B
ET
A C
EL
L M
AS
SB
ET
A C
EL
L M
AS
S
DIABETES“PRE”-
DIABETES
GENETICPREDISPOSITION
INSULITISBETA CELL INJURY
NEWLY DIAGNOSED DIABETES
MULTIPLE ANTIBODY POSITIVEMULTIPLE ANTIBODY POSITIVE
GENETICALLY AT RISK
(?Precipitating Event)Progressiveloss insulinrelease
Glucosenormal
Overt immunologicabnormalities
Normal insulin release
Autoantibodies/Autoreactive B Cells Contribute to NOD Diabetes
• Immunoglobulin knockout prevention NOD DMSerreze et al, J. Immunol 1998, 161:3912-3918
• I-Ag7 on B cells needed for NOD diabetes.Noorchashm et al, J. Immunol 1999, 163, 743-750
• Anti-Insulin VH125 Heavy Chain Increases diabetes in NOD mice.Hulbert et al, J. Immunol, 2001, 167: 5535-5538
• Transplacental autoantibodies accelerate NOD diabetes.Greeley et al, Nature Immunol.
Progression to Diabetes increases with number of Autoantibodies(GAD, ICA512, Insulin)
T1D in NOD mice is T cell dependentT1D in NOD mice is T cell dependent
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
8-9 wks 13 wks 18 wks
NO
D S
CID
NO
D S
CID
NO
DN
OD
0%
11%
70%
*Diabetic T cell transfer into normal or immunodeficient NOD*T cell depletion studies*Combined contributions of CD4+ and CD8+ T cells:
- Lessons from knockouts*CD4+ and CD8+ T cell clones can induce T1D alone.
Clones/Lines CD4/CD8 Source Antigen TCR Tetramer Transgenic Comment Author
BDC2.5 CD4 NOD Spleen Unknown V 4, V1 Yes Yes Haskins
BDC6.9 CD4 NOD Spleen Unknown V 4, V13.1 Yes Chromosome 6 Haskins
BDC6-4.3 CD4 NOD Islet InsulinB:9-23
V13.3-J53 Wegmann
BDC12-2.4 CD4 NOD Islet InsulinB:9-23
V13.3-J53 Wegmann
2H6 CD4 PeripancreaticLymph Node
InsulinB:12-25
V14 TGF betaProtective
Zekzer
5A CD4 ImmunizedSplenocytes
GAD524-543
Zekzer
4.1 CD4 Unknown Yes Yes Santamaria
Phogrin-15 CD4 ImmunizedLymph Node
PhogrinPeptide 2
V 8, V10 Kelemen
Phogrin -12 CD4 ImmunizedLymph Node
PhogrinPeptide 7
V 5,V11.3 Kelemen
G9C8 CD8 NOD Islet InsulinB:15-23
Yes Wong
8.3 CD8 NOD Islet NRP-A7mimotope
V17-J42 Yes Yes PerforinindependentFas mediated
Santamaria
A/4 CD8 NOD Islet V8-J52 Yes Serreze
Lines CD4CD4CD8
GAD: 530-543GAD: 524-538GAD: 546-554
V4
V12
“Driver”Protective
QuinnQuinnQuinn
Autoantigens: Lessons from diabetogenic T cells.
Diverse T cell response - epitope spreading/cascade?
Checkpoints in T1D development
Checkpoint 1 Insulitis (peri)
-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-3-4 weeks of age, non-destructive-Th2 dominated
Checkpoint 2 Beta cell loss & diabetes- T cells gain more aggressive effector mechanisms: Th1/Th2 balance, expression of Fas Ligand on CTLs, direct cytotoxicity.-Loss of protective mechanisms:
-Protective cytokines, Regulatory T cells-Amplification : Epitope spreading-10-12 weeks of age, destructive-Th1 dominated
Diabetogenic MHC I-Ag7• The unusual H-2g7 MHC haplotype of NOD mice:
Kd, I-Ag7,I-Enull, Db: Idd1 on chromosome 17.
• I-Ag7 and some HLA-DQB alleles: encode serine, alanine, or valine at position 57 and mediate T1D susceptibility
• Aspartic acid at position 57 is associated with resistance.
• Mutations to Aspartic acid reduce disease incidence but does not reduce insulitis.
• Homozygosity is required for disease:– Possible requirement for a threshold of MHC-peptide complexes for tolerance
induction.– T1D incidence increases with HLA haplotype combinations (DR2/3)
DQB1*0402Asp57
Leu56
-chain
-chain
Defective Central ToleranceDiabetogenic MHC I-Ag7
1. I-Ag7 haplotype is poor peptide binder.
2. Failure to efficiently negatively select autoreactive T cells
3. Failure to positively select Treg cells.
Abnormal peripheral T cell Tolerance in NOD mice.
• Hyporesponsive T cell responses:– TCR induced proliferation and cytokine production (IL-2 / IL-4).– Deficient PKC/Ras/MAPK pathway
– Weak MLR response
• Deficient frequency of NK-T cells:– IL-4 producing cells
– Th1/Th2 balance
• Aberrant regulatory T cell network.
T cell immunoregulation in the NODEvidence
• Delay between insulitis onset and diabetes• Prediabetic T cells prevent adoptive transfer of
disease into NOD.scid mice.• Thymectomy• Cyclophosphamide• Treg cells are numerous and heterogeneous
Balance of effector and regulatory Balance of effector and regulatory mechanisms determines peripheral tolerancemechanisms determines peripheral tolerance
Type 1 insulin dependent autoimmune diabetes
-islet Ags
nTreg
CD4+Foxp3+
Functional deficiency in CD4+CD25+ Treg cells in autoimmunity ?
nTreg
Self-reactiveTeff cell
Autoimmune disease:
Organ-specific - T1D, MS/EAE, Sjogren’s, ThyroiditisSystemic - SLE, APS, RA
CD4+CD25+
-islet Insulin
Normal
T1D
Age
T1D
Aberrant activation of effector T cells?
HealthT1D
Health
The Non-Obese Diabetic (NOD) mouse.
Model of spontaneous type 1 insulin-dependent diabetes (T1D).
Abnormal T cell tolerance to Abnormal T cell tolerance to -islet antigens-islet antigens
CD4+CD25+ nTreg cells in T1D.
•Functional deficiency in CD4+CD25+ Treg cells:– NOD mice succumb to T1D more rapidly in their absence– Delayed administration blocks disease.– NOD mice deficient for B7.1/2, CD40, and CD28 molecules have a more aggressive disease course.
•CD4+CD25+ Treg cells are absent
• Faulty “signals” in NOD mice?•Development, activation requirement, survival or function?
Adoptive transfer NOD model of T1D.
DiabetogenicTeff CD4+CD25-
ProtectivenTreg CD4+CD25+
Diabetes?
NOD.TCR -/-
Wild-Type
BDC2.5 Islet-specific
V4+CD4+ TCR Tg
NOD
Are there functional deficiencies in CD4+ nTreg cells in NOD?
0
20
40
60
80
100
120
0 5 10 15 20 25 30
Days post-transfer
Dia
bet
es i
nci
den
ce (
%)
WholeDep++++++
BDC2.5Teff Treg
+ -+ +- +
0
20
40
60
80
100
0 5 10 15 20 25 30
;
Thymus
Peripheral
Days post-transfer
Dia
bet
es
inci
de
nc
e (%
)D
iab
ete
s in
cid
en
ce
(%)
BDC2.5Teff Treg
+ -+ +
Functional CD4+CD25+ nTreg cells in NOD mice.
Age-dependent loss in nTreg cells?
Roland Tisch JEM 2005
Immunomonitoring of nTreg cells in health and disease.
nTreg X,Y,ZnTreg
Peripheral CD4+ T cells expressing CD25
Activated nTregActivated effectorsAnergized effectors
Induced Treg
HealthPre-clinical or
symptomatic disease
Immune activation
Normal Peri-insulitis Insulitis/T1D
Neonatal Adult
NOD BDC2.5
Are there quantitative differences in the cellular frequency ofCD4+ nTreg cells in NOD mice?
PancreaticLN
Non-draining
LN
CFSE
BDC2.5
BDC2.5 + Treg
PancreaticLN
Non-drainingLN
CD4+CD25+ nTreg cells do not affect the activation or proliferation
of diabetogenic T cells.
CD69
Resistance to T1D correlates with an increased infiltrate of CD4+ Foxp3+ nTreg cells
in pancreatic environments.
- nTreg function+ nTreg cells
- nTreg cells
Use of nTreg cells for the cure of T1D.
Primary Established
Tarbell et al. JEM 2004
Genetic determinants of nTreg cell development in
NOD mice?
Foxp3 dependent development of nTreg cells.
Deficiency of Foxp3+ nTreg cells promotes T1D.
Primary cause or consequence?
Inherited Susceptibility Loci: Both MHC and non-MHC genes are required.
LOCUS CHROMOSOME CANDIDATE GENES
IDDM1 6p21 HLA-DQ\DR
IDDM2 11p15 INS VNTR
IDDM3 15q26 D15s107
IDDM4 11q13 MDU1, ZFM1, RT6, FADD/MORT1, LRP5
IDDM5 6q24-27 ESR, MnSOD
IDDM6 18q12-q21 D18s487, D18s64, JK (Kidd locus)
IDDM7 2q31 D2s152, IL-1, NEUROD, GALNT3
IDDM8 6q25-27 D6s264, D6s446, D6s281
IDDM9 3q21-25 D3s1303
IDDM10 10p11-q11 D10s193, D10s208, D10s588
IDDM11 14q24.3-q31 D14s67
IDDM12 2q33 CTLA-4, CD28
IDDM13 2q34 D2s137, D2s164, IGFBP2, IGFBP5
IDDM14 ? NCBI # 3413
IDDM15 6q21 D6s283, D6s434, D6s1580
IDDM16 ? NCBI # 3415
IDDM17 10q25 D10s1750-D10s1773
Insulin Gene (INS)
Class I VNTR26-63 repeats
Predisposing
IDDM2
Insulin Gene (INS)
Class III VNTR140-200 repeats
IDDM2
Protective
The IDDM2 Locus
VNTR = Variable Number of Tandem Repeats • VNTR stimulates INS steady-state transcription in ß-cells
• VNTR length inversely correlates with INS mRNA levels in ß-cells in vivo
• Class III VNTR alleles = LOWER (~30%) INS transcription than predisposing class I VNTR alleles
• Class III VNTR alleles = Higher thymic INS transcription than predisposing alleles
Low incidence of T1D in Low incidence of T1D in
Idd3Idd3 recombinant congenic NOD mice recombinant congenic NOD mice
ChrChr
22
33
44
55
66
77
88
99
1100
11
1122 11
33 1144 11
551166
1177 11
88 1199XX
1111
20%20% Wicker LS et al. J Exp Med Wicker LS et al. J Exp Med 19941994Lyons PA et al. Genome Res Lyons PA et al. Genome Res 20002000
B6B6
..B6-B6-Idd3Idd3
NODNOD
1%1%
B6B6
NOD.NOD.B6-chr3B6-chr3
80%80%