The Generation of Retrogenic Mice Insertion: TCR alpha only or alpha_P2A_beta Phoenix cells (293T...
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Transcript of The Generation of Retrogenic Mice Insertion: TCR alpha only or alpha_P2A_beta Phoenix cells (293T...
Insertion: TCR alpha only oralpha_P2A_beta
Phoenix cells(293T cells transfected with pCL-Eco)
pMIG
pMIG
pMIG
SCID orC-KOBM cells
SCID
3’LTR
5’LTR
IRE
S
GFP
Ψ
pMIG
MIG
CMV-env (Mo-MuLV)RSV-gag-pol (Mo-MuLV)
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
Rag-/- isletsOnly islets + type B
CD11c islets
InsulinomaType A
Insulin granules
Type B
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 I-A(g7) peptide-binding groove.
Chr. 14
Chr. 6
V
J
V
D
J
Peptide
Antigen Presenting Cell
Antigen Recognition by CD8+ T Cells
T cellreceptor
V V
Tcell
class IMHC
2m
cellT. DiLorenzo
V(D)J Rearrangement
1011 -1016 different possible
combinations.
Kuby Immunology
I-Ag7I-Ag7
PeptidePeptide
AA Peptide Side ChainsAA Peptide Side ChainsAA Peptide Side ChainsAA Peptide Side Chains
TEETH
GROOVE
“TEETH”
T Cell Receptor Analogy
Child with IPEX syndrome
Awaiting Bone Marrow Transplant
9 Months of Age
IPEX: Immune Dysfunction, Polyendocrinopathy, Enteropathy, X-linked
• 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: Bottazzo
-cells
PancreaticLymph Node
T cells
ISLET
A MODEL
Ag
APC
t
tt
Mathis/Benoist
TRAV5D-04+TRAJ53 Restriction?
Insulin B:9-23: SHLVEALYLVCGERG?
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
?
MAN NOD MOUSE
IAg7DR3/DR4
Homann 2006,JCI
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
T Cell Receptor
Natural T Regulatory Cells
Antigen Specific T Reg
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
Amino Acid Sequence of Mouse 1 and 2 and Human Insulin
B:9-23
0 10 20 30 40 50 600
20
40
60
80
100
ins1-, ins2-, Tg+ (n=25, P<0.01)
ins1+, ins2-, Tg+ (n=25)
Weeks of age
% D
iab
ete
s F
ree
Lack of progression to diabetes of NOD mice lacking both insulin native genes.
Ins1-, ins2-: n=Ins1+, ins2-: n=
2525
1014
2123
11
24 Life table update 5/19/05
Nakayama Nature 435:220,2005
Insulin is the primary antigen and precedes IGRP in hierarchy of autoantigens
Epitope and antigen spreading,expansion
Diabetes
Insulin specific T cells
Insultis
Krishnamurthy et al JCI:116:3258, 2006
Is there a primary antigen or immune response to multiple antigens required for autoimmunity?
Epitope and antigen spreading, expansion
Diabetes
T cells specific for one antigen
Diabetes
Expansion of T cells
T cells specific for multiple antigens
Insulitis Insulitis
OR
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, 2005Structure 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
P1 P4 P9
I-Ag7 v,e,q I,L A,s D,E
12% 20% 30,11% 45%
P6
DQ8 E,d A,S A,V,s E,D
27,17% 19% 20% 60,25%
Preferred AA in Bound Peptides
Wiley Nat Immunol
% amino acid at position
NOD Mouse anti-islet T Cell Clones/transgenics/retrogenics
Clone CD4/CD8 Source Antigen TCRα/β Tetramer TransgenicRetrogenicComment AuthorBDC2.5 CD4 Spleen 7D-6/2 yes yes yes 10.1 same mimotope HaskinsBDC10.1 CD4 Islet 17/20 yes yes DM+++ retrogenic HaskinsBDC6.9 CD4 Islet 5D-4*01/2 yes yes Strain specific, chr 6 HaskinsNY4.1 CD4 Islet 5D-4*04/ yes yes yes DM transgenic Santamaria
Phogrin-13 CD4 Immun LN IA2β 640-659 13-2/3 yes insulitis retrogenic HuttonPhogrin-18 CD4 Immun LN IA2β 755-777 10/16 yes insulitis retrogenic Hutton10.23 CD4 IA2 676-688 13-4/4 yes Hutton
2H6 CD4 Panc LN insulin B:12-25 21/31 yes TGFβ protection ZekzerBDC12-4.1 CD4 Islet insulin B:12-22 5D-4*04/1 yes yes DM retrogenic WegmannBDC12-4.4 CD4 Islet insulin B:12-22 5D-4*04/5 yes DM retrogenic WegmannBDC 8-.1.1 CD4 Immun LN insulin B:12-22 5D-4*04/15 yes DM retrogenic WegmannBDC12-1.19 CD4 Islet insulin B:9-23 13-1/19 DM retrogenic WegmannBDC12-2.35 CD4 Islet insulin B:9-18 7-4/13-1 DM retrogenic WegmannBDC6-4.3 CD4 Islet insulin B:9-23 5D-4*04/1 WegmannBDC12-2.4 CD4 Islet insulin B:9-16 Wegmann6C5 CD4 islet 9D-4 young NOD Fathman
5A CD4 Immun Spl GAD ZekzerB16.3 CD4 spleen GAD65 pp286–300 6-2/5 yes suppress DM McDevittBW5147 CD4 spleen GAD65 pp206–220 3/13-2 suppress DM McDevittPA15.14B CD4 spleen GAD pp206–220 6D-6/13-3 yes no effect retrogenic VignalliIA4 CD4 GAD pp217–236 6-6/2 yes no effect retrogenic TischPA17.9G7 CD4 GAD pp284–300 6-2/4 yes no effect retrogenic VignalliPA18.10F10 CD4 GAD pp510–524 7-4/4 yes no effect retrogenic VignalliPA18.9H7 CD4 GAD pp524–538 21/15 yes no effect retrogenic Vignalli530.45.19 CD4 GAD pp530–543 (5D-4/2) yes no effect retrogenic Sercarz
G9C8 CD8 Islet insulin B:15-23 yes Wong
8.3 CD8 Islet IGRP 206-214 yes yes Perforin independent, Fas Santamaria
Peakman
A2 CD8 PPI:15-24
Durinovic-Bello
DRB1*0401 CD4 73-90
Gottlieb
B:9-23 DQ8
Peakman
DRB1*0401 C19-A3
Hafler
DRB1*0401 A1-15
Expanded T cells from pancreatic lymph nodes of type 1 diabetic subjects recognize an insulin epitopeKent 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 RestrictedInsulin 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 HLA-A*0201-restricted T cell responses to Insulin Jarchum and
DiLorenzo J Immunol 2010 vol 184
Ins B:10-18
Humanized HLA-A2 Mice lacking Insulin 2!
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 H-2b.
The frequency and immunodominance of Islet-specific 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
New Responses
PROINSULIN
PREPROINSULIN
“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
Female NOD Mice Peripheral Blood
Avidin
Kd
NRP-V7 Peptide (KYNKANVFL)
Kd
Kd
Kd
Tetramer Analysis
0
0.2
0.4
0.6
0.8
1
1.2
5 10 14 18 21 24 27 30
Age (weeks)% N
RP
-V7
te
tra
me
r+ C
D8
+ c
ell
s
Diabetes
0
0.2
0.4
0.6
0.8
1
1.2
5 9 12 15 18 21 24 27 30
Age (weeks)% N
RP
-V7
te
tra
me
r+ C
D8
+ c
ell
s
No Diabetes
Tan et al., JCI 1/2003
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
CD8+ CTL
CTL
Screening of Peptide Fractions by 51Cr Release Cytotoxicity Assay Method Used to Discover IGRP
APC
T. DiLorenzo
GAD, glutamic acid decarboxylase; IAPP, islet amyloid polypeptide
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
Antigens for CD8+ T Cells in Type 1 Diabetes Patients
DiLorenzo
Antigen Position MHC T cells How identified
DMK 138-146 H-2Db AI4; isletPositional scanning
libraries
IGRP 206-214 H-2Kd 8.3; isletMHC 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
Insulin 1/2 B15-23 H-2Kd G9C8; islet Expression cloning
DMK, dystrophia myotonica kinaseGAD, glutamic acid decarboxylaseIGRP, islet-specific glucose-6-phosphatase catalytic subunit-related protein
Antigens for Islet-infiltrating CD8+ T Cells in NOD Mice
DiLorenzo
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 34-42(B10-18); Proins 41-50(B17-26);Proins 42-51;Prins44-51; but also B chain-C-peptide (49-57)
• Multiple class I alleles, including A1 and B8
Peptide 2: Amino acids 643-658Peptide 7: Amino acids 762-777
C terminus
PTP
GADPSADATEAYQEL (rat) GADPSADATEAYQEL (mouse) GGDPGADATAAYQEL (human)
KNRSLAVLTYDHSRI (rat)KNRSLAVLTYDHSRI (mouse)KNRSLAVLTYDHSRV (human)
TM
N terminus
T cell epitopes on the diabetes autoantigen Phogrin (IA-2beta) are conservedamong different species