type 1 diabetes no cure; therapy is insulin for life; physiologic glycaemic control never achieved...
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Type 1 diabetesType 1 diabetes
• No cure; therapy is insulin for life; physiologic glycaemic control never achieved
• No cure; therapy is insulin for life; physiologic glycaemic control never achieved
•Incidence increasing by ~5% every 5 years; costs ~£1 billion of UK NHS budget
•Incidence increasing by ~5% every 5 years; costs ~£1 billion of UK NHS budget
•Excess morbidity and mortality•Excess morbidity and mortality
Anti-insulin: health
Anti-insulin: disease
Peakman
CD8 T-cell
HLA I
•Infiltrating CD4+, CD8+ T cells•Anti-T cell therapies are effective•Islet cell autoantibodies disease
CD4 T-cell
HLA II
TCR
Epitope
CD4Treg
Type 1 diabetes is T cell mediatedType 1 diabetes is T cell mediated
Isletautoantigen
APC
Peakman
The Immune System
Innate• Rapid Microbial
Defense• Adaptive Immune
System Activation
Acquired (Adaptive)• Long-lived
Microbial Defense• Neoplasm
surveillance• Autoimmunity,
Transplantation Rejection & Atopy
BDC
The Innate Immune System
• Antimicrobial Peptides (e.g., Defensins, Cathelicidins)
• Phagocytes (Macrophages, Neutrophils, Monocytes, Dendritic Cells)
• Pattern Recognition Receptors• Alternative Complement System• NK Cells• B1B Cells*
* Aspects of both the innate and adaptive immune system
BDC
Selected Pattern Recognition Receptors: Toll-like ReceptorsTLR: Selected
Ligands:Role in Immunity: Localization:
TLR1 PGN, Zymosan, Lipoproteins
Antifungal & Antibacterial
Dendritic Cells,
Macrophages,
T Cells,
B Cells,
Epithelium
TLR2
TLR6
TLR4 LPS Antibacterial
TLR5 Flagellin
TLR11 ?
TLR9 CpG Antibacterial & Antiviral
TLR3 dsRNA Antiviral
TLR7 ssRNA
TLR8 ssRNA
TLR10 ? ?
Selected Pattern Recognition Receptors: Other Families
Receptor Selected Ligands Role in Immunity LocalizationNOD ProteinsNOD1 PGN (Gm-) Antibacterial CytoplasmicNOD2 PGN (Gm + & -) Antibacterial CytoplasmicCD14 LBP:LPS, PGN Antibacterial (with TLR4) Serum &
Phagocyte CellSurface
C-type LectinsMacrophageMannose Receptor(MMR), DC-SIGN,DEC-205
Glycoproteins orGlycolipids
Antibacterial, Antiviral,Antifungal
Macrophage,DCs
Surfactant A, D(Collectin Family)
LPS,Lipoproteins,Oligosaccharides
Opsonization of Bacteria,Virus & Fungi; CytokineStimulation; Apoptotic CellClearance
Soluble in theLungs
MBP/MBL Mannose groupson bacterialcarbohydrates
Complement Activation(Antibacterial & Antiviral)
Serum
ScavengerReceptorsSR-A,CD36
LPS, LTA, PGN Antibacterial; ApoptoticCell Clearance
Macrophages,Endothelium
Systematic Mouse ligand Human ligand Receptor Referencesname (alias) (alias)
CCL1 TCA-3/I-309 I-309 CCR8CCL2 JE/MCP-1 MCP-1 CCR2 Bertuzzi et al., 2004; Bradley et al., 1999; Cardozo et al., 2001; Cardozo et al., 2003;
Chen et al., 2001; Frigerio et al., 2002; Giarratana et al., 2004; Grewal et al., 1997; Kutlu et al., 2003; Nomura et al., 2000; Schroppel et al., 2005; Yang et al., 2004
CCL3 MIP-1 MIP-1 CCR1 & 5 Bradley et al., 1999; Cameron et al., 2000; Giarratana et al., 2004; Lohmann et al., 2002CCL4 MIP-1 MIP-1 CCR5 Bradley et al., 1999; Cameron et al., 2000; Lohmann et al., 2002)CCL5 RANTES RANTES CCR1, 3 & 5 Bradley et al., 1999; Carvalho-Pinto et al., 2004; Frigerio et al., 2002; Weber et al., 2006CCL6 C10 unknown CCR1CCL7 MARC/MCP-3MCP-3 CCR1, 2 & 3 Bradley et al., 1999; Matos et al., 2004CCL8 MCP-2 MCP-2 CCR3 & 5CCL9/10 MIP-1 unknown CCR1CCL11 Eotaxin-1 Eotaxin-1 CCR3CCL12 MCP-5 unknown CCR2 Bradley et al., 1999CCL13 unknown MCP-4 CCR2 & 3CCL14 unknown HCC-1 CCR1 & 5CCL15 unknown HCC-2/MIP-1d CCR1 & 3CCL16 unknown HCC-4 CCR1 & 2CCL17 TARC TARC CCR4 Giarratana et al., 2004; Kim et al., 2002CCL18 unknown PARC unknownCCL19 ELC MIP-3b/ELC CCR7 Bouma et al., 2005a; Bouma et al., 2005bCCL20 MIP-3/LARC MIP-3a/LARC CCR6 Cardozo et al., 2003CCL21 SLC/6Ckine SLC/6Ckine CCR7 Bouma et al., 2005b; Giarratana et al., 2004CCL22 MDC MDC CCR4 Giarratana et al., 2004; Kim et al., 2002CCL23 unknown MIPIF-1/MIP-3 CCR1CCL24 Eotaxin-2 Eotaxin-2 CCR3CCL25 TECK TECK CCR9CCL26 unknown Eotaxin-3 CCR3CCL27 CTACK CTACK CCR10CCL28 MEC MEC CCR3 & 10CXCL1 KC GRO CXCR2 Cardozo et al., 2001; Matos et al., 2004CXCL2 MIP-2 GRO CXCR2CXCL3 unknown GRO CXCR2CXCL4 PF4 PF4 CXCR3BCXCL5 LIX ENA-78 CXCR2 Matos et al., 2004CXCL6 unknown GCP-2CXCL7 TCK-1 NAP-2 CXCR2CXC8 unknown IL-8CXCL9 MIG MIG CXCR3 Christen et al., 2003; Frigerio et al., 2002; Matos et al., 2004CXCL10 IP-10 IP-10 CXCR3 Baker et al., 2003a; Baker et al., 2003b; Bradley et al., 1999; Cardozo et al., 2001;
Cardozo et al., 2003; Christen et al., 2004; Christen et al., 2003; Ejrnaes et al., 2005; Frigerio et al., 2002; Giarratana et al., 2004; Morimoto et al., 2004; Nicoletti et al., 2002; Rhode et al., 2005; Shimada et al., 2001
CXCL11 I-TAC I-TAC CXCR3 Cardozo et al., 2003CXCL12 SDF-1/PBSF SDF-1/ CXCR4 Dubois-Laforgue et al., 2001; Kawasaki et al., 2004; Kayali et al., 2003CXCL13 BLC BLC/BCA-1 CXCR5CXCL14 BRAK BRAK unknownCXCL15 Lungkine unknown unknownCXCL16 SR-PSOX SR-PSOX CXCR6XCL1 lymphotactin SCM-1/ATAC XCR1 Bradley et al., 1999; Weber et al., 2006XCL2 unknown SCM-1b XCR1
CX3CL1 fractalkine fractalkine CX3CR1 Cardozo et al., 2001
CXCR1 & 2
CXCR1 & 2
Table 1. T1D, chemokines and their receptors (modified after Lut et al., 2006). Chemokines with a putative role in T1D pathogenesis are identified by gray background shading.
The Adaptive Immune System
• Cell-mediated Immunity (Cytotoxicity)• T cells
• CD4+ (Th1 & Th2)
• CD8+ (Tc1 & Tc2)
• Humoral Immunity (Antibody production)• B Cells
BDC
Th1 and Th2 CD4+ T Cells
Th1Th1• IL-12 induces differentiation• Cytokine Production:
Interferon-Interleukin-2
• Intracellular Pathogens • Macrophage Activation• Delayed Type
Hypersensitivity
Th2Th2• IL-4 induces differentiation• Cytokine Production:
Interleukin-4Interleukin-5Interleukin-13
• Extracellular Pathogens• B Cell activation & IgE• Eosinophil responses• Immediate Type
HypersensitivityBDC
T cell signaling molecules and autoimmunity
(Mustelin T, et al. Mol Immunol. 2004)
Human T1D loci (Ref1)
MHC : λs ≈ 3Insulin : odds 1.9CTLA4 : odds 1.2PTPN22 odds 1.7
Cblb : Komeda rat(Yokoi N, Nat Genet, 2002)
Pten: Cre-loxP knock-out(Suzuki A, Immunity, 2001)
Zap70: Sakaguchi mice(Sakaguchi N, Nature, 2003)
Concannon P et al. Diabetes. 2005 Oct;54(10):2995-3001. H. Ueda
2 light chains ( or )2 heavy chains (5 isotypes: IgG, M, A, D, E)2 Binding sites (Divalent)Secreted into circulationBinds Soluble Antigen
2 Chains / (95%) or / (5%)
1 Binding site (Monovalent)Membrane Bound, Not SecretedBinds Antigen Complexed with MHC
Zap 70
fyn lck
V V
C C
CH1
CH2
CH3
CH2
CH3
CH1
CL CL
VL VL
VH VH
Igg Igg
Blk, Fyn or Lyn
B and T Lymphocyte Antigen Receptors
BDC
Human Leukocyte Antigen
human MHC
cell-surface proteins
important in self vs. nonself distinction
present peptide antigens to T cells
CLASS I: A,B,C CLASS II: DR,DQ,DP
HLAJ. Noble
DQB1*0402
Asp57
Leu56
-chain
-chain
BDC BDC
Topology of Self-peptide/MHC Binding by Ob.1A12 TCR
HA1.7Ob.1A12
Red: TCRYellow: TCR
Autoimmune (MBP Peptide+DR2) Anti-viral (HA Peptide+DR1)
Hahn, Wucherpfenning et al. Nature Immunology 6:490-496, 2005
Ob.1A12 HA1.7
Hahn, Wucherpfenning et al. Nature Immunology 6:490-496, 2005
Autoimmune (MBP Peptide+DR2)
Anti-viral (HA Peptide+DR1)
Red: TCRYellow: TCR
The Human Leukocyte Antigen Complex (6p21.31)
DP DQ DR B C A
Class II (1.1 Mb)Class III
Class I (2.2Mb)
Complement and Cytokines
Class I-like genesand pseudogenes
Frequent Recombination
Recombinationis Rare
TelomereCentromere
Recombinationis Rare
(0.7Mb)
BDC
32
•Binds 8-10mers•Expressed on most Nucleated cells•Presents Cytosolic Proteins to CD8+ T cells
•Binds 13-25mers•Expressed on APCs, Macs, B cells, activated T cells•Presents Vesicular Proteins to CD4+ T cells
Class I Class II
HLA Class I and II Molecules Have a Distinct Structure and Function
BDC
0501 0201
0301 0302
cis
cis
DQA1 DQB1
trans
DQA1*0301/DQB1*0201 DQA1*0501/DQB1*0302
DQA1*0501/DQB1*0201
DQA1*0301/DQB1*0302
Maternal
Paternal
Cis and Trans- Class II Dimerization
BDC
HLA-Peptide: TCR
CDR1
CDR2CDR3
CDR3CDR2
CDR1
NH3+
COO-
TCR
alpha
TCR
beta
2 Helix 1 Helix
BDC
Avidin
DQDQ
DQ
“Tetramer” for T Cell Analysis
DQ PEPTIDE
BDC
MHC peptide
HLA Class II tetramer (DR0401-hGAD555-567)
Leucinezippers spacers
streptavidin
W.W.Kwok & G.T.Nepom, BenaroyaResearch Institute at Virginia Mason
Antigen
APCAPC
Endocytosis
T Cell Receptor
Peptide
MHC II
CD4CD4++ T cellT cellCD4CD4++ T cellT cell
T cell Recognition of Antigen on an APC
CD4CD4++ T cellT cellCD4CD4++ T cellT cell
T Cell Receptor
Peptide
LPS
TLR4
Antigen Presenting Cell (APC)Antigen Presenting Cell (APC)
MHC II
“Signal 1”
“Signal 2”
“Signal 3”
IL-1
IL-6
IL-12
CD28
B7
IL-12 Receptor
Signal 1: Specificity
Signal 2: Activation
Signal 3: Differentiation
T cell Activation by an Activated APC
Absence of Signal 2
Activation
Clonal Anergy or Deletion
TCR MHC
APC
Tolerance
T Cell
TCR MHC
APC
Signal 1 + Signal 2
B7
T Cell
cytokines
CD28
The 2-Signal Model of Lymphocyte Activation
BDC
CD2
APC APC MHC II
B7 (CD80/86)
APC and T cell Interactions
CTLA-4
CD40L
CD28
B7 (CD80/86)
TCR
CD40
CD58 (LFA-3)
CD4CD4++ T CellT Cell
Activation
Activation
Recognition
Adhesion
Activation
V
CC
V
p56 lck
peptide
CD3CD28
CD40
LFA-3
LFA-1
ICAM-1MHC IIB7
CD40L
CD4
CD45TCR
CD2
CD4+ T Cell
APC/ B cell
Molecular Interactions of Helper T Cells and APC
B7
CTLA-4
VLA-1
Collagen
CD80/CD86
L. Chess 2002
T cell activation is regulated by signals derived from the TCR /CD3/CD4 complex and the CD40L and CD28/CTLA-4 co-stimulatory
molecules
CD3
MHC class II
TCR
Peptide antigen
CD4+ T Cell
Antigen Presenting Cell (APC)
V V
C C
CD4
Antigen specific TCR signals
CD40
CD40L
signal
CD28/CTLA-4
CD80 (B7.1)/CD86 (B7.2)
lck
Co-stimulatory signals
(- ) / [+]
[+]
L. Chess 2002
PIP2
IP3 + DAG
PLC1
Ca++
NFAT activation
calcineurin
TCR
CD4 CD45CD28
MAPK
Shc
Ras
SOS
Grb2
PKC
NFB
TCR signalingTCR signaling
(ION)(PMA)
PTK
Zap70 LckLck
TecFyn
Fathman
MHC/peptide
TCR
Qa-1/V
Activated CD4+ T cell
RestingCD4+ T cell
Down-regulation of Activated CD4+ T Cells
T cell activation induces expression of functional T cell surface molecules
Regulatory CD8+ T cell
TCR (anti-Qa-1/V)
Activated CD8+ T cell
(+) (-)
TCR
APC
TCR
Late Activated CD4+ T cell
VLA-1Collagen
CD40L
Migration of sites of
inflammation
Induction and activation of B cells
APCs
CD25
L. Chess 2002
Immunological tolerance
• Definition: – specific immune unresponsiveness to an antigen that
is induced by exposure of lymphocytes to that antigen (tolerogen vs immunogen)
• Significance:– All individuals should be tolerant of their own
antigens (self-tolerance); breakdown -->autoimmunity– The induction of tolerance could be exploited to treat
autoimmune diseases– Mechanisms of tolerance must first be understood
Fathman
Mechanisms of unresponsiveness to self antigens
• Central tolerance– Immature self-reactive T lymphocytes that recognize self antigens
in the thymus undergo negative selection (deletion)
• Peripheral tolerance– Mature self-reactive T lymphocytes that escape central tolerance
and recognize self antigens in peripheral tissues can be inactivated (anergyanergy), killed (deletion) or regulated (suppressed)
• “Clonal ignorance”– Mature self-reactive lymphocytes do not respond to self antigens
in non-inflamed settings
Fathman
TH1 CD4+ cells
TH2 CD4+ cells
CD8 or CD4 suppressor precursor
CD8 or CD4 suppressor effector
Activated CD4 T cells
peptide/APC
Regulatory immunityCD4/CD8 interactions
(- )
(- )
The Control of Activated CD4+ T Cells by Regulatory T cells
Apoptosis
Resting CD4 T cells
IL-12/IFN-
IL-4
NKT cells/ CD4+CD25+ cellsCD4+CD25- cells
(- )
(- )
IL-10 IFN-
L. Chess 2002
Regulatory T Cell Subsets
Regulatory T cell Murine Markers Proposed Mechanisms of InhibitionSuppressor Cell CD8+ Recognition of Qa-1:peptide on activated CD4+ T
cells induction of cytotoxicityNatural Treg CD4+, CD25+
CTLA-4+,GITR+, Foxp3+(intracellular)
Cell-contact dependent but not antigen-specific;Ligation of B7 on effector cells; IL-2sequestration; CTLA-4 interaction with IDO tolerogenic DCs; IL-10 & TGF-beta production
Adaptive Treg CD4+, CD25-,Foxp3-
Cell-contact dependent but not antigen-specificinhibition
Tr1 CD4+, CD45Rblo Cell-contact independent; IL-10 & IL-4 secretionTh3 CD4+, CD45Rblo Cell-contact independent; TGF-beta secretionInvariant NKT cell Invariant TCR
(V14-J281),CD4+, CD8-,NK1.1+
CD1d:glycolipid complex recognition; IL-10secretion
Regulatory T Cells in Autoimmunity
CD4+CD45RBlo TGF, IL10 Colitis by CD45RBhi
Th3 TGF,+/-IL10,IL4
EAE, glomeruloneph, MG,clone DM
TR1 IL10, normalTGF,no IL4
IBD by CD45RBhi ,Respond IL-15
CD4+CD25+(? =TR1)
CTLA4 Thymectomy autoimmuneConstituitive CTLA4
Other CD4+ IL2, IFN MBP EAE model
CD8+ Oral tolerance intestine
CD4-CD8-TCR Fas Skin Allograft
Gamma/Delta Gut, Nasal Insulin
NKT IL4,IL10,IFN,TGF
CD1 Activated
BDC Roncarolo et al. Curr Opinion Immunol 2000
XPID: X-linked Polyendocrinopathy, Immune dysfunction and Diarrhea
Foxp3 Gene Essential CD4-CD25 T Reg
• XLAAD: Autoimmunity Allergic Dysregulation
• Defect in scurfin protein (gene = Foxp3/JM2)or “scurfy mouse”
• Immunopathogenesis relates to a deficiency of T regulatory cells
-Scurfy x Nude: No Autoimmunity-CD4+ T cells into Nude: Disease-Bone Marrow into irradiated: No Disease-Mixed Chimera: No Disease
BDC
Requirements for the development of an autoimmune disease
Nature Immunology (9): 759-761 (2001) Fathman
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
L. Chess 2002
MHC/self-peptide CD4
MHC/VCD4
CD4+ Vx T cell APC
Activated autoreactive CD4+TCR Vx TH1 cell
TCR Vx TCR Vx
(1) expansion of CD4+, autoreactive TH1 cells specific for autoantigens
(2) migration and infiltration of these self reactive CD4+ TH1 cells into tissues and induction of inflammation and autoimmunity
(3) induction of regulatory cells which control the growth and activation of the pathogenic autoreactive repertoire of CD4+ T cells
Induction of CD4+ TH1 mediated autoimmunity:
A paradigm for the pathogenesis of rheumatoid arthritis, multiple sclerosis and
type I diabetes
L. Chess 2002
1984:Subset Participants Immunology in Diabetes Rome