FOR A GIVEN SPECIES MHCORGAN SPECIFIC AUTOIMMUNE DISEASE

FOR A GIVEN SPECIES(? )/MHC/ORGAN SPECIFIC AUTOIMMUNE DISEASE • One Critical Protein • One Critical Peptide • One Critical Register • ? Germline Encoded Critical TCR Segment THUS: Targeting Trimolecular Complex Possible for Prevention HYPOTHESIS THE NOD EXAMPLE

TCR rearrangement Vα Vβ Jα Dβ Jβ

T Cell Receptor Gene Segments Antigen Presenting Cell HLA Molecule Peptide V V D J Chr. 14 J Chr. 6

T cell Recognition of Antigen on an APC Antigen Endocytosis CD 4+ T cell T Cell Receptor APC Peptide MHC II Trimolecular Complex

The Trimolecular Complex NOD MOUSE TCR MHC PEPTIDE INS B: 9 -23

MHC B: 9 -23 TCR Liu et al Diabetes 2012 Zhang et al, J. Diabetes 2011 Michels et al, J. Immunol 2011 MHC B: 9 -23 MHC TCR B: 9 -23 Deletion of T cells expressing specific TCR alpha or TCR beta genes Antibody binds to MHC/peptide complex and blocks it from interacting with TCR Small molecule prevents from binding/dislodges the B: 9 -23 peptide from MHC Interfering with formation of the trimolecular recognition complex

“Stages” in Development of Type 1 A Diabetes (? Precipitating Event) Beta cell mass Genetic Predisposition Overt immunologic abnormalities Normal insulin release Progressive loss insulin release Glucose normal Overt diabetes C-peptide present Age (years) Minimal C-peptide Eisenbarth, 2012

n. POD 6052 -02 Tail: 12 yo 1 year diabetes -Lobular Pseudoatrophic Islets Glucagon/anti-CD 3 Insulin and Ki 67

Type 1 diabetes Anti-insulin: health • No cure; therapy is insulin for life; physiologic glycaemic control never achieved Anti-insulin: disease • Excess morbidity and mortality • Incidence increasing by ~5% every 5 years; costs ~£ 1 billion of UK NHS budget Peakman

Type 1 diabetes is T cell mediated • Infiltrating CD 4+, CD 8+ T cells • Anti-T cell therapies are effective • Islet cell autoantibodies disease CD 4 T-cell CD 8 T-cell CD 4 Treg TCR Epitope HLA II HLA I APC Peakman Islet autoantigen

The Immune System Acquired (Adaptive) Innate • Rapid Microbial Defense • Long-lived Microbial Defense • Adaptive Immune System • Neoplasm surveillance Activation • 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 • B 1 B Cells* * Aspects of both the innate and adaptive immune system BDC

Selected Pattern Recognition Receptors: Toll-like Receptors TLR: Selected Ligands: TLR 1 PGN, Zymosan, Antifungal & Lipoproteins Antibacterial TLR 2 Role in Immunity: TLR 6 TLR 4 LPS Antibacterial TLR 5 Flagellin TLR 11 ? TLR 9 Cp. G Antibacterial & Antiviral TLR 3 ds. RNA Antiviral TLR 7 ss. RNA TLR 8 ss. RNA TLR 10 ? ? Localization: Dendritic Cells, Macrophages, T Cells, B Cells, Epithelium

Selected Pattern Recognition Receptors: Other Families

The Adaptive Immune System • Cell-mediated Immunity (Cytotoxicity) • T cells • CD 4+ (Th 1 & Th 2) • CD 8+ (Tc 1 & Tc 2) • Humoral Immunity (Antibody production) • B Cells BDC

Th 1 and Th 2 CD 4+ T Cells Th 1 Th 2 • IL-12 induces differentiation • • Cytokine Production: • Interferon- Interleukin-2 • Intracellular Pathogens • • Macrophage Activation • • Delayed Type Hypersensitivity • • IL-4 induces differentiation Cytokine Production: Interleukin-4 Interleukin-5 Interleukin-13 Extracellular Pathogens B Cell activation & Ig. E Eosinophil responses Immediate Type Hypersensitivity BDC

T cell signaling molecules and autoimmunity Human T 1 D loci (Ref 1) MHC : λs ≈ 3 Insulin : odds 1. 9 CTLA 4 : odds 1. 2 PTPN 22 odds 1. 7 Cblb : Komeda rat (Yokoi N, Nat Genet, 2002) Pten: Cre-lox. P knock-out (Suzuki A, Immunity, 2001) Zap 70: Sakaguchi mice (Sakaguchi N, Nature, 2003) (Mustelin T, et al. Mol Immunol. 2004) Concannon P et al. Diabetes. 2005 Oct; 54(10): 2995 -3001. H. Ueda

B and T Lymphocyte Antigen Receptors VH VH V CH 1 VL VL CL CL CH 2 Ig /Ig CH 3 V e z z e d C C Ig /Ig fyn lck Zap 70 Blk, Fyn or Lyn 2 light chains ( or ) 2 heavy chains (5 isotypes: Ig. G, M, A, D, E) 2 Binding sites (Divalent) Secreted into circulation Binds Soluble Antigen 2 Chains / (95%) or / (5%) 1 Binding site (Monovalent) Membrane Bound, Not Secreted Binds Antigen Complexed with MHC BDC

J. Noble HLA 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

DQB 1*0402 -chain Leu 56 -chain BDC Asp 57 BDC

Hahn, Wucherpfenning et al. Nature Immunology 6: 490 -496, 2005 Topology of Self-peptide/MHC Binding by Ob. 1 A 12 TCR Autoimmune (MBP Peptide+DR 2) Ob. 1 A 12 Anti-viral (HA Peptide+DR 1) HA 1. 7 Red: TCR Yellow: TCR

Hahn, Wucherpfenning et al. Nature Immunology 6: 490 -496, 2005 Ob. 1 A 12 HA 1. 7 Anti-viral (HA Peptide+D R 1) Autoimmune (MBP Peptide+DR 2) Red: TCR Yellow: TCR

The Human Leukocyte Antigen Complex (6 p 21. 31) Class II (1. 1 Mb) DP Class III Class I (2. 2 Mb) (0. 7 Mb) DQ DR B C A Telomere Centromere Frequent Recombination is Rare Complement and Cytokines Class I-like genes and pseudogenes Recombination is Rare BDC

HLA Class I and II Molecules Have a Distinct Structure and Function • Binds 8 -10 mers • Expressed on most Nucleated cells • Presents Cytosolic Proteins to CD 8+ T cells • Binds 13 -25 mers • Expressed on APCs, Macs, B cells, activated T cells • Presents Vesicular Proteins to CD 4+ T cells 1 2 1 1 2 3 2 2 Class II BDC

Cis and Trans- Class II Dimerization Maternal DQA 1 DQB 1 0501 0201 cis DQA 1*0501/DQB 1*0201 Paternal 0301 0302 cis trans DQA 1*0301/DQB 1*0302 DQA 1*0301/DQB 1*0201 DQA 1*0501/DQB 1*0302 BDC

HLA-Peptide: TCR NH 3+ 2 Helix 1 Helix 1 R CD TCR alpha C 2 R D 3 R CD 2 R D 3 C R CD 1 R CD COO- TCR beta BDC

“Tetramer” for T Cell Analysis DQ DQ PEPTIDE DQ Avidin DQ BDC

T cell Recognition of Antigen on an APC Antigen Endocytosis CD 4+ T cell T Cell Receptor APC Peptide MHC II

T cell Activation by an Activated APC IL-1 IL-6 IL-12 Receptor CD 28 “Signal 3” LPS B 7 CD 4+ T cell T Cell Receptor “Signal 2” TLR 4 “Signal 1” Peptide MHC II Antigen Presenting Cell (APC) Signal 1: Specificity Signal 2: Activation Signal 3: Differentiation

The 2 -Signal Model of Lymphocyte Activation Absence of Signal 2 T Cell APC TCR MHC Tolerance Clonal Anergy or Deletion Signal 1 + Signal 2 CD 28 T Cell B 7 APC TCR MHC cytokines Activation BDC

APC and T cell Interactions CTLA-4 B 7 (CD 80/86) CD 28 Activation Recognition TCR MHC II Activation APC CD 58 (LFA-3) Activation CD 40 CD 2 Adhesion CD 40 L CD 4+ T Cell

Molecular Interactions of Helper T Cells and APC CD 4+ T Cell CTLA-4 CD 28 p 56 lck CD 3 CD 40 L C C V CD 2 z zh h d e TCR V CD 45 LFA-1 VLA-1 peptide B 7 CD 80/CD 86 CD 40 MHC II LFA-3 ICAM-1 Collagen APC/ B cell L. Chess

T cell activation is regulated by signals derived from the TCR /CD 3/CD 4 complex and the CD 40 L and CD 28/CTLA-4 co-stimulatory molecules CD 4+ T Cell Antigen specific TCR signals Co-stimulatory signals (- ) / [+] lck d e CD 3 z z hh C C CD 28/ CTLA 4 V CD 40 L [+] , TCR Peptide antigen CD 40 CD 80 (B 7. 1)/ CD 86 (B 7. 2) V CD 4 MHC class II signal Antigen Presenting Cell (APC) L. Chess

TCR CD 4 TCR signaling CD 28 PLC 1 Zap 70 Lck Fyn Tec (PMA) Shc PTK SOS Ras IP 3 + DAG Ca++ Lck Grb 2 PIP 2 (ION) CD 45 PKC MAPK calcineurin NF B NFAT activation Fathman

T cell activation induces expression of functional T cell surface molecules Activated CD 4+ T cell MHC/peptid e Induction and activation of B cells APCs CD 40 L TCR APC TCR Resting CD 4+ T cell Late Activated CD 4+ T cell TCR CD 25 (+) Qa-1/V (-) VLA-1 Collagen TCR (anti-Qa-1/V ) Activated CD 8+ T cell Regulatory CD 8+ T cell Down-regulation of Activated CD 4+ T Cells Migration of sites of inflammation L. Chess

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 (anergy), anergy killed (deletion) or regulated (suppressed) • “Clonal ignorance” – Mature self-reactive lymphocytes do not respond to self antigens in non-inflamed settings Fathman

The Control of Activated CD 4+ T Cells by Regulatory T cells NKT cells/ CD 4+CD 25+ cells CD 4+CD 25 - cells Apoptosis peptide/APC (- ) TH 1 CD 4+ cells IL-12/ IFN- (- ) IL-10 IL-4 Resting CD 4 T cells IFN- (- ) Activated CD 4 T cells (- ) TH 2 CD 4+ cells Regulatory immunity CD 4/CD 8 interactions CD 8 or CD 4 suppressor effector precursor L. Chess

Regulatory T Cell Subsets

Regulatory T Cells in Autoimmunity BDC Roncarolo et al. Curr Opinion Immunol 2000

XPID: X-linked Polyendocrinopathy, Immune dysfunction and Diarrhea Foxp 3 Gene Essential CD 4 -CD 25 T Reg • XLAAD: Autoimmunity Allergic Dysregulation • Defect in scurfin protein (gene = Foxp 3/JM 2) or “scurfy mouse” • Immunopathogenesis relates to a deficiency of T regulatory cells -Scurfy x Nude: No Autoimmunity -CD 4+ 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

Immunopathophysiology of Diabetes Dendritic cell/ APC CD 2 CD 4+ Cell (TH 0 ) IL-12 DR 3, DR 4, , DQ 8/insulin , , TCR peptide IFN- IL-4 CD 4+ Cell (TH 2 ) CD 40 L CD 40 Macrophage/dendritic cell Fc R Fas. L perforin CD 40 L Activated TH 1 CD 4+ T Cell CD 8+ CTL IL-1, TNF, LT, NO, PGE-2 IL-4 CD 40 L B Cell ? anti-insulin, GAD ab anti. Mog ? Antibody mediated injury cell death islet cells L. Chess

Induction of CD 4+ TH 1 mediated autoimmunity: A paradigm for the pathogenesis of rheumatoid arthritis, multiple sclerosis and type I diabetes (1) expansion of CD 4+, autoreactive TH 1 cells specific for autoantigens MHC/self-peptide CD 4 TCR V x CD 4+ V x T cell APC CD 4 MHC/V TCR V x Activated autoreactive CD 4+ TCR V x TH 1 cell (2) migration and infiltration of these self reactive CD 4+ TH 1 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 CD 4+ T cells L. Chess

1984: Subset Participants Immunology in Diabetes Rome