The role of T cells in the pathogenesis of rheumatic disease (.. with a focus on RA)

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2 cells in health and rheumatic diseases -Sheraton Hotel, Ankara, 13 March 2009 he role of cells in the pathogenesis of rheumatic disease (.. with a focus on A) Andrew P. Cope MD PhD Arthritis esearch Campaign Professor of heumatology Academic Department of heumatology Division of mmunology, nfection and nflammatory Diseases King s College School of Medicine King s College London andrew.cope@kcl.ac.uk

3 A working model of disease pathogenesis for A environmental triggers diagnosis of A much joint destruction Healthy population immune response autoantibodies progression from UA to A no disease arthritis onset A remission genes spontaneous remission non-specific arthritis little joint destruction genotype serotype phenotype

4 Checkpoints defining the immunobiology of A initiation persistence tobacco smoke pathways perturbing immune regulation pathways regulating joint integrity 4 trauma Susceptible host Onset of arthritis Chronic disease infection initiation factors 1 innate immunity adaptive i.r. to modified self antigens effector cells mediating persistence HEAPY checkpoints defining the evolution and progression of A

5 Overview of the Lecture 1. What triggers the adaptive immune response in A?

6 What is the molecular basis of adaptive immunity? hymic selection Maintenance of cells in the periphery dendritic cell self ligand CD4/8 cec MHC C APC multiple ligands DP hy cell clonotypic receptor positive selection a repertoire of self-reactive cells C tickling of self-reactive cells Lymphocytes are referential to self-ligands!

7 mmune recognition of infectious non-self (as opposed to non-infectious self ) inducible DC pathogen derived peptide antigen CD80/86 Signal 2 CD28 constitutive MHC C Signal 1 constitutive How are costimulatory molecules induced?

8 Pathogen Associated Molecular Patterns (PAMPs) and Pattern ecognition eceptors (Ps) Exogenous ligands Lipoproteins dsna (bacteria, (viruses) mycoplasma, mycobacteria) LPS hsp Flagellin (bacteria) ssna (viruses) synethetic compounds ssna CpG DNA? Factors from uropathogenic bacteria L1/6 L2 L3 L4 L5 L7 L8 L9 L10 L11 DANGE SGNALS

9 mmune recognition of infectious non-self (as opposed to non-infectious self ) inducible DC pathogen derived peptide antigen L signal CD80/86 Signal 2 MHC Signal 1 constitutive CD28 C constitutive How are costimulatory molecules induced?

10 Pathogen Associated Molecular Patterns (PAMPs) and Pattern ecognition eceptors (Ps) Exogenous ligands Lipoproteins dsna (bacteria, (viruses) mycoplasma, mycobacteria) LPS hsp Flagellin (bacteria) ssna (viruses) synethetic compounds ssna CpG DNA? Factors from uropathogenic bacteria L1/6 L2 L3 L4 L5 L7 L8 L9 L10 L11 necrotic cells dsna Hsp60,70 HMGB1 Gp96 HA, HS FN ssna Endogenous ligands ssna DNA chromatin-gg complexes

11 L signal transduction pathways adapted from O Neill, 2006

12 nhibition of L dependent production of inflammatory mediators NFa (pg/ml) Uninfected Adβ-gal AdMyD88dn AdMaldn AdκBα MMP-3 (ng/ml) Uninfected Adβ-gal AdMyD88dn AdMaldn AdκBα MMP-13 (ng/ml) Uninfected Adβ-gal AdMyD88dn AdMaldn AdκBα Sacre et al, 2007 Am J Path 170:

13 Overview of the Lecture 1. What triggers the adaptive immune response in A? 2. What do cells see?

14 Da chain a-helix C-terminus Disease associated DB1*0401 b86 Gly b67 Leu b70 Gln b71 Lys }Pocket 4 b71lys + P4Asp - Non-associated DB1*0402 b86 Val b67 le b70 Asp b71 Glu }Pocket 4 N-terminus collagen ( ) Db chain a-helix

15 Proposed hierarchy of HLA-DB1 alleles in determining disease severity in A *0401/*0404 *0401/*0401 *0404/*01 *0401/*01 *0401/X *0404/X *01/*01 nodular vasculitis *01/X X/X non-erosive erosive seronegative disease seropositive erosive disease (from Weyand et al, 1995)

16 Defining HLA-D4 restricted cell responses to cartilage antigens in transgenic mice lacking mouse MHC class HCgp39 in FA Dab1*0401 Dab1*0402 harvest draining 10d fuse with C neg thymoma as fusion partner clone hybridomas evaluate antigen & peptide specific responses

17 Dab1*0401 and Dab1*0402 present completely distinct sets of HCgp-39 peptide epitopes to CD4+ cells % all responding hybrids Dab1*0401 (n = 250) Dab 1*0402 (n = 151) HCgp-39 peptide Cope et al, 1999 Arthritis heum 42:

18 a. [3H] thymidine incorporation (cpm) he immune response is different (1) b S -3.4 S -3.6 *0401 *0402 DB1*04 subtype FN g (pg/ml) *0401 *0402 DB1*04 subtype medium HCgp-39 HCgp-39 peptide pool -d pool Cope et al, 1999 Arthritis heum 42:

19 he immune response is different (2) c. FNg d. NFa FNg (pg/ml) NF (pg/ml) *0401 (C-line) *0401 (3C4) * *0401 (C-line) *0401 (3C4) *0402 DB1*04 subtype DB1*04 subtype Cope et al, 1999 Arthritis heum 42:

20 Defining the key autoantigenic determinants DC MHC class CD28 C eff B disease specific autoantibodies antigen experienced effector cells

21 What have A-specific autoantibodies taught us? stress reactivity to modified self

22 What have A-specific autoantibodies taught us? stress reactivity to modified self citrullinated proteins arginine fi citrulline neoepitopes

23 What have A-specific autoantibodies taught us? inflammation stress mφ, PMN infiltration reactivity to modified self PAD2/4 Ca 2+ deimination citrullinated proteins vimentin, fibrin, histones, a-enolase inflammation specific Schellekens, Vossenaar, ak, van Venrooij et a

24 What have A-specific autoantibodies taught us? inflammation stress mφ, PMN infiltration reactivity to modified self PAD4 genetic polymorphism Suzuki et al, Nat Gen 2003 PAD2/4 Ca 2+ mna stability deimination citrullinated proteins vimentin, fibrin, histones, a-enolase inflammation specific Schellekens, Vossenaar, ak, van Venrooij et a

25 What have A-specific autoantibodies taught us? inflammation stress? anti-ccp B cell response gg1 synovial plasma cells H mφ, PMN infiltration reactivity to modified self cell response PAD4 genetic polymorphism Suzuki et al, Nat Gen 2003 PAD2/4 Ca 2+ mna stability deimination inflammation specific citrullinated proteins vimentin, fibrin, histones, a-enolase HLA class disease specific

26 Arg not favoured Cit permissive at P4 Da chain a-helix C-terminus P4Asp - b71lys + Disease associated DB1*0401 b86 Gly b67 Leu b70 Gln b71 Lys }Pocket 4 Non-associated DB1*0402 b86 Val b67 le b70 Asp b71 Glu }Pocket 4 N-terminus collagen ( ) Db chain a-helix

27 Overview of the Lecture 1. What triggers the adaptive immune response in A? 2. What do cells see? 3. Have any immunogeneticstudies been informative?

28 Gene polymorphism signal input Functional impact HLA-DB1 CA PAD4 DC hymic selection Cell survival PPN22 h differentiation PD1 CD28 C Migration CLA4 CD25 SA4 eff Effector responses mmune regulation modified signal output

29 Overview of the Lecture 1. What triggers the adaptive immune response in A? 2. What do cells see? 3. Have any immunogeneticstudies been informative? 4. What do A cells look like?

30 Prediction: 1.Activated phenotype 2.Features of prior antigen exposure 3.Extensive proliferative activity 4.Expansions of selected (high affinity) clones 5.eactive to tissue specific (cartilage, synovium, bone) antigens 6. Features of distinct hlineage differentiation 7. ntrinsic survival advantage 8. Potent migratory capacity 9. mpaired regulatory function

31 he reality: effector response costimulation L-4 L-6 L-15 L-23 NF COSL ANKL mnf OX40 CD28 NF + antigen recognition L-10 + Bcl-2 lo Bcl-XL lo C Cz dim A synovial cell L-17 + FNg + H CD45O,CD45B dim CD44,CD69,CD62L lo CC7 lo VLA-1,VLA-4 LFA-1 memory effector CC4,CC5,CC6, CXC3,CXC4,CXC5 CX3C1 migration La 1 b 2 NKG2D CD28 null senescence CD25 neg CD27 neg CD40L neg Cope AP 2008 Arthritis es her

32 Overview of the Lecture 1. What triggers the adaptive immune response in A? 2. What do cells see? 3. Have any immunogeneticstudies been informative? 4. What do A cells look like? 5. Multiple pathways of cell effectorfunction

33 he C/CD3 antigen receptor complex C/CD3 complex Cz bright Cz Cz dim CD3e

34 Cz dim cells are enriched for effector h cell subsets (A) 70 P < (B) 60 Data.027 CD4 + %FNg expressing cells Cz Cz dim SSC-W L-17 0 Cz bright Cz dim (stim: PMA and ionomycin) Zhang et al, Blood 2007

35 egulatory CD4 + cell subsets reside in the Cz bright population (A) 50 P < (B) 100 P < %L-10 expressing cells %Foxp3 + cells Cz bright Cz dim 0 Cz bright Cz dim Zhang et al, Blood 2007

36 Migration of Cz dim cells across activated endothelium upper chamber lower chamber 88.5% 25% healthy donor PBL Cz bright Cz 11.5% 75% Cz dim CD3 NF EC gelatin transwell % cells migrating p < p < p > bright dim bright dim bright dim CD3 CD4 CD8 Cz expressing subset Zhang et al, Blood 2007

37 Accessory signals are intact in Cz dim cells 1500 FNg L-10 cytokine production (pg/ml) Cz dim cells plus: 0 CHO/vector: CHO/CD80: CHO/CD86: Cz dim cells generated by stimulation with L-2, L-6 and NFa in collaboration with Fionula Brennan

38 Analysis of contact dependent effector responses z bright z dim NFa (pg/ml) monocytes 3:1 5:1 7:1 cells:monocytes (Cz bright ) 0 monocytes cells monocytes + LPS 3:1 5:1 7:1 cells:monocytes (Cz dim )

39 Cz dim cells are enriched at sites of inflammation 80 PB % Cz dim cells SF 0 PB SF SM CD3 + cells Cz CD3e Zhang et al, Blood 2007

40 Persistence of Cz dim effector cells after infliximab therapy 11.1% 88.9% 45.4% 54.6% CD3 NF blockade PHENOYPE % CD3 + Cz dim cells Cz circulating Cz dim memory, activated CC/CXC profile integrins senescence Ag engagement effector cells Cz circulating Cz dim 0 PB SF SM NF blockade synovial tissue synovial tissue high disease activity low disease activity

41 KEY MESSAGES 1. he phenotype and function of PB and synovial cells points to a key role for inflammation and aging in the pathogenic process. 2. Expression of costimulatorymolecules can be exploited for therapeutic process. 3. Antigen determinants for cells remain poorly define. Extended autoantibody profiling is likely to provide insights in the future. 4. cell effector pathways are complex. he precise role of h1, h2 and h17 cells requires further investigation. 5. Deeper understanding of the basis for defective immune regulation is urgently required. 6. Genome wide association studies have provided new clues to the perturbations of adaptive immunity in chronic inflammatory autoimmune diseases such as A.

42 ountergulatory etworks ACUE PHASC CHONC L inhibitors L-4, L-10, GFb, L-13, L-11, L-2 L-1a, snf-, L-10, L-18bp, OPG, adiponectin Lb tissue damage, L-12, L-23 L ligands L-15, L-18 FLS 1, NF, L-6, L-22 GE2, bfgf, PDGF GF, VEGF, GFb COX, LOX L transmigration of monocytes PMN, mast cells and NK cells FNs + survival factors CXCL5/8/9/10/12/13, CCL2/3/5/20/21 CX3CL1 activation of endothelium, adhesion molecules CD80/86 CD28 EALY L-2, L-4, L-13, GFb L-15 GM-CSF eff DC C L-2 CD40L LAE CD40 FNg L-17 ANES NF La/b L-10 L-2 deficiency + L-6 NAON ANGEN MODE NFLAMMAON MODE GFb L-10 GFb L-10 reg BLyS APL B L-6 L-10 chemokines AutoAb reg L-1, L-6, NF L-15, L-32 GM-CSF oncostatin M M-CSF, VEGF Chemokines adipokines HMGB1 L-10 B MΦ eff NF M-CSF OPG CD28 null Cz dim osteoclasts FLS L-17 + ANKL Adipo NF L-1 oncostatin M PGE 2 tissue response adipokin MMP ADAM ADAM chrondrocy features of inflammation and repair

43 Credits C signalling Joanna Clark Zhuoli Zhang Pia somäki Karolina Aleksiyadis Nina Panesar Federica Marelli-Berg Claudia Monaco Enrico Ammirati Alex Annenkov Yuti Chernajovsky Cz genetics Claire Gorman Andrew ussell im Vyse mna stability Andy Clark (3 U) SKG mouse studies Xiang Wu Oli Somenzi harsana harmalingam Shimon Sakaguchi Funding Wellcome rust arc MC EU