Modulation of human antigen-specific T cell response - Institute of Neuroimmunology
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Modulation of human antigen-specific T cell response therapeutic implications for multiple sclerosis Institute of Neuroimmunology CHARITÉ CAMPUS MITTE - UNIVERSITÄTSMEDIZIN BERLIN INSTITUTE OF NEUROIMMUNOLOGY CLINICAL AND EXPERIMENTAL NEUROIMMUNOLOGY INSTITUTE OF NEUROIMMUNOLOGY CLINICAL AND EXPERIMENTAL NEUROIMMUNOLOGY Research areas: Funding: Neurology (Imaging Techniques) German Research Council (DFG) through grants allocated to collaborative research centres „Sonderforschungsbereich“ (SFB) in Medicine Patient Clinical Trials Biochemistry (Cell Signaling; Lipid Raft Signaling; Proteomics; Post-translational modifications) Animal models (Experimental Autoimmune Encephalitis) Immunology (Tolerance in Allergy, Autoimmunity and Transplantation; Infectious Immunology) INSTITUTE: 1. Outpatient clinic (Clinicians, Study nurse, Data bank) 2. MRI area 3. Laboratory/Animal Faclility (PhD, PhD students and Technicians) An interaction between the specialized areas exists such that the data is efficiently transferable. In particular experimental immunoregulatory agents are directly passed on to the Clinic in early Phase II studies In vitro experiments Genetics & Molecular Biology (Genetic Analysis of Multiple sclerosis in EuropeanS) SFB507: Mechansisms of immune-mediated chronic CNS damage SFB625: Modulation of T cell response by statins – a new therapeutic strategy in MS Federal Ministry for Research and Education “Bundesministerium für Bildung und Forschung” (BMBF) Joint German-Israeli Research Program (intergovernmental agreement concluded between the Israeli Ministry of Science and Technology (MOS) and the BMBF) CLINICALLY- BENEFICIAL AGENT X •T cell response/signaling pathways •CNS protective mechanisms Ex vivo analyses MS patients RESPONSE MARKERS •Gene expression •T cell response Outlook: TARGET-SPECIFIC STRATEGIES target gene induction/silencing In vivo analyses EAE model 1 CNS MULTIPLE SCLEROSIS (MS) APC inflammation demyelination axonal damage autoimmune nature APOPTOSIS dysregulation activation myelin specific T T apoptosis CNS CNS damage cytotoxic factors T APC activation myelin specific T APC T activation myelin specific T T T apoptosis apoptosis CNS damage cytotoxic factors T APC activation activation myelin specific myelin specific T T Bcl-XL MS patients T apoptosis T apoptosis J Neuroimmunol. 2002 2 Regulation of activated T cells by apoptosis T activation myelin specific Bcl-XL MS patients T apoptosis J Neuroimmunol. 2002 J Neuroimmunol. 2002 Regulation of activated T cells by apoptosis therapeutic options for MS: 2.0 2 Healthy Controls Bcl-XL:β-actin ratio MS Patients Bcl-XL ß-actin 1,5 1.5 Rx Rx 1 1.0 activation myelin specific 0,5 0.5 T MS patients T apoptosis 0 0.0 0 ,4 MS Patients Healthy Controls Rx J Neuroimmunol. 2002 Antigen-specific T cell lines therapeutic options for MS: plasma 3 antigen eg. MBP mononuclear cells density gradient erythrocytes 1 A B SPLITS C1-C6 1 2 3 4 5 6 7 8 9 10 11 SPLITS C7-C12 12 A A B B C C D D E E F F G G H H 1 1 2 2 3 C1 C2 C3 C1 C2 C1 C2 C3 C1 C2 C3 C1 C2 C3 3 4 5 4 6 7 5 8 9 6 1 2 C1 C1 C1 11 C5 C6 C7 C8 C9 C10 C11 C12 C4 C5 C6 C7 C8 C9 C10 C11 C12 C4 C5 C6 C7 C8 C9 C10 C11 C12 C4 C5 C6 C7 C8 C9 C10 C11 C12 C5 C6 C7 C8 C9 C10 C11 4 5 6 7 8 9 10 11 12 C D E C1 C1 C1 F C1 C1 G C1 C1 C1 C1 C1 C1 C1 C1 H 10 11 12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 2 C3 3 C4 4 C5 5 C6 6 C7 7 C8 8 C9 9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 primary culture 14 days incubation with antigen 7 10 C4 3 8 9 10 11 12 12 Rx Rx activation myelin specific T MS patients T A C3 apoptosis B C D C4 C12 E C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 expanded further weekly restimulation with specific antigen F G H ANTIGEN Rx NO ANTIGEN 3 Apoptotic ligand TRAIL downregulates Ca2+ influx through CRAC channels of antigen-specific T cells therapeutic options for MS: Rx TRAIL T activation myelin specific T apoptosis Rx CRAC – calcium release activated calcium channels J Immunol. 2002 J Immunol. 2002 Apoptotic ligand TRAIL inhibits proliferation and cell cycle progression of human (auto)antigen-specific T cells p27Kip1 TRAIL G1 100 S R cell cycle TRAIL G2 M Stimulation Index CDK4 MBP-stimulated FZ2 80 60 40 20 0 0 ng/ml T activation myelin specific 100 ng/ml 300 ng/ml Stimulation Index therapeutic options for MS: 50 aCD3/aCD28-stimulated FZ2 40 30 20 10 0 0 ng/ml 30 ng/ml 100 ng/ml 300 ng/ml TRAIL TRAIL T apoptosis MBP TRAIL Rx J Immunol. 2002 TRAIL and its receptors TRAIL TR1 (DR4) TR2 (DR5) TR3 (DcR1) J Immunol. 2002 TRAIL receptors in human brain TR4 (DcR2) death domain FLIP ? caspa se-8 amplifying intrinsic loop executioner caspases APOPTOSIS J Neurosci. 2002 4 Lack of TRAIL but presence of its receptors in CNS TRAIL is not expressed in normal human brain neuron AP C CD4 microglia/astrocyte oligodendrocyte BRAIN PLACENTA TRAIL TR-1, TR-2 J Neurosci. 2002 Induction of TRAIL-mediated glioma cell death by human T cells survival of LN18 [%] 80 TRAIL 60 40 TRAIL TRAIL 20 3:1 1:1 3:1 1:1 3:1 1:1 3:1 1:1 3:1 1:1 activation 0 AV4 AV4+ anti-CD95L AV4+ anti-TNF AV4+ rhTR-2:Fc myelin specific AV4+ anti-CD95L +anti-TNF +rhTR2:Fc T T apoptosis J Neuroimmunol. 2002 J Immunol. 2002 Inhibition of T cell proliferation and cell cycle progression Inhibition of T cell proliferation by atorvastatin by HMG-CoA reductase inhibitor atorvastatin therapeutic options for MS: CDK4 atorvastatin p27Kip1 S R G1 cell cycle G2 M activation myelin specific aCD3/CD28 atorvastatin T T apoptosis J Exp Med 2003 Inhibition in proliferation of antigen-specific T cells: α-CD3/α-CD28 dose-dependent (IC50: 3-5 µM) J Exp Med 2003 5 No modulation of early T cell activation (Ca2+ influx) by atorvastatin Beneficial effect of atorvastatin in EAE Vehicle Atorvastatin J Exp Med 2003 J Exp Med 2003 Induction of T cell anergy by atorvastatin HMG-CoA HMG-CoA reductase Mevalonate IPP DMAP GPP FPPsynthase FPP FPTase Isoprenylation of GTP-binding protein (Ras, Rho) Squalene GGPTase GGPP Cholesterol Statins are HMG-CoA reductase inhibitors Induction of T cell anergy by atorvastatin negative cell cycle regulator p27Kip1 degradation: GTPases? induction of tolerance by inhibitory cytokines such as IL-10 (Boussiotis et al., Blood 2001) negative cell cycle regulator p27Kip1 degradation: GTPases? T cell anergy (Boussiotis et al., Nat Med 2000) Induction of T cell anergy necessitates IL-10 signaling negative cell cycle regulator p27Kip1 induction of tolerance by inhibitory cytokines such as IL-10 (Boussiotis et al., Blood 2001) 6 Atorvastatin induces a subset of cells with a regulatory phenotype suppressive capacity of atorvastatin-treated cells inhibitory cytokines/direct cell-cell contact Phosphorylation of extracellular signal-related kinase 1 by atorvastatin Rapid phosphorylation of extracellular signal-related kinase 1 MAPK Erk1 rapid phosphorylation following atorvastatin sustained expression (>24h) Robust phosphorylation of Erk 1 mediated via MEK MEK inhibition by U0126 delays Erk 1 phosphorylation by atorvastatin Phosphorylation of Erks is necessary for anergy induction MEK inhibition restores normal T cell response 7 Chemotaxis of human lymphocytes after atorvastatin exposure Transwell migration assay 5 3 Number of Migrating Cells [10] 2-3 hr incubation activated autoreactive T cells chemokine receptor, CXCR4 4 3 2 1 0 chemokine gradient - 30 µM - atorvastatin + 10 µM 20 µM 30 µM 50 µM atorvastatin 50 µM vehicle control SDF [125ng/ml] chemokine, CCL19 Chemotaxis of human MBP-specific lymphocytes towards CCL19 Chemotaxis of human MBP-specific lymphocytes towards CCL19 Chemotaxis of human lymphocytes after atorvastatin exposure 3 Number of Migrating Cells [10 ] 25 20 15 10 5 Rho 0 - no SDF - 1µM atorva 10µM atorva 25µM atorva 25µM 25µM atorva + atorva + 100µM FPP 100µM GGPP 25µM atorva + 200 µM Mev Isoprenylation Rho GGPTase GGPP Geranylgeranylated Rho 125ng/ml SDF 8 GEF GDP Rho GEF GDP GTP Rho GTP Rho Rho D RB GAP GAP RBD Rho PH D CR Kinase PH CRD Kinase Inactive ROCK GEF GDP Rho Rho D RB GAP PH GEF GDP GTP D CR GTP Rho Rho D RB GAP Kinase PH D CR Kinase Rho MLCP MLCP stress fibre assembly stress fibre assembly cell contraction cell contraction MLC MLC ROCK ‘n’ Rho expression in human PBMCs (ROCK cleavage) kDa Atorvastatin inhibits ROCK cleavage (induction of Rho A) kDa 1 2 3 4 150 p160ROCK 150 p160ROCK 25 RhoA 25 RhoA β-Actin 50 50 37 β-Actin LW [h] Con A [h] 6 18 6 42 18 72 42 96 72 37 96 Con A (4d) A25 (4d) 9 Atorvastatin inhibits ROCK cleavage (induction of Rho A) ROCK cleavage is restored following mevalonate IP: Rho A kDa 150 p160ROCK kDa ConA (4d) A25 [d] 2 3 4 2 3 4 kDa WB 150 p160ROCK 21 RhoA 50 37 β-Actin ConA (4d) A25 [d] 2 3 4 2 3 p160ROCK 150 ConA 4d A25 [d] mevalonate [d] 1 2 3 4 1 2 3 4 1 2 3 4 4 TAKE HOME MESSAGE control aCD3/aCD28 aCD3/aCD28 atorvastatin 1. immunomodulatory activities of TRAIL and atorvastatin – potential in treating autoimmune disease; therapeutic f-actin activity of atorvastatin 2. multi-faceted atorvastatin - imp. mechanism for EAE/ME ¾ selective targets for inducing tolerance in autoimmune disease or protecting vs. CNS damage in MS ROCK merged Institute of Neuroimmunology, Charité Berlin Germany Ivo Bendix Carmen Infante-Duarte Timour Prozorovski Orhan Aktas Alina Smorodtchenko Bibiane Seeger Susan Pikol Frauke Zipp 10
