Signaltransduktion in T-Zellen Vorlesung Immunologie 16.5.12 Friederike Berberich-Siebelt Abteilung für Molekulare Pathologie Pathologisches Institut Universität Würzburg 1
Possible T cell responses effector memory Th17 2
Fragestellung Warum proliferiert, differenziert oder stirbt die T-Zelle? Unter welchen Umständen reagiert die T-Zelle bzw. eben nicht (Anergie)? Welche Signale bestimmen die Differenzierungsrichtung (T-helfer 1, 2, 17 oder T-regulatorische Zelle)? Was entscheidet über die Entwicklung zur Effektor- bzw. zur Gedächniszelle? 3
Structures of the pre-tcr and αβtcr Open circles in the cytoplasmic domains of the CD3,,, and ζ chains denote tyrosine residues within the immunoreceptor tyrosine-based activation motif (ITAM). Horizontal bars within pt TCR, TCR TCR and ζ ζ represent disulfide bonds. 4
Cytoplasmic domains of TCR and BCR and FcR contain ITAMs black rectangles: Immunoreceptor tyrosine-based activation motif (D/E)XXYXXL(X)6-8YXXL 5
Innate and adaptive immunity Dranoff G (2004) 4 Cytokines in cancer pathogenesis and cancer therapy. CD - cluster of differentiation CD4 - T cells (helper/inducer), monocytes, myeloblasts, NK cell lymphoma CD8 - T cells (suppressor/cytotoxic), large granular lymphocytes 6
Innate and adaptive immunity are both specific innate fast pattern recognition = specific adaptive slow antigene recognition = highly specific memory 7
Innate and adaptive immune receptors integrate signalling pathways innate BCR adaptive TCR Rawlings et al. Nature Reviews Immunology 6, 799 812 (2006) 8
Functions of costimulators in T cell activation 9
Formation of the immunological synapse SMAC = SupraMolecular Activation Cluster A. Kupfer (U. Colorado) 10
Formation of microclusters and immunological synapse 11
Essential Role of Ubiquitin and TSG101 Protein in Formation and Function of the csmac Santosha Vardhana, Kaushik Choudhuri, Rajat Varma, Michael L. Dustin Volume 32, Issue 4, 23 April 2010, Pages 531-540 P P P P P Ub P Ub Ub recognition by ESCRT-I component TSG101 P Ub Lysosomal degradation 12
Intracellular signaling events during T cell activation 13
Early tyrosine phosphorylation in T cell activation 14
Two classes of PTK = Phospho Tyrosine Kinases 1. src family (Lck, Fyn) Lck binds no-covalently to CD4 and CD8 2. Syk/ZAP70 (Syk in B cells, ZAP70 in T cells) ZAP70 binds to P-Tyr when ITAM is phosphorylated by Lck Lck SH2 (src homology domain 2) binds to P-tyr Y505 and forms a closed, inactive conformation. Phosphorylation of Y394 activates Lck. 15
Csk and CD45 regulate Lck Csk is a kinase that negatively regulates Lck by phosphorylation of Y505; CD45 phosphatase positively regulates Lck by putting it in primed state (in resting T cells and thymocytes) 16
Activation of Lck and ZAP70 Lck transphosphorylates itself phosphorylates ITAM ZAP70 binds to ITAM ZAP70 is activated by Lck ZAP70 phoshorylates LAT, SLP76 17
The Ras-Map kinase pathway in T cell activation 18
Activation of T cells induces phosphorylation of ERK TPA Iono N.mab + + + + + + + + + + + + -P-ERK light exposure -P-ERK dark exposured -ERK 55-44- 33-25- 55-44- 33-25- TCR signals (anti-cd3/cd28) can be bypassed by TPA/PMA (phorbol ester) that activates PKC and ionomycin (calcium ionophore). 19
Signals via membrane inositol phospholipid metabolism TPA = DAG analogon ionomycin = Ca 2+ ionophore Downloaded from: StudentConsult (on 26 January 2007 09:13 AM) 2005 Elsevier 20
PKC-θ (theta) is essential for TCR signalling ELISA Sun et al. (2000) Nature 404, 402-407. 9 PKC genes (PKC α,β,γ,δ,ε,θ,ζ), but deletion of this gene alone leads to defective T cell activation. 21
Activation of transcription factors in T cells 22
A general scheme of NF B activation 23
Mitogen-activated protein kinase scaffold proteins in immune signalling Scaffold proteins and immune-cell signalling Andrey S. Shaw & Erin L. Filbert Nature Reviews Immunology 9, 47-56 (January 2009) 24
The role of PKC enzymes in IKK NF B activation downstream of antigen receptors 25
NF B activation is abolished when PKCθ is absent EMSA: electromobility shift assay/ band shift Sun et al. (2000) Nature 404, 402-407. 26
Activation of transcription factors in T cells 27
Scaffold proteins of the calcium signalling pathway Scaffold proteins and immune-cell signalling Andrey S. Shaw & Erin L. Filbert Nature Reviews Immunology 9, 47-56 (January 2009) 28
Family and activation of NFAT transcription factor Ca 2+ Serfling E, Klein-Hessling S, Palmetshofer A, Bopp T, Stassen M, Schmitt E. (2006). Eur J Immunol. NFAT transcription factors in control of peripheral T tolerance. Ca 2+ Ca 2+ Ca 2+ Macian F (2005). 29 NFAT proteins: key regulators of T-cell development and function.
NFAT-activated programmes of gene expression: T-cell activation vs. T-cell anergy Ca 2+ + other signals NFAT + AP1 + productive activation Unopposed Ca 2+ NFAT + w/o anergy 30
ITIMs are Immunoreceptor Tyrosine Inhibitory Motifs V/I/xYxxL/V, present in co-receptors that negatively regulates ITAM receptors. 31 SHP-1 and SHP-2 phosphatases bind to CTLA-4
CD28 stimulation leads to few changes in gene expression relatively to CD3 (just more of the same genes) Microarray analysis Diehn et al. (2002) Proc. Natl. Acad. Sci. USA 99, 32 11796-11801.
CD28 regulates glucose metabolism through PI3Kinase-AKT and up-regulates Glut1 glucose transporter 33
Signal 1 leads to increased demand for energy, signal 2 enhances glycolysis & energy for growth/proliferation 34
Signal 3: Cytokine Milieu Determining CD4 + T Cell Differentiation and Conversion (Pu1+IRF4) (Bcl6) 35 Liang Zhou, Mark M.W. Chong, and Dan R. Littman (2009). Immunity
CD4 + T helper cells different to express a specific subset of lymphokines Th 0 1 2 17 Restim. -+ -+ -+ -+ -+ 0 1 2 17 -+ -+ -+ -+ -+ Th17 IL-17 IL-4 IL-5 IL-10 IL-13 Th2 GAPDH IL-2 IFN- Th1 L32 GAPDH RNase protection assay for lymphokines 36
NFAT and T-helper-cell differentiation Macian F. (2005) Nat Rev Immunol. 5 NFAT proteins: key regulators of T-cell development and function. 37
The JAK STAT pathway Ke Shuai & Bin Liu (2003) 3, 900-911. Regulation of JAK-STAT signalling in the immune system 38
The negative regulation of the JAK/STAT pathway Ke Shuai & Bin Liu (2003) 3, 900-911. Regulation of JAK-STAT signalling in the immune system 39
Transcriptional master regulators facilitate differentiation of Th1, Th2, Th17 or itregs 40
Foxp3, the master regulator of (murine) Treg cells 1998: Gene key may unlock immune system diseases The 'scurfy' immune system gene has been found in mice, a defective gene which could act as an on-off switch for the immune system. Foxp3 Foxp3 CD4 CD25 Treg lineage upregulation of Foxp3 in Thymus ntregs medullary thymic epithelium upregulation of Foxp3 in Periphery itregs TGF 41
Foxp3 positive regulatory T cells Foxp3 TGF (+ RA) IL2 Foxp3 42 HG Mills, Nat. rev. Immunol. 2004
Control of the murine Foxp3 gene itreg generation in periphery Treg lineage stability cellular memory module Foxp3 induction within thymus Zheng Y, Josefowicz S, Chaudhry A, Peng XP, Forbush K, Rudensky AY (2010) Nature. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Xu L, Kitani A, Strober W (2010). Mucosal Immunol. Molecular mechanisms regulating TGF-beta-induced Foxp3 expression. 43
Intracellular signaling and important transcription factors 44
T cell activation involves several steps: 1. Close proximity of CD4 or CD8 to TCR 2. Activation of PTK (protein tyrosine kinase) 3. Plasma membrane inositol phospholipid hydrolysis 4. Increased intracellular Ca 2+ and activation of PKC (protein kinase C) 5. Activation of transcription factor NFAT 6. Activation of transcription factor NFκB 7. Activation of MAP kinases and transcriptional activation of AP-1 (fos and jun) 8. Transcription of cytokine genes (e.g. IL-2 through binding of NFAT/AP-1 and NFκB to the IL-2 promoter/enhancer) 45
Grundsätzlich die Möglichkeit für Master- und/oder Doktorarbeiten Molekulare und zelluläre Immunologie (Mausmodelle) Dr. Friederike Berberich-Siebelt Pathologisches Institut (Abteilung für Molekulare Pathologie / Prof. E. Serfling) Josef-Schneider-Str. 2 97080 Würzburg Tel.: 31 81208 path230@mail.uni-wuerzburg.de 46