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Signaltransduktion in T-ZellenVorlesung Immunologie 16.5.12

Friederike Berberich-SiebeltAbteilung für Molekulare Pathologie

Pathologisches InstitutUniversität Würzburg

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Th17

effector memory

Possible T cell responses

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Fragestellung

Warum proliferiert, differenziert oder stirbt die T-Zelle?

Unter welchen Umständen reagiert die T-Zelle bzw. eben nicht (Anergie)?

Welche Signale bestimmen die Differenzierungs-richtung (T-helfer 1, 2, 17… oder T-regulatorische Zelle)?

Was entscheidet über die Entwicklung zur Effektor- bzw. zur Gedächniszelle?

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Structures of the pre-TCR and αβTCR

Open circles in the cytoplasmic domains of the CD3, , , and ζ chains denotetyrosine residues within the immunoreceptor tyrosine-based activation motif (ITAM).Horizontal bars within pT–TCR, TCR–TCR and ζ–ζ represent disulfide bonds.

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Cytoplasmic domains of TCR and BCR and FcR contain ITAMs

black rectangles: Immunoreceptor tyrosine-based activation motif(D/E)XXYXXL(X)6-8YXXL

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Innate and adaptive immunity

CD - cluster of differentiationCD4 - T cells (helper/inducer), monocytes, myeloblasts, NK cell lymphomaCD8 - T cells (suppressor/cytotoxic), large granular lymphocytes

Dranoff G (2004) 4Cytokines in cancer pathogenesis and cancer therapy.

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Innate and adaptive immunity are both specific

innate adaptivefast slowpattern recognition = specific antigene recognition = highly specific

memory

8Rawlings et al. Nature Reviews Immunology 6, 799–812 (2006)

Innate and adaptive immune receptors integrate signalling pathways

8Rawlings et al. Nature Reviews Immunology 6, 799–812 (2006)

BCR TCRadaptiveinnate

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Functions of costimulators in T cell activation

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Formation of the immunological synapse

SMAC =SupraMolecular Activation Cluster

A. Kupfer (U. Colorado)

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Formation of microclusters and immunological synapse

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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

P

P

Ub

Ub

Ub

Ub recognition byESCRT-I component TSG101

Lysosomal degradation

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Intracellular signaling events during T cell activation

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Early tyrosine phosphorylation in T cell activation

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Two classes of PTK = Phospho Tyrosine Kinases1. 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

LckSH2 (src homology domain 2) binds to P-tyr Y505

and forms a closed, inactive conformation.Phosphorylation of Y394 activates Lck.

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Csk and CD45 regulate LckCsk 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)

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Activation of Lck and ZAP70

Lck transphosphorylates itselfphosphorylates ITAM ZAP70 binds to ITAM

ZAP70 is activated by LckZAP70 phoshorylates LAT, SLP76

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The Ras-Map kinase pathway in T cell activation

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Activation of T cells induces phosphorylation of ERK

TPAIono

N.mab

55-44-33-25-

+ + + +

55-44-33-25-

+ + + ++ + + +

-P-ERKlight exposure

-P-ERKdark exposured

-ERK

TCR signals (anti-CD3/CD28) can be bypassed byTPA/PMA (phorbol ester) that activates PKC

and ionomycin (calcium ionophore).

20Downloaded from: StudentConsult (on 26 January 2007 09:13 AM)

© 2005 Elsevier

Signals via membrane inositol phospholipid metabolism

TPA = DAG analogon

ionomycin =Ca2+ ionophore

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PKC-θ (theta) is essential for TCR signalling

9 PKC genes (PKC−α,β,γ,δ,ε,θ,ζ),but deletion of this gene alone leads to defective T cell activation.

ELISASun et al. (2000) Nature 404, 402-407.

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Activation of transcription factors in T cells

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A general scheme of NFB activation

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Mitogen-activated protein kinase scaffold proteins in immune signalling

Scaffold proteins and immune-cell signallingAndrey S. Shaw & Erin L. FilbertNature Reviews Immunology 9, 47-56 (January 2009)

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The role of PKC enzymes in IKK–NFB activation downstream of antigen receptors

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NFB activation is abolished when PKCθ is absent

EMSA: electromobility shift assay/„band shift“Sun et al. (2000) Nature 404, 402-407.

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Activation of transcription factors in T cells

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Scaffold proteins of the calcium signalling pathway

Scaffold proteins and immune-cell signallingAndrey S. Shaw & Erin L. FilbertNature Reviews Immunology 9, 47-56 (January 2009)

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Family and activation of NFAT transcription factor

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.

Macian F (2005). NFAT proteins: key regulators of T-cell development and function.

Ca2+

Ca2+

Ca2+

Ca2+

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NFAT-activated programmes of gene expression:T-cell activation vs. T-cell anergy

Ca2+ + other signalsNFAT + AP1 +…

productive activation

Unopposed Ca2+

NFAT + w/o

anergy

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ITIMs are Immunoreceptor Tyrosine Inhibitory Motifs

V/I/xYxxL/V, present in co-receptors that negatively regulates ITAM receptors.SHP-1 and SHP-2 phosphatases bind to CTLA-4

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CD28 stimulation leads to few changes in gene expression relatively to CD3

(just more of the same genes)

Microarray analysisDiehn et al. (2002) Proc.Natl. Acad. Sci. USA 99,11796-11801.

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CD28 regulates glucose metabolism through PI3Kinase-AKT and up-regulates Glut1 glucose transporter

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Signal 1 leads to increased demand for energy, signal 2 enhances glycolysis & energy for growth/proliferation

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Signal 3: Cytokine Milieu Determining CD4+ T Cell Differentiation and Conversion

Liang Zhou, Mark M.W. Chong, and Dan R. Littman (2009). Immunity

(Bcl6)

(Pu1+IRF4)

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CD4+ T helper cells different to express a specific subset of lymphokines

Restim. - + - + - + - + - +Th 0 1 2 „“ 17

- + - + - + - + - +0 1 2 „“ 17

IL-17

IL-4IL-5IL-10IL-13

IL-2

IFN-

L32GAPDH

GAPDH

RNase protection assay for lymphokines

Th2

Th1

Th17

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NFAT and T-helper-cell differentiation

Macian F. (2005) Nat Rev Immunol. 5NFAT proteins: key regulators of T-cell development and function.

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The JAK STAT pathway

3, 900-911.Regulation of JAK-STAT signalling in the immune system

Ke Shuai & Bin Liu (2003)

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Ke Shuai & Bin Liu (2003)3, 900-911.

Regulation of JAK-STAT signalling in the immune system

The negative regulation of the JAK/STAT pathway

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Transcriptional master regulators facilitate differentiation of Th1, Th2, Th17 or iTregs

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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.

Foxp

3

Foxp

3

CD4 CD25

upregulation of Foxp3 in Thymus nTregsmedullary thymic epithelium

Treg lineageupregulation of Foxp3 in Periphery iTregsTGF

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Foxp3 positive regulatory T cells

Foxp3

Foxp3TGF (+ RA)

IL2

HG Mills, Nat. rev. Immunol. 2004

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Control of the murine Foxp3 gene

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.

iTreg generation in periphery Foxp3 induction within thymusTreg lineage stabilitycellular memory module

Xu L, Kitani A, Strober W (2010). Mucosal Immunol.Molecular mechanisms regulating TGF-beta-induced Foxp3 expression.

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Intracellular signaling and important transcription factors

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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 Ca2+ 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)

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Grundsätzlich die Möglichkeit für Master- und/oder Doktorarbeiten

Molekulare und zelluläre Immunologie(Mausmodelle)

Dr. Friederike Berberich-SiebeltPathologisches Institut

(Abteilung für Molekulare Pathologie / Prof. E. Serfling)Josef-Schneider-Str. 2

97080 WürzburgTel.: 31 81208

path230@mail.uni-wuerzburg.de