Haut (Epithel) Gewebe (Haut) Komplement aktivierung MΦ. Mast zelle. Blutgefäß. Öffnung Tight junctions. Induktion Adhäsions moleküle

Transkript

1 Blutgefäß Gewebe (Haut) Haut (Epithel) Komplement aktivierung MΦ DCs Mast zelle Histamin PGs Zytokine (z.b. TNFa, IL-1) Chemokine (z.b. IL-8 = CXCL8) Vaso dilatation Öffnung Tight junctions Influx C +AK Induktion Adhäsions moleküle Influx Granulozyten PAMPs = pathogen associated molecular patterns DAMPS = danger/damage associated molecular patterns PRRs = pattern recognition receptors

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3 Activation of inflammation by Danger is required to initiate an immune response apoptotic cell death necrotic cell death Pathogen (PAMPS) NO danger Damage (DAMPS) DC DC Ag presen tation T soluble Mediators (e.g. cytokines) Ag presen tation T costimulation

4 MHC class I HLA-A, B und C MHC class II = HLA-DR, DP und DQ Presentation of endogenes Antigens (host, Tumors, Viruses) all nucleated cells Recognition by cytotoxic T-Lymphozyten (CD8+) Presentation exogenes Antigens (Bakterien, Pilze etc.) Only on specialized Antigen-presenting cells (=APCs) (DCs, Makrophages; Monocytes, B-cells) Recognition by T-Helper Lymphozyten (CD4+) Crosspresentation (viral epitopes for naive CD8+ T cells)

5 MHC-Molecules are polymorphic There are several alleles for each Gene insight the population.

6 How many different MHC-I molecules are expressed on each cell? How diverse are your individual MHC molecules?

7 How many different MHC-I molecules are expressed on each cell? How can we nevertheless achieve to present a wide range of different epitopes?? Only Anchor-amino acids must fit Example of HLA-I Allel specific Peptides Are we able to present each theoretical possible epitope?

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9 Immature DC Mature DC Principal function Antigen capture Antigen presentation Expression of FcR, Mannose-R ++ - Expression of Co-stimulatory Molecules (CD80/CD86) - or low ++ MHCII half life 10h 100h number of surface molecules x10 6

10 Figure 5-17

11 (Alternative) Antigenpresentation of (Glyco)lipids via CD1 CD1d Glycolipids CD1c lipids CD1a, b, c lipids NKT cells IL-4, IFN-g gd-t cells IFN-g CD1-restricted ab-t cells IFN-g

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13 Adaptive immune response / specific Antigen recognition needs GOD Generation of diversity

14 T H -Zellen: Bindung von MHC-II an TCR/CD4 T C -Zellen: Bindung von MHC-I an TCR/CD8

15 Other manifestations of GOD Sea urchin Lamprey (Neunauge) Jawless fish Increased number of germline encoded genes which encode for pathogen recognizing receptors Increased number of somatical encoded pathogen recognizing receptors by gene fragment rearrangement J. P. Rast et al., Science 314, (2006) Pancer, Z. et al. Nature 430, (2004).

16 Generation of TCR diversity

17 T-cells mature in the thymus T cell precursors (CD3-CD4-CD8-) blood BM positive selection negative selection Naive, mature CD3+ T cell (either cd4+ or cd8+)

18 Generation of 10 8 naive, mature T cells/day!! (re-generation after T cell depletion) Thymus activity and size decreases with age

19 TCR gene structure

20 Somatic Recombination Figure 4-12

21 Recombination is achieved via RAG1 +2 and specific recoginition sites RSS recoginzed by DNA-Recombinases: recombination-activating genes: RAG1 and RAG2

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23 Depletion /re-arrangement can be detected by Southernblot in T- and B-cells

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25 Mechanism of DNA-rearrangement Enzymes: Recognition sites: DNA-Recombinases: recombination-activating genes: RAG1 and RAG2 2 nonamer-heptamer sequences (ggtttttgt-23bp-cactgtg) Deletion Inversion Lack of RAG-genes: severe combined immunodeficiency = SCID Additional diversity by junctional diversity : removal (nucleases) and random addition (TdT) (terminal deoxyribonucleotidyl transferases) of nucleotides at junctions between V-D and D-J

26 TCR-rearrangement In thymic cortex

27 Combinatorial diversity of antigen receptores Figure 4-13

28 After productive TCR-rearangement - Positive selection for self MHC-recognition in thymic cortex Positive Selection in cortex: CD3 +, CD4 +,CD8 +, TCR + Zellen Low affinity binding to either MHCI or II leads to positive selection and fate decision between CD4 + or CD8 +

29 thymocyte Rearrangement of g/d genes productive Positive selection Epidermal tissues non productive Rearrangement of b genes Productive (Pre-TCR with pre-tcra) Rearrangement of a genes Non productive Allelic exclusion Rearrangement of b genes from second allel productive Non productive Productive (TCRab) Coexpression of CD4 AND CD8 Low affinity interaction With MHCI OR MHCII + self peptide No OR high affinity interaction With MHCI OR MHCII + self peptide MHC-restriction Positive selection Mature, naiv T cell

30 Change of surface expression markers during maturation

31 Negative selection for self peptide/mhc-recognition in thymic medulla = CENTRAL TOLERANCE negative selection in medulla: TCRs with high affinity to self-peptide/mhc-complexes (expressed on medullary epithelial and dendritic cells) are depleted (by apoptosis)

32 How can we generate central tolerance against non-thymic self proteins?

33 transcription factor AIRE: induces expression of many (but not all) non-thymic Proteins = self antigens Lack of AIRE = lack of central tolerance = severe autoimmunity

34 Expression of RAGs and TdT is tightly restricted to pro- and pre- T and B cells Wrong rearrangements (e.g. between different chromosomes) leads to chromosomal translocations fusion proteins e.g. Bcr-abl, MLL-AF9 ectopic expression of transcription factors e.g. Lmo2 causes Leukaemias

35 Diagnostics of TCR (or BCR) repertoire monoclonal oligoclonal polyclonal

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37 Classical mice with thymus defects

38 What happens if you would have a defect in MHCII expression?

39 What happens if you express a transgenic TCR? Majority of maturated T cells express single TCR

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42 Homing to lymphnodes

43 Homing to lymphnodes

44 T cell activation

45 Dendritic cells Dendritic cells T-cells

46 Activation of inflammation by Danger is required to initiate an immune response apoptotic cell death necrotic cell death Pathogen (PAMPS) NO danger Damage (DAMPS) DC DC Ag presen tation T soluble Mediators (e.g. cytokines) Ag presen tation T costimulation

47 CD4 und CD8 als Korezeptoren Lck CD4 erkennt MHC-II eine Polypeptidkette mit 4 Ig-ähnlichen Domänen D1 bindet an a2/b2-domäne von MHC-II wird von T-Helfer-Zellen exprimiert CD8 erkennt MHC-I Dimer aus a und b-kette mit je 1 Ig-ähnlichen Domäne, über eine hochglykosylierte (und deshalb gestreckten) Polypeptidkettenabschnitt mit dem Membransegment verbunden bindet an a 3 /a 2 -Domäne von MHC-I wird von zytotoxischen T-Zellen exprimiert Lck CD8 + / die lymphocyte-specific protein tyrosine kinase (lck) ist eine mit CD4/CD8 assoziierte Tyrosinkinase

48 Aktivierung von T-Zellen durch Antigen-präsentierende Zellen (APC) Aktivierung der T-Zelle durch drei Signale über TCR/CD4(CD8)... MHC-II(MHC-I)- Peptid Kostimulation über CD28... B7.1/7.2 über Cytokine: Differenzierung in eine der CD4 + -Subpopulationen

49 Kinapsen und immunologische Synapsen Synapse: längerer stabiler Kontakt ( bis 12 h) mit smac adhesion LFA-1 CD2 (LFA-2) ICAM-1,2 LFA-3 (CD 58) central supramolecular activation complex (c-smac) mit Signalmolekülen für die T-Zellaktivierung (TCR, CD28,...) peripheral supramolecular activation complex (p-smac) mit Integrinen TCR signaling CD4 or CD8 T cell TCR APC antigen/mhc Co-stimulation CD28 CD40L B7-1,2 CD40 Kinapse: transienter Kontakt T-Zelle mit DC (s bis min)

50 TCR Lyk Anker (an die Innenseite der Plasmamembran) N-Terminus (Kontakt mit CD4, CD8) P SH3-Domäne SH2-Domäne Aktivierendes Tyrosin Inhibierendes Tyrosin

51 T-cell signalling NF-kB Ca 2+ Calcineurin NF-AT

52 T-cell activation - Signals 1+2 Signal-1 Signal-2 Raf Ras Rac DAG Ca 2+ MAPK ERK JNK PKC Calcineurin TFs AP-1 NF-kB NF-AT IL-2 + CD25

53 Polyklonale Aktivierung von Lymphozyten Superantigene: einige bakterielle (z. B. Exotoxine von Staphylokokken und Streptokokken) und retrovirale Proteine können CD4-T-Zellen polyklonal (2 20 % aller Klone) aktivieren Superantigene binden an die äußere Oberfläche (nicht die Peptid-Bindungsstelle) von MHC-II und an den konstanten Teil des TCR und verbinden damit die APC und die T-Zelle. Die im Übermaß gebildeten Zytokine (Zyytokin-Sturm) wirken immunsuppressiv und/oder können einen toxischen Schock (z. B. TNF-a) auslösen in vitro simuliert man DAG mit PMA und Ca 2+ -Release mit Ionomycin experimentell

54 TCR activation induces IL-2 and high affinity IL-2-receptor TCR IL-2 IL-15 IL-2R a b g a Ras PI3K IL-2 a a IL-2Ra =CD25 STAT 5 p38 MAPK MK2 Akt/PKB TOR transcription translation / mrna stability Effector cytokines cell cycle, Proliferation

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56 zweite Welle von inhibitorischen bzw. kostimulatorischen Molekülen nach Aktivierung wird CTLA-4 synthetisiert, das eine höhere Affinität zu B7 besitzt als CD28, aber inhibitorisch wirkt; dadurch wird die proliferative Antwort begrenzt Nature Reviews Immunology, 1 (2001), activated T-cell ICOS (inducible T-cell costimulator, homologous to CD28) is expressed on activated, but not resting T cells. Its ligand, B7-H2 (LICOS) on APCs is upregulated by TNF-α or LPS. In contrast to CD28 mediated costimulation, ICOS pathway preferentially regulates effector function of T cells APC activated T-cell Sharpe, A.H., Freeman, G.J.: The B7 CD28 superfamily. Nature Reviews Immunology 2 (2002),

57 cytokine memory memory expression - speedy - simultaneous - signal independent (only TCR) IL-12/IL-4 antigen presenting cell IL-2 IL-4 INF-g IL-10 antigen presenting cell CD80/86 CD28 MHCII TCR MHCII TCR Th days Th1/2 hours D R F Z

58 Grafik aus: Janeway et al., Immunobiology 5th ed.

59 Effector mechanisms of cytotoxic T cells

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61 Induktion einer adaptiven Immunantwort erfordert Aktivierung von Antigen-präsentierenden Zellen (APCs) IL-12 IL-2 TH1 IFNg TNFa IL-1 IL-6 TGFb IL-23 IL-21? chronische Entzündung TH17 IL-17 TNFa IL-22

62 naïve TH0 IL-12 TH1 t-bet STAT4 IFNg IFNg TNFa TH2 GATA-3 STAT6 Abwehr intrazellulärer Pathogene TH17 ROR-gt STAT3 Tregs FoxP3 Physiologische Funktion Pathogene Wirkung (akute) systemische Entzündung no IL-12 IL-2 IL-4 IL-13 Abwehr von Parasiten Allergie TGFb TGFb IL-6 IL-23 IL-17 TNFa IL-22 Abwehr extrazellulärer Pathogene chronische Entzündung Autoimmunität IL-10 TGFb CTLA4 Periphere Toleranz

63 How do we measure T cell activation?

64 Proliferation assays A CD4 + T cells pre-cultured with anti-cd4 CD4 + T cells pre-cultured without anti-cd4 - + CD4 + responders - + CD4 + responders Regulation of anti-cd3 responses p 0.05 p CD4 + responders only CPM B without anti-cd4 with anti-cd4 CD4 + naïve responders only CD4 + T cells pre-cultured with or without anti-cd4 + CD4 + naïve responders Cell counts CFSE

65 cell count CD4 FITC Intracellular cytokine staining A Unstimulated 2.96% IL-12/ % 3.98% 30.79% IL-12/18 + TGF-b 5.65% 10.89% IFN-g PE B No pre-treatment (fresh) 24h acd3/acd28 pre-treated - IL-10 + IL-10 Key Name Key PBMC.001 Name PBMC.001 Key Name Key Name TCR Tcells.005TCR Tcells.005

66 Pharmaceutical inhibitors of T cell activation