Molekulare Immunologie

Transkript

1 Molekulare Immunologie Dynamik der Lymphozytenaktivierung PD Dr. Ottmar Janssen

2 Veränderung des Zytoskeletts Dynamik der Lymphozytenaktivierung Aufbau des intrazellulären Signalnetzwerkes

3 Veränderung des Zytoskeletts Dynamik der Lymphozytenaktivierung Aufbau des intrazellulären Signalnetzwerkes TZR Detergent-löslich Zytoskelett-assoziierter TZR Detergent-unlöslich Raft-assoziierter TZR Detergent-unlöslich

4 Die Immunologische Synapse Triple immunofluorescence labeling of an Ag-specific T helper B cell conjugate after 34 h of interaction. Ig labeling (blue) shows the B cell. The tubulin labeling (red) shows the MTOC in the T cell facing the cell contact, and a mitotic spindle in the B cell, indicating that the small resting splenic B cell was induced to proliferate. The labeling for IL5 (in green) shows a massive enrichment next to the cell contact.

5 Die Immunologische Synapse The receptor ligand interactions cause the formation of SMACs on both the T cell and the APC. Although the SMACs on both cells present an identical mirror image, their molecular contents are very different.

6 SMAC = Supramolecular Activation Cluster

7 Dynamik der Aktivierung SMACs lokale Veränderung der Membranfluidität lokale Veränderung der Kinaseaktivität lokaleveränderung des Zytoskeletts Ilangumaran et al., 2000 Modell: Abraham Kupfer

8 SMACs SMACS: Supramolecular Activation Clusters

9 Die Sekretorische Immunologische Synapse The secretory synapse. a,b CTL (left): target (right) conjugate stained with antibodies against cathepsin D (blue), LFA- 1 (green) and talin (red) c,d CTL (left): target (right) conjugate stained with antibodies against granzyme A (green), Lck (red) and actin (blue). Single confocal sections are shown in a and c, and reconstructions of serial sections in the z- axis to show the interface between the two cells are shown in b and d. e Diagram showing spatial organisation of proteins observed during granule secretion.

10 Die Immunologische Synapse

11 Die Immunologische Synapse Membrane acquisition by CTL CTL: target conjugates are stained with antibodies to H2D d (target MHC I) (red), and anti cathepsin D (a e) or lamp1 (f) to label SL (green).

12 Die Immunologische Synapse Detection of specific pmhc complexes on the surface of APCs. APCs are pulsed with biotinylated peptides and the resulting biotin pmhc is labeled with streptavidin phycoerythrin, which can be detected with 3D fluorescence microscopy. T Zellen können einen einzigen (!) Peptid-Liganden erkennen 10 Peptides sind erforderlich, um in CD4 + T-Zellen eine maximale Ca 2+ Antwort zu induzieren und eine stabile Synapse auszubilden 3-4 Peptides sind erforderlich, um in CD8 + T-Zellen eine maximale Ca 2+ Antwort zu induzieren und eine stabile Synapse auszubilden

13 Die Immunologische Synapse CDR3 beta can undergo large conformational changes (>15 Å 2 ) upon binding pmhc. X-ray crystallography studies of unbound and pmhc bound TCR show by comparison large structural changes in CDR3 beta. Two-step model of TCR binding Schematic representation of TCR binding as a twostep process that consists of TCR contact with MHC helices, followed by folding of the CDR3 loops around the peptide to achieve the final stable state.

14 Die Immunologische Synapse

15 Die Immunologische Synapse Blockade of antigenic stimuli leads to breakup of T cell APC interaction. Addition of anti-pmhc antibody rapidly blocks synapse PI3K activity and causes T-cell detachment. T lymphocytes were infected with retrovirus expressing ph(akt)-yfp and shown is the effect of adding anti-pmhc antibody to a 5-h old conjugate which was imaged at the indicated time points using 3D fluorescence microscopy.

16 Dynamik der Aktivierung Antigen blockade affects the degree of T-cell activation in a time-dependent manner. (A) Effect of antigen blockade on IL-2 production. T-cell blasts were mixed with peptide-pulsed APC, added anti-pmhc antibody at indicated time points and secreted IL-2 was determined by ELISA. (B) Effect of antigen blockade on number of cell divisions. T-cell blasts were labeled with CFSE and mixed with peptide-pulsed APC and antipmhc antibody at indicated time points and cells were analyzed by flow cytometry 120 h after cell pooling. Positive control in both experiments was left untreated.

17 Die sekretorische IS in NK-Zellen IS zwischen NK Zelle und Zielzelle MHC protein accumulates at the intercellular contact between NK cells and target cells. The MHC protein is visualised by the attachment of Green Fluorescent Protein (GFP). At the intercellular contact or 'immune synapse' the MHC protein is organised into a ring around a central patch of adhesion protein, LFA-1. See Reference 1 for details. We are currently investigating the formation of immune synapses and how they control intercellular recognition.

18 Die sekretorische IS in NK-Zellen Acquisition of MHC class (H-2D d ) in the inhibitory rodent NK cell IS. The rat NK cell line RNK16 transfected with Ly49A (NK cell) were mixed with EL-4 lymphoma cells transfected with H-2D d -GFP (target) in a 1:1 ratio. The figure shows "green" D d -GFP molecules, visible as green dots in the NK cell, after an interaction between RNK16 cells expressing inhibitory Ly49A receptors with EL-4-D d -GFP cells. No transfer occurred when the RNK16 cells lacked Ly49A or in the presence of antibodies against either the Ly49 receptors or H-2D d -GFP

19 Die Inhibitorische IS in NK-Zellen The inhibitory human NK cell IS. The human tumor cell line YTS expressing KIR2DL1 and the EBV transformed human B cell line transfected with HLA-Cw6-CFP and tubulin-yfp were co-incubated for 30 min The cells were then fixed with Cytofix/Cytoperm, and labeled simultaneously with anti-cd43 mab, and rabbit anti-ezrin polyclonal antibody. After washing the cells, the bound antibodies were detected with Alexa Fluor 568 goat anti-mouse IgG and Alexa Fluor 350 goat anti-rabbit IgG, respectively. After further washing, the fixed conjugates were then incubated with Alex Fluor 633 phalloidin to stain filamentous (f)-actin. The first five images in panel are taken from a single plane through the middle of the conjugate, showing clustering of HLA-Cw6-CFP at the intercellular contact, indicative of an inhibitory NK cell IS. It is clear that ezrin and CD43 are excluded from the region where HLA-C clusters.

20 Viruspartikel-Transfer durch die IS Organization of HTLV-1 proteins and host cell proteins in the viral synapse. Isolated lymphocytes naturally infected with HTLV-1 express the Gag protein, shown in red (a) and the Env protein, also shown in red (b) in an unpolarized manner. Contact with another cell induces polarization of the HTLV-1 Gag protein (d) within 40 min, and transfer to the other cell within 2 h (e). The polarized Gag protein (red) frequently accumulates at the intercellular contact in the center of a ring of talin (green) (c), which resembles the appearance of the IS.

21 T-Zellen