NK Zelle LGL (grosser granulärer Lymphozyt) 3 Aktivitäten: K (Killer) - ADCC (Ab-dependent cellular cytotoxicity) NK LAK - Lymphokin-aktivierter Killer Spontanes Killing von Tumor- und virusinfizierten Zellen Hybridresistenz Fc  III Rezeptor / CD16: K Aktivität NK Rezeptoren: NK (und LAK Aktivität) Marker: CD56+ (90%) CD3- TZR- NKp46

HSZ Lymphoide VZ T + NK VZB VZ B Lymphozyt T Lymphozyt (Thymus) NK Zelle

Funktionen: Virusabwehr in früher Infektionsphase Tumor-Killing „Hybridresistenz“, Problem Knochenmarktransplantationen  -IFN Produktion Aktivierung und Killing reguliert durch NK Rezeptoren ADCC Funktion in Interphase natürliches / spezifisches Immunsystem

Hybridresistenz A Maus B Maus AB Bone Marrow Transplantation xx Killing von No-Self ?

Missing-self hypothesis: MHC class I KIRs: Ig-like NKG2A/CD94: lectin-like Ig-domain lectin-domain NKG2ACD94 KIR2DKIR3D dimer HLA-CHLA-B HLA-E ITIM Ligands on normal cell NK or NK/T c

Activating NK receptors KIR-S: Ig-like NCRs: Ig-like NKG2C/CD94: lectin-like Ig-domain lectin-domain NKG2C CD94/ KIR2SKIR3S HLA-C?HLA-B?HLA-E ITAM Ligands on virus-infected or tumor cell NKG2E CD94/ HLA-E NKG2D MICA/B DAP12 DAP10 NK or NK/T

Adapted from: C. Bottino et al. / Molecular Immunology 41 (2004) 569–575 Human HLA class I-specific receptors:

NK killing capacity and activation -Killing mechanisms: Perforin/Granzymes, FasL, cytokines -CD56 dim and CD56 bright NKs differ in trafficking and killing capacity -Different cell lines as targets in in vitro systems: High sensitivity: e.g. K-562 (myeloerythroid cell line) Moderate sensitivity: e.g. MOLT-4 (T-cell line) Low sensitivity: e.g. GAC-2 (gastric cancer cell line) - NK cells can be activated via IL-2 proliferate and show increased killing capacity Adapted from: T L Whiteside and R B Herberma, Clinical and Diagnostic Laborytory Immunology, 1994 Vol.1 No2 K-562 Fresh K-562 Fresh Tumor Tumor /++ J.T. Thornthwaite,1972 NK LAK IL-2

Introduction: NK cell killing mechanisms Adapted from: M Uhrberg, Leukemia (2005) Vol.19 1) Killing via Perforin channels pathmicro.med.sc.edu/bowers/ctl-10c.jpg 2) Killing via Cytokines, Fas-L

Introduction: Determination of NK cell Activity Target cell Eu- labeled NK Eu- labeled NK Coculture Europium-Release-Killing-Assay Adapted from: M Uhrberg, Leukemia (2005) Vol.19 Flow Cytometry: e.g. CD107a

NKp46 CD107a FSC SSC PB-NKPB-NK + K562 CD107a-Activity-Assay Determination of the killing capacity of NK cells Establishment of: Europium-Killing-Assay

Flow cytometry - FACS FSC: forward scatter, size of cells SSC: side scatter, granularity of cells Video

NK cell activity in healthy persons Donor B: 11 % NKp46 staining in Lymphocyte-Population Donor A: 5 % NKp46 staining in Lymphocyte-Population FSC SSC APC PBMCs isolated from whole blood via Ficoll gradient centrifugation

NKG2-A and -C expression on PB-NK NKG2C NKG2A FSC SSC

IL-1, TNF , LPS, AGE, oxLDL, Stimulation interleukins, adhesion molecules, pro-coagulant, iNOS, COX, anti-apoptotic Response ? specific and essential signals leading to gene regulation ? hypoxia VEGF Ang2 Angiogenesis Sprouting Neo-vessel formation Inflammation Immune cell transmigration Pro-coagulation

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NF  B Signalweg I  B wird an Ser/Thr phosphoryliert, ubiquitinyliert und im Proteasom proteolytisch abgebaut TRADD ( TNFR associated death domain) TRAF (TNFR associated factor) IKKK (I  B-Kinase-Kinase) IKK (I  B-Kinase) (beide Ser/Thr Kinasen) I  B (Inhibitor von NF  B) NF  B (nuclear factor  B Gene)

Figure Molecular Biology of the Cell (© Garland Science 2008)

Figure 19-19b Molecular Biology of the Cell (© Garland Science 2008)

Blocking NF-  B by rAdVs expressing dnIKK2 inhibits Inflammatory EC functions Wrighton et al., JEM (1996) Oitzinger et al., Blood (2001) Expression of cell adhesion molecules: Inhibition of monocyte adhesion: