2 Outline What are they Where are they Where they come from How do they workHow their responses are controlledThe features of their inhibitory and activation receptor familiesReceptor ligands – some of them onlyReceptor signalling and synapseUterine NK cells in pregnancyViral responses to NK recognition
3 NK terminology Abbreviations ERK extracellular signal-regulated kinase GM-CSF granulocyte macrophage colony-stimulating factorIFN interferonITAM immunoreceptor tyrosine-based activation motifITIM immunoreceptor tyrosine-based inhibition motifKIR killer cell immunoglobulin-like receptorKLR killer cell lectin-like receptorLAT linker for the activation of T cellsNK cell natural killer cellPI-3K phosphatidylinositol-3 kinasePLC phospholipase CSLAM signaling lymphocyte activation moleculeSAP SLAM-associated proteinSH Src homologySHP SH-containing tyrosine phosphataseSHIP SH2 domain-containing inositol-5 phosphataseTCR T-cell antigen receptorMICA – MHC class I associated chain A
4 Cytotoxic T cells and NK cells Antigen specificMHC-restrictedRequires priming(takes days to respond)MemoryAntigen non-specificMHC non-restrictedPriming not required(rapid response, hours)No memory
5 Classical NK activityK562 is a human erythroleukemia cell line grown from a 53 year old female CML patient in blast crisis. It was the first human myeloid leukaemia line to be immortalised and has been in continuous culture since 1974.It expresses no MHC class I or MHC class II.It is very effectively lysed by human allogeneic peripheral lymphocytes.This cell line “defined” NK activity because it required no MHC, long before NK cells or receptors were discovered.
6 NK cells Large granular lymphoid cells ~5-15% human PBL ~2-3% mouse spleen cellsNon MHC dependent cytotoxicitystandard targets - K562 for human- YAC-1 for mouseProduce lots of IFNg, TNFaCD3- CD56+ CD16+ (human)CD3- NK1.1+ (mouse)On this slide you can see a NK cell attached to a target cell. In Fumio’s lab we are mainly interested in the interaction of NK cells with their target cells. Although NK cells have been described almost twenty years ago we are just now getting an idea of how this interaction is working. But still a lot of problems are unsolved. How does a NK cell know which cell it is supposed to kill which one not? How do NK cells learn self-tolerance and target-recognition? Which receptors are involved in that interaction? Some of this questions I want to adress in my project.But first of all, I would like to give a brief introduction to NK cells and their biology.A cytotoxic cell starts screening a target cell like virusprogramm. What is going to happen?
7 Natural Killer (NK) Cells From the bone marrowLymphoid but lack most markers for T and B cellsDo not develop through the thymusExpress CD56, a specific NK markerExpress a receptor for Fc portion of IgG, called FcgRIII (CD16)Cytokines (IL-2) promote differentiation into lymphokine-activated killer (LAK) cells
8 Effector MechanismsMechanism of killing similar to those of CD8 T cells – perforins and granzymes.Susceptibility of target is inversely proportional to expression of class I MHC .The more MHC class I expressed, the less the target is killed! Notion of inhibitory receptors
9 NK Effector Mechanisms (continued) IgG-coated target cells recognized by FcgRIII (CD16) are killed by antibody-dependent cell-mediated cytotoxicity (ADCC)Lymphokine-activated killer cells (LAK) kill broader range of cells than NK cells, via IL2, IL15, IL18, IFNa/b, IL12
10 NK cells – where they are BloodSpleenBone marrowLiverPlacenta (uterine NK)Lungs?Gut?
12 NK cells roles Constant scanning for “health” of other cells Inhibitory receptor activity dominatesKill using perforin and granzymeRecruit other cells – cytokinesActivate - inflammatory cytokinesRegulate/suppress – during pregnancy
13 NK killing mechanisms NK NK NK NK target target target target Direct cytotoxicityADCC(Antibody DependentCell MediatedCytotoxicity)TRAIL(TNF-RelatedApoptosis-Inducing Ligand)TNFaNKNKNKNKCD16targettargettargettarget
14 Recognition of foreign vs self by cytotoxic T cells CTL‘self + foreign’.killpeptideMHCcellSelf MHC +self peptideSelf MHC +foreign peptideforeign MHC +foreign peptideNo MHC
15 Recognition of foreign vs self by NK cells (missing self)peptideMHCcellSelf MHC +self peptideSelf MHC +foreign peptideforeign MHC +foreign peptideNo MHC
25 3 forms of NK inhibitory receptors Vivier&Anfossi 2004“Inhibitory NK-cell receptors: witness of the past, actors of the future”, Nature Reviews Immunology, 4, p190
26 NK receptors for MHC class I Immunoglobulin familyLectin-like familyLIR (ILT) (1)KIR (>13)CD94/NKG2 (4)HumanLy49 (>10)CD94/NKG2 (3)Mouse
27 KIR family and their ligands Natural killer cells and their receptorsMiddleton et al (2002)Transplant Immunology 10 (2002) 147–164
28 KIR inhibitor signalling Vivier and Anfossi 2004“Inhibitory NK-cell receptors on T cells:witness of the past, actors of the future” Nature Reviews Immunology, 4, p190
29 KIR Ig-like domains Natural killer cells and their receptors No ITIMNo ITIMNatural killer cells and their receptorsMiddleton et al (2002)Transplant Immunology 10 (2002) 147–164
30 Inhibition or activation from the same ligand but different receptor Natural killer cells and their receptorsMiddleton et al (2002)Transplant Immunology 10 (2002) 147–164
31 NK cell receptors p30,p44, p46 Natural Cytotoxicity Receptors Moretta 2002“NK cells and DC: rendezvous in abused tissues” Nature Reviews Immunology, 2, p957
32 NK cell receptors Vivier and Anfossi 2004 “Inhibitory NK-cell receptors on T cells:witness of the past, actors of the future” Nature Reviews Immunology, 4, p190
33 DAP12 association and signalling Natural killer cell receptor signalingLanier (2003)Current Opinion in Immunology 15:308–314
34 DAP12 or DAP10 signalling Natural killer cell receptor signaling Lanier (2003)Current Opinion in Immunology 15:308–314
35 Some NK receptors can associate with other signalling receptors Natural killer cell receptor signalingLanier (2003)Current Opinion in Immunology 15:308–314
36 NK cell activating receptors We know much about inhibitory receptors on NK cells, buthow about activating receptors?MICA/MICB - ligandsLy49D, H?More activating receptors are to be found.
37 The balance of activation and inhibition signals Multiple inhibitory (ITIM) and activation (ITAM) receptors exist on the same cell??How do these balance each other?Essentially inhibitory signals override activation signals, so activation and killing requires an absence of inhibitory signalling.A powerful homeostatic mechanism that provide single NK cells with multipotent functions.
38 Activation induces actin polymerisation at the pSMAC pole Activation induces actin polymerisation at the pSMAC pole. Assymetric spreading and granule transportFrom Krzweski & Strominger, 2008
39 Microtubule polarisation allows granules to migrate to synapse and fuse with membrane From Krzweski & Strominger, 2008
40 Inhibition prevents the formation polar MTOC Inhibition prevents the formation polar MTOC. Leads to symmetrical spreading and loss of synapseFrom Krzweski & Strominger, 2008
41 Videos – effect of inhibitory signal These videos compare NK cells that have been stimulated byJust LFA-1 alone or in combination with an activating ligand MHC class I related chain A (MICA) which is a ligand for NKG2D receptor. The videos examine f-actin – GFP.LFA-1 stimulation results in asymmetric spreading, motion and long dwell synpase formation while MICA induces symmetrical spreading and stopping and loss of synapse.MICA expression is regulated by a number of viruses (e.g. HCMV – UM142).
42 NKT cells – Vα14J α18 TcR recognises CD1d Godfrey et al 2004“NKT cells: what’s in a name?” Nature Reviews Immunology, 4, p231
43 Maternal-fetal HLA class I and NK receptors Trowsdale & Betz (2006) Nature Immunology 7 p
45 MCMV – best studied model MHC-like protein produced by CMVBalb/c strain is susceptible to CMVC57Bl/6 strain is resistant to CMVNK cells lack the Ly49H activating receptorConfers susceptibility to MCMVNothing to activate NK cells. Virus infects unchallenged.NK cells have Ly49H activating receptorConfers resistance to MCMVBinds the viral m157 NK cells which effectively kill CMV infected cells
46 Modulation of MHC class I antigen presentation by cytomegalovirus proteins Lodoen & Lanier (2005) Nature Reviews Microbiology 3 p50-69
47 Direct NK recognition of MCMV-infected cells Lodoen & Lanier (2005)Nature Reviews Microbiology 3p50-69
48 HCMV UL40 peptide binds HLA-E to prevent anti-HCMV response
49 Downregulation of NKG2D ligands by HCMV UL16 Lodoen & Lanier (2005) Nature Reviews Microbiology 3 p50-69
50 Other NK-cell inhibitory receptors that do not have MHC ligands Kumar & McNerney (2005) Nature Reviews Immunology 5 p
51 Potential pathogen ligands for MHC independent inhibitory receptors RolePossible receptorFunctionEpstein-Barr virusIncreases CD48 expression2B4Activation of NK cellsNeisseria spp, Salmonella typhimurium, H. influenzae, Moraxella, Mouse HVBinds CEACAM1 for entryCEACAM1Inhibition of T cells, unknown effect on T cellsHepatitis C virusEnvelope protein E2CD81Inhibits NK cellsFowlpox, cowpox, vaccinia, myxoma, African swine fever, rat CMVC-type lectin homologuePossibly NKR-P1UnknownPoxvirus, variola, vaccinia, myxoma virusCD47 homologuePossibly-SRP-b2N. meningitidis, H. influenza, E. coli, T. cruziSialic acidPossibly SIGLEC7 or 9Kumar & McNerney (2005) Nature Reviews Immunology 5 p
52 Key points NK receptors are extremely diverse NK cells survey the “health” of other cells.NK cells have dual signalling receptors – inhibitory signal dominatesInhibitory receptors KIR family, LIR family, CD94/NKG2A, Ly49.Activating receptors Lots of them e.g. CD94/NKG2DInhibitory - ITIMs bind (SHP) phosphatases which stop cascade. SH2 domains. Typically found on long intracellular receptor domainsActivation - ITAMs bind PTK (src kinases fyn, lck, shk) initiates kinase cascade. SH domains. Typically found on adaptor proteins (DAP) for shorter intracellular receptors.
53 Key points Humans = KIR (Ig-like) & LIR (lectin-like) receptors Mouse only have KIRLigands for KIRs are MHC class I molecules – in general!HLA – C and HLA – E seem to be the special ligands for KIRs.Missing self - no MHC class I means no inhibitory signal.NK have potent Cytokine and/or Cytotoxic ResponsesuNK Suppressor/regulatory control immune system during pregnancyNK-T cells are a unique subset of T cells that recognise a single ligand. (aGalCer/CD1) Va24-Ja18 paired with Vb11 in humansViruses have ways of “maintaining” the balance of inhibitory vs activating ligands.