Natural Killer Cell Receptors MedSci 708 Outline •What are they •Where are they •Where they come from •How do they work •How their responses are controlled •The features of their inhibitory and activation receptor families •Receptor ligands – some of them only •Receptor signalling and synapse •Uterine NK cells in pregnancy • Viral responses to NK recognition NK terminology Abbreviations ERK extracellular signal-regulated kinase GM-CSF granulocyte macrophage colony-stimulating factor IFN interferon ITAM immunoreceptor tyrosine-based activation motif ITIM immunoreceptor tyrosine-based inhibition motif KIR killer cell immunoglobulin-like receptor KLR killer cell lectin-like receptor LAT linker for the activation of T cells NK cell natural killer cell PI-3K phosphatidylinositol-3 kinase PLC phospholipase C SLAM signaling lymphocyte activation molecule SAP SLAM-associated protein SH Src homology SHP SH-containing tyrosine phosphatase SHIP SH2 domain-containing inositol-5 phosphatase TCR T-cell antigen receptor MICA – MHC class I associated chain A Cytotoxic T cells and NK cells Cytotoxic T cells NK cells • Antigen specific • MHC-restricted • Requires priming (takes days to respond) • Memory • Antigen non-specific • MHC non-restricted • Priming not required (rapid response, hours) • No memory Classical NK activity • K562 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. NK cells • Large granular lymphoid cells • ~5-15% human PBL • ~2-3% mouse spleen cells • Non MHC dependent cytotoxicity • standard targets - K562 for human - YAC-1 for mouse • Produce lots of IFNg, TNFa • CD3- CD56+ CD16+ (human) • CD3- NK1.1+ (mouse) Natural Killer (NK) Cells • From the bone marrow • Lymphoid but lack most markers for T and B cells • Do not develop through the thymus • Express CD56, a specific NK marker • Express a receptor for Fc portion of IgG, called FcgRIII (CD16) • Cytokines (IL-2) promote differentiation into lymphokine-activated killer (LAK) cells Effector Mechanisms • Mechanism 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 NK Effector Mechanisms (continued) • IgG-coated target cells recognized by FcgRIII (CD16) are killed by antibody-dependent cellmediated cytotoxicity (ADCC) • Lymphokine-activated killer cells (LAK) kill broader range of cells than NK cells, via IL2, IL15, IL18, IFNa/b, IL12 NK cells – where they are • Blood • Spleen • Bone marrow • Liver • Placenta (uterine NK) • Lungs? • Gut? BM NK and Thymus T-cell Repertoire(s) NK cells roles 1. 2. 3. 4. 5. 6. Constant scanning for “health” of other cells Inhibitory receptor activity dominates Kill using perforin and granzyme Recruit other cells – cytokines Activate - inflammatory cytokines Regulate/suppress – during pregnancy NK killing mechanisms Direct cytotoxicity ADCC (Antibody Dependent Cell Mediated Cytotoxicity) NK TRAIL (TNF-Related ApoptosisInducing Ligand) TNFa NK NK NK target target target CD16 target Recognition of foreign vs self by cytotoxic T cells CTL ‘self + foreign’. kill peptide MHC cell Self MHC + self peptide Self MHC + foreign peptide foreign MHC + foreign peptide No MHC Recognition of foreign vs self by NK cells NK (missing self) peptide MHC cell Self MHC + self peptide Self MHC + foreign peptide foreign MHC + foreign peptide No MHC NK - ADCC Antibody dependent cell cytoxicity NK -Receptor mediated killing MHC locus of mouse and man Mouse H-2 Chr12 Class II region K A Class III region E Class I region D L TNF Human HLA Chr6 Class II region DP DQ DR a b a b a b Class III region Class I region B C A E Complement TNF ~ 10 Mb H G F NK cells – how they kill Inhibitory receptors block the activating receptors – lack of MHC or blocking ligand NK Inhibitory receptor NK NK Activation receptor Ligand Self MHC HLA-C,E Target Blocking ligand No MHC Target Target Human NK cell receptors for HLA NK cell in vivo development Natural killer cell receptor signaling Lanier (2003) Current Opinion in Immunology 15:308–314 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 NK receptors for MHC class I Immunoglobulin family LIR (ILT) (1) Lectin-like family KIR (>13) CD94/NKG2 (4) Human Ly49 (>10) Mouse CD94/NKG2 (3) KIR family and their ligands Natural killer cells and their receptors Middleton et al (2002) Transplant Immunology 10 (2002) 147–164 KIR Ig-like domains No ITIM No ITIM Natural killer cells and their receptors Middleton et al (2002) Transplant Immunology 10 (2002) 147–164 Inhibition or activation from the same ligand but different receptor Natural killer cells and their receptors Middleton et al (2002) Transplant Immunology 10 (2002) 147–164 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 DAP12 association and signalling Natural killer cell receptor signaling Lanier (2003) Current Opinion in Immunology 15:308–314 DAP12 or DAP10 signalling Natural killer cell receptor signaling Lanier (2003) Current Opinion in Immunology 15:308–314 Some NK receptors can associate with other signalling receptors Natural killer cell receptor signaling Lanier (2003) Current Opinion in Immunology 15:308–314 NK cell activating receptors We know much about inhibitory receptors on NK cells, but how about activating receptors? MICA/MICB - ligands Ly49D, H ? More activating receptors are to be found. 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. Activation induces actin polymerisation at the pSMAC pole. Assymetric spreading and granule transport From Krzweski & Strominger, 2008 Microtubule polarisation allows granules to migrate to synapse and fuse with membrane From Krzweski & Strominger, 2008 Inhibition prevents the formation polar MTOC. Leads to symmetrical spreading and loss of synapse From Krzweski & Strominger, 2008 Videos – effect of inhibitory signal These videos compare NK cells that have been stimulated by Just 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). NKT cells – Vα14J α18 TcR recognises CD1d Godfrey et al 2004 “NKT cells: what’s in a name?” Nature Reviews Immunology, 4, p231 Maternal-fetal HLA class I and NK receptors Trowsdale & Betz (2006) Nature Immunology 7 p241-246 Viral proteins affecting NK-cellmediated immunity Lodoen & Lanier (2005) Nature Reviews Microbiology 3 p50-69 MCMV – best studied model m157 MHC-like protein produced by CMV Balb/c strain is susceptible to CMV C57Bl/6 strain is resistant to CMV NK cells lack the Ly49H activating receptor Confers susceptibility to MCMV Nothing to activate NK cells. Virus infects unchallenged. NK cells have Ly49H activating receptor Confers resistance to MCMV Binds the viral m157 NK cells which effectively kill CMV infected cells Modulation of MHC class I antigen presentation by cytomegalovirus proteins Lodoen & Lanier (2005) Nature Reviews Microbiology 3 p50-69 Direct NK recognition of MCMVinfected cells Lodoen & Lanier (2005) Nature Reviews Microbiology 3 p50-69 HCMV UL40 peptide binds HLA-E to prevent anti-HCMV response Downregulation of NKG2D ligands by HCMV UL16 Lodoen & Lanier (2005) Nature Reviews Microbiology 3 p50-69 Other NK-cell inhibitory receptors that do not have MHC ligands Kumar & McNerney (2005) Nature Reviews Immunology 5 p363-374 Potential pathogen ligands for MHC independent inhibitory receptors PATHOGEN Role Possible receptor Function Epstein-Barr virus Increases CD48 expression 2B4 Activation of NK cells Neisseria spp, Salmonella typhimurium, H. influenzae, Moraxella, Mouse HV Binds CEACAM1 for entry CEACAM1 Inhibition of T cells, unknown effect on T cells Hepatitis C virus Envelope protein E2 CD81 Inhibits NK cells Fowlpox, cowpox, vaccinia, myxoma, African swine fever, rat CMV C-type lectin homologue Possibly NKR-P1 Unknown Poxvirus, variola, vaccinia, myxoma virus CD47 homologue Possibly-SRP-b2 Unknown N. meningitidis, H. influenza, E. coli, T. cruzi Sialic acid Possibly SIGLEC7 or 9 Unknown Kumar & McNerney (2005) Nature Reviews Immunology 5 p363-374 Key points • NK receptors are extremely diverse •NK cells survey the “health” of other cells. •NK cells have dual signalling receptors – inhibitory signal dominates • Inhibitory receptors KIR family, LIR family, CD94/NKG2A, Ly49. • Activating receptors Lots of them e.g. CD94/NKG2D • Inhibitory - ITIMs bind (SHP) phosphatases which stop cascade. SH2 domains. Typically found on long intracellular receptor domains • Activation - ITAMs bind PTK (src kinases fyn, lck, shk) initiates kinase cascade. SH domains. Typically found on adaptor proteins (DAP) for shorter intracellular receptors. Key points • Humans = KIR (Ig-like) & LIR (lectin-like) receptors • Mouse only have KIR • Ligands 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 Responses • uNK Suppressor/regulatory control immune system during pregnancy • NK-T cells are a unique subset of T cells that recognise a single ligand. (aGalCer/CD1) Va24-Ja18 paired with Vb11 in humans • Viruses have ways of “maintaining” the balance of inhibitory vs activating ligands.