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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.

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Presentation on theme: "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."— Presentation transcript:

1 Natural Killer Cell Receptors MedSci 708

2 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

3 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

4 Cytotoxic T cells and 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 Cytotoxic T cells NK cells

5 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.

6 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 IFN , TNF  CD3 - CD56 + CD16 + (human) CD3 - NK1.1 + (mouse)

7 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 Fc  RIII (CD16) Cytokines (IL-2) promote differentiation into lymphokine-activated killer (LAK) cells

8 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

9 NK Effector Mechanisms (continued) IgG-coated target cells recognized by Fc  RIII (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, IFN , IL12

10 NK cells – where they are Blood Spleen Bone marrow Liver Placenta (uterine NK) Lungs? Gut?

11 BM NK and Thymus T-cell Repertoire(s)

12 NK cells roles 1. Constant scanning for “health” of other cells 2. Inhibitory receptor activity dominates 3. Kill using perforin and granzyme 4. Recruit other cells – cytokines 5. Activate - inflammatory cytokines 6. Regulate/suppress – during pregnancy

13 NK killing mechanisms ADCC (Antibody Dependent Cell Mediated Cytotoxicity) CD16 TRAIL (TNF-Related Apoptosis- Inducing Ligand) TNF  NK target Direct cytotoxicity target NK

14 Recognition of foreign vs self by cytotoxic T cells Self MHC + self peptide Self MHC + foreign peptide foreign MHC + foreign peptide No MHC peptide MHC cell kill ‘self + foreign’. CTL

15 Self MHC + self peptide Self MHC + foreign peptide foreign MHC + foreign peptide No MHC peptide MHC cell Recognition of foreign vs self by NK cells (missing self) NK

16 NK - ADCC Antibody dependent cell cytoxicity

17 NK -Receptor mediated killing

18  EHGF Class II regionClass I region Class II regionClass III regionClass I region TNF DP DQDR AE Complement MHC locus of mouse and man TNF Class III region Mouse H-2 Chr12 ~ 10 Mb Human HLA Chr6 K D L B C A

19 NK cells – how they kill

20 NK Inhibitory receptors block the activating receptors – lack of MHC or blocking ligand NK Target Inhibitory receptor Activation receptor Self MHC HLA-C,E Ligand Target No MHC Target Blocking ligand

21 Human NK cell receptors for HLA

22 NK cells – how they kill (like CTL)

23 NK cell in vivo development Natural killer cell receptor signaling Lanier (2003) Current Opinion in Immunology 15:308–314

24 NK cells in the periphery

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 CD94/NKG2 (4) KIR (>13) Human LIR (ILT) (1) Ly49 (>10) CD94/NKG2 (3) Mouse NK receptors for MHC class I Immunoglobulin family Lectin-like family

27 KIR family and their ligands Natural killer cells and their receptors Middleton 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 Middleton et al (2002) Transplant Immunology 10 (2002) 147–164 No ITIM

30 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

31 NK cell receptors Moretta 2002 “NK cells and DC: rendezvous in abused tissues” Nature Reviews Immunology, 2, p957 p30,p44, p46 Natural Cytotoxicity Receptors

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 signaling Lanier (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 signaling Lanier (2003) Current Opinion in Immunology 15:308–314

36 NK cell activating receptors Ly49D, H ? We know much about inhibitory receptors on NK cells, but how about activating receptors? More activating receptors are to be found. MICA/MICB - ligands

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. Assymetric spreading and granule transport From 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. Leads to symmetrical spreading and loss of synapse From Krzweski & Strominger, 2008

41 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).

42 NKT cells – V α 14J α1 8 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 p241-246

44 Viral proteins affecting NK-cell- mediated immunity Lodoen & Lanier (2005) Nature Reviews Microbiology 3 p50-69

45 MCMV – best studied model m157 MHC-like protein produced by CMV C57Bl/6 strain is resistant to CMVBalb/c strain is susceptible 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

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 3 p50-69

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 p363-374

51 Potential pathogen ligands for MHC independent inhibitory receptors Kumar & McNerney (2005) Nature Reviews Immunology 5 p363-374 PATHOGENRolePossible receptorFunction Epstein-Barr virusIncreases CD48 expression2B4Activation of NK cells Neisseria spp, Salmonella typhimurium, H. influenzae, Moraxella, Mouse HV Binds CEACAM1 for entryCEACAM1Inhibition of T cells, unknown effect on T cells Hepatitis C virusEnvelope protein E2CD81Inhibits NK cells Fowlpox, cowpox, vaccinia, myxoma, African swine fever, rat CMV C-type lectin homologuePossibly NKR-P1Unknown Poxvirus, variola, vaccinia, myxoma virus CD47 homologue Possibly-SRP-  2 Unknown N. meningitidis, H. influenza, E. coli, T. cruzi Sialic acidPossibly SIGLEC7 or 9Unknown

52 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.

53 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) V  24-J  18 paired with V  11 in humans Viruses have ways of “maintaining” the balance of inhibitory vs activating ligands.


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