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

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 IFNg, TNFa
CD3- 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 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

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 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
Blood
Spleen
Bone marrow
Liver
Placenta (uterine NK)
Lungs?
Gut?

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

12. NK cells roles 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

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

14. 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

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

16. NK - ADCC Antibody dependent cell cytoxicity

17. NK -Receptor mediated killing

18. MHC locus of mouse and man
Mouse H-2 Chr12
Class II region
Class III region
Class I region K A E D L
TNF
Human HLA Chr6
Class II region
Class III region
Class I region DP DQ DR
B C A a b a b a b E H G F
Complement
TNF
~ 10 Mb

19. NK cells – how they kill

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

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

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
No ITIM
No ITIM
Natural killer cells and their receptors
Middleton 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 receptors
Middleton 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 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
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.

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 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
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 – 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

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

45. MCMV – best studied model
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

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 p

51. Potential pathogen ligands for MHC independent inhibitory receptors
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
N. meningitidis, H. influenza, E. coli, T. cruzi
Sialic acid
Possibly SIGLEC7 or 9
Kumar & 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 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) Va24-Ja18 paired with Vb11 in humans
Viruses have ways of “maintaining” the balance of inhibitory vs activating ligands.