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.