natural killer cells andrew makrigiannis laboratory of molecular immunology ircm
TRANSCRIPT
Natural Killer (NK) Cells
• Found predominantly in blood, liver, spleen, and lung. Rare in recirculating lymphocyte pool
• Non-T, Non-B lymphocyte
• Distinct Marker Combinations
– CD3-, Ig-, NK1.1+, DX5+
• Distinct Morphology
– large granular lymphocytes
• Cytotoxic without prior sensitization
• Multiple roles in Innate Immunity
– tumor surveillance
– viral immunity
– Th1 vs Th2 via IFN-
IMPORTANCE
• Elevated NK cell-mediated activation or cytotoxicity seen in infections of:
• Arenavirus (Lymphocytic choriomeningitis virus)• Herpesvirus (Murine cytomegalovirus and Herpes
simplex virus)• Orthomyxoviruses (Influenza virus)• Picornavirus (Coxsackie virus)
• Peak NK cell responses in first several hours or days• Peak T and B cell responses take more than a week
to develop
IMPORTANCE
• Case study 1: Four related patients with specific NK cell deficiency resulting in Epstein Barr virus-driven lymphoproliferative disorder and viral-based respiratory illnesses.
• Case study 2: 13 year old with complete lack of NK cells
– Initial overwhelming chicken pox infection and varicella pneumonia
– Antibody and T cell responses were intact
– Later developed primary life-threatening HCMV infection and a severe HSV infection.
Other case studies: Low or no NK cell cytotoxic activity linked with increased sensitivity to: HSV, EBV, HCMV, papilloma virus.
Also, NK cell defects occurn late in HIV infection.
PERFORIN
-found exclusively in the cytoplasmic granules of killer cells
-CTL and NK from perforin KO mice have major cytotoxic defects.
-Perforin KO mice are more susceptible to tumours and infection.
-pore forming protein of plasma membrane or endosome allowing granzymes to enter target cell cytoplasm
-granulysin (humans) thought to function similarly to perforin
GRANZYME B
-protease located in cytoplasmic granules of killer cells
-cleaves after aspartate residues that have the sequence: Ile/Val-Gly/Met/Glu-X-Asp-X-Gly
-caspase 3 and 8 are direct substrates for granzyme B
-caspases are cysteine proteinases involved in apoptosis
-other granzymes in granules (granzyme A, K, M)
NK CELL CYTOKINES
Interferon gamma (IFN-):
- activation, growth and differentiation of T, B, NK cells and macrophages
-promotes Th1 differentiation
-enhances MHC expression on APC
-some direct anti-viral activity
NK CELL CYTOKINES
Tumour necrosis factor alpha (TNF-):
-paracrine and endocrine mediator of inflammation
-regulates growth and differentation of a wide variety of cells
-selectively cytotoxic for many transformed cells
NK CELL CYTOKINES
Granulocyte/macrophage colony stimulating factor (GM-CSF):
-survival and growth factor of hematopoeitic stem cells (HSCs)
-differentiation and activating factor for granulocytic and monocytic cells
-growth factor for endothelial cells, erythroid cells, megakaryocytes and T cells
Human
Cs-s
Cs-s
Cs-s
NH2
Cs-s
Cs-s
NH2
KIR
Both
MHC INHIBITORY RECEPTORS
Mouse
NH2 NH2
Ly49
NH2
NH2
CD94/NKG2A
SHP-1 SHP-2 SHIP
normal cell
H-2Dd
Ly49G
granules
NKG2D
-
NK cell
tumor cell
Rae1- +
NK cell
INHIBITORY RECEPTOR FUNCTION
Natural Killer Gene Complex (NKC)
Nkrp1 Nkg2 Ly49
Mouse chr. 6
Human chr. 12
(Yokoyama and Plougastel, 2003, Nat. Rev. Immunol., 3:304)
A, B, C, E, F, G, I, J, M, O, S, T, and V
COOH COOH
MURINE INHIBITORY LY49
NH2 NH2
SH
2S
H2
PT
Pa
se
SHP-1 Cytotoxicity Cytokines
ITIM
COOHCOOH
R54R54
NH2 NH2
DD
YxxL(x)6-8YxxL
YxxL(x)6-8YxxL
-PO4
-PO4
Zap70
DAP12
Syk
SH
2S
H2
Cat
SH
2S
H2
Cat
D, H, L, P, R, U, and W
MURINE ACTIVATING LY49
Cytotoxicity Cytokines Chemokines
Why are Ly49s important?
• Functional analogues of KIRs
• Regulate NK cell reactivity to self
– Inhibitory Ly49 - Autoimmunity
– Activating Ly49 - Pathogens
• Resistance to MCMV, Ectromelia, HSV, and
Leishmania have been mapped to the Natural Killer
Gene Complex.
Class I MHC Binding by C57BL/6 Ly49Family Members
A Dd, k B ?C Db, d, k, Kb,d (peptide receptive)
D Dd, r, sp
E ?
F ?
G Dd, Ld
H m157 (MCMV)
I Db
J ?
Ly49 Putative Ligand
CentromereTelomere
Organization of the Ly49 Gene Family in B6 Mice
Functional genePseudogene
Transcriptional orientation
a c m j l g i n h k d f x e q
The ‘129’ Inbred Mouse Strain
• Embryonic stem cells used for gene deletion models.
• 129 mice more susceptible to intracellular pathogens
than B6 mice.
• 129 mice show impaired allogeneic and xenogeneic
graft rejection.
• 129 NK cells show unusual reactivity with anti-Ly49
mAb.
• Many substrains: 129P3, 129X1, 129S1-6 etc.
iBALB
ui129
kB6
nB6
hB6
u129
s129fB6
e129
eB6eBALB
ec2129
ec1129
g129
gBALBgB6t129
r129
dB6 lr129
pd129
p129
yBALB xB6
mB6
lBALB
aB6aBALBo129
v129
q2129
q3129
q1129
qB6
qBALB
b129
bB6
bBALB
iB6
i2129
cB6
cBALB
jB6
i1129
0.05
100
99
100100
100
81
9998
73100
9810086
9762
D
L
AG
B
QE
H
I
Ly49Subfamilies
0.05
s129fB6
e129eB6
ec2129
ec1129
ui129
kB6
nB6
hB6
u129
iB6
i2129
jB6
cB6
i1129
q2129
q3129
q1129
qB6
b129
bB6
r129dB6lr129
pd129
xB6
p129
mB6lBALB
o129
v129
aB6g129
gB6
t129
Exon 2
iB6ui129
kB6
nB6
hB6u129
s129fB6e129eB6
ec2129
ec1129
g129
gB6 t129
r129
dB6
lr129
pd129
xB6
p129
mB6
lBALBo129
v129
aB6
q2129
q3129
q1129qB6
b129bB6
i2129jB6cB6
i1129
0.05
Total coding region
0.05
q2129
q3129
q1129
qB6
iB6
i2129
jB6
cB6
i1129
ui129
kB6
nB6
hB6
u129
s129
fB6
e129eB6ec2
129
ec1129
b129bB6r129
dB6
lr129
pd129xB6
p129
g129
gB6
t129
mB6lBALB
o129
aB6
Exon 3
0.05
b129bB6
mB6lBALB
g129
gB6t129
o129v129
aB6
q2129 q3
129
q1129
qB6
ui129kB6nB6
hB6
u129
iB6
i2129jB6
cB6
i1129
s129
fB6e129
eB6ec2
129
ec1129
r129
dB6lr129
pd129
xB6
p129
Exon 4
0.05
mB6
lBALB
g129
gB6
t129o129
v129
aB6
r129dB6 lr129
pd129
xB6
p129
ui129
nB6
hB6
u129
iB6i2129
jB6
cB6
i1129kB6
s129fB6
e129
eB6
ec2129
ec1129
Exon 6
0.05
ui129
nB6
hB6
u129
iB6
i2129
jB6
cB6
i1129
s129
fB6
e129
eB6
ec2129
ec1129
o129
v129
aB6
r129
dB6
lr129
pd129
xB6
p129 mB6
lBALB
g129
gB6
t129
Exon 5
0.05
ui129nB6 hB6 u129iB6
i2129
jB6
cB6
i1129
kB6
s129
fB6
e129
eB6
ec2129
ec1129
mB6
lBALB
g129
gB6
t129
o129v129
aB6
r129dB6
lr129
pd129
xB6p129
Exon 7
v129
Phylogenetic Analysis Reveals Hybrid Ly49 Genes
Hybrid Ly49
Stalk CRDTMIC Stalk CRDTMICStalk CRDTMIC
Stalk CRDTMIC Stalk CRDTMIC
Ly49HLy49L
Ly49G Ly49D Ly49C
129S6
ec1
q3
ec2
q2
t
v
ppd
o
i2
gi1uiu
r
s
lr
e
q1
ancestr
al
m
j
l
k
C57BL/6
a
c
gin
h
d
f
x
e
q
Ly49 Haplotype Evolution
The BALB/c Mouse
- Common animal model for immune studies
- Susceptible to MCMV
- Decreased ability to kill CHO tumor cells
- Known BALB/c Ly49: a, c, g, and l
C57BL/6
129
e f d k h i g l j ax m cn
q1 e q2 e/c2 l/r s t e/c1 r u u/i i1 g p/d i2 ov p
q
q e i g l c
0 100 200 300 400 500 600
x aBALB/c
700 kb
q3
Ly49 Gene Content in Three DivergentMouse Haplotypes
functional pseudogene unknown
0
100
200
300
400
500
600
700
kb
CEN
TEL
o
i2p
pd
g
i1uiur
ec1q3
ts
lrec2
q2
v
e
129S6
a
cmjlg
inhk
dfx
e
q
q1
C57BL/6
a
cl
giy
e
q
BALB/c
ThreeLy49Haplotypes
A G C D F E
A G C B F E
CD69CD94/NKG2
CD69 CD94/NKG2
B6
BALB
Q D K H L MCD69CD94/NKG2
B6E X NF GI CJ A
Q LCD69 CD94/NKG2
BALB
E Y GI C A
Nkrp1/Clr
Ly49
99.7 100 94.1
98.495.993.897.996.592.699.699.1
98.710095.7
Cen
Cen
Tel
Tel
Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
Farag, S. S. et al. Blood 2002;100:1935-1947
Figure 1.
Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.
Farag, S. S. et al. Blood 2002;100:1935-1947
Figure 4.
FURTHER READINGFigures and tables were taken from the following:
What does it take to make a natural killer?Nat Rev Immunol. 2003 May;3(5):413-25.
Natural killer cells, viruses and cancer.Nat Rev Immunol. 2001 Oct;1(1):41-9.
How do natural killer cells find self to achieve tolerance?Immunity. 2006 Mar;24(3):249-57.
Immune functions encoded by the natural killer gene complex.Nat Rev Immunol. 2003 Apr;3(4):304-16.
Cytotoxic T lymphocytes: all roads lead to death.Nat Rev Immunol. 2002 Jun;2(6):401-9.
Natural killer cell receptors: new biology and insights into the graft-versus-leukemia effect.Blood. 2002 Sep 15;100(6):1935-47.