Natural Killer Cells

Andrew MakrigiannisLaboratory of Molecular ImmunologyIRCM

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.

NK CELL DEVELOPMENT

(MAKING OF A KILLER)

NK CELL DEVELOPMENT

In Vivo

NK CELL DEVELOPMENT

In Vitro

NK CELL DEVELOPMENT

NK CELL DEVELOPMENT

NK CELL DEVELOPMENT

‘LICENSE TO KILL’

ARMING OF NK CELLS

‘LICENSING’ HYPOTHESIS

‘LICENSING’ HYPOTHESIS

NK CELL FUNCTIONS

CYTOTOXIC MECHANISMS

(THE ‘WEAPONS’)

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)

GRANULE EXOCYTOSIS

GRANULE EXOCYTOSIS

GRANYZME B: APOPTOSIS

GRANZYME A KILLING

TRANSMEMBRANE DEATH RECEPTORS

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

RECEPTORS FOR TARGET CELL RECOGNITION

(VICTIM SELECTION)

Antibody-dependent cell-mediated cytotoxicity (ADCC)

ACTIVATING RECEPTORS & ADAPTORS

ACTIVATING RECEPTORS

NKG2D MECHANISM

NKG2D LIGANDS

TUMOURS vs. NKG2D

CMV vs. NKG2D (part I)

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

MHC INHIBITORY RECEPTORS

normal cell

H-2Dd

Ly49G

granules

NKG2D

-

NK cell

tumor cell

Rae1- +

NK cell

INHIBITORY RECEPTOR FUNCTION

CMV vs. NK CELLS (part II)

m157(MHC-like)

MOUSE NK CELL RECEPTORS

Natural Killer Gene Complex (NKC)

Nkrp1 Nkg2 Ly49

Mouse chr. 6

Human chr. 12

(Yokoyama and Plougastel, 2003, Nat. Rev. Immunol., 3:304)

NKC GENE ORGANIZATION

Stalk CRD

1 2 3 4 5 6 7

TMIC

NKC GENE AND PROTEIN STRUCTURE

NKC RECEPTOR PROTEIN STRUCTURE

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

Generation of the Complex Ly49 Repertoire

FunctionalPseudogene

Y

Y Y

YY

YY

Y

Y

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.

The 129S6 Ly49 Cluster Sequence

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

0

100

200

300

400

500

600

700

kb

CEN

TEL

a

cmjlg

inhk

dfx

e

q

C57BL/6

a

cl

giy

e

q

BALB/c

CHO

MCMV

1

2

3

4

5

Human KIR Haplotypes

(Trowsdale et al., 2001, Immunological Reviews, 181:20)

q e[ ]

i g[ ]

c a

Framework Ly49 Genes

Non-MHC Inhibition of NK cells

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

THERAPEUTIC POTENTIAL

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.