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NK Cells
Experimental Basis of Immunology
January 17, 2007
W.H. Chambers, Ph.D.
G.17e Hillman Cancer Center
623-3218
[email protected]
I. Introduction
• Natural Killer (NK) Cells were first described in the early 1970’s by R.
Herberman; R. Kiessling; and G. and E. Klein
• Defined as a functional entity, i.e. cell capable of recognizing and
killing tumor cells without prior exposure
• Represent a component of the non-adaptive immune system
• Defined in the early 1980’s as having a large granular lymphocyte
(LGL) morphology (Reynolds, et al., 1981)
• Represent a heterogeneous population of cells with diverse functions
• Can be best defined phenotypically as CD3-, CD16+, CD56+, CD122+,
CD158+, CD161+
Innate capacity of lysis
Large granular lymphocytes
CD3-, CD16+, CD56+, CD122+, CD158+, CD161+
II. Pathway of NK Cell Differentiation: Topics
• Differentiation of NK cells in the fetus
• Differentiation of NK cells in adults
• Terminal differentiation of mature
NK cells
NK Cell Differentiation
• Derive from, and require normal, intact bone marrow for
functional maturation
• Represent one of the major lymphocyte populations [T, B,
NK, NK-T] – ~5% of cells among PBLs
• Present in athymic [nude] mice and rats
• Present in scid mice, and in RAG-1 and RAG-2 knockout
mice
• Can be distinguished from other lymphocytes by the
absence of clonally distributed, receptors derived via gene
rearrangements
NK Progenitors: Fetus
Fetal thymus
p-T
Fetal liver
HSC
Fetal blood
CLP
c-T/NKP
c-kit+
Thy-1CD25CD161c-
c-kit+
Thy-1CD25CD161cCD19B220lo
Modified from Lian and Kumar, 2002
p-T/NK
c-kit+
Thy-1+
CD25CD161c+
p-T/NK
c-kit+
Thy-1+
CD25CD161c+
T
c-kit+
Thy-1+
CD25+
CD161c-
p-NK
c-kitThy-1+/CD25CD161c+
.
.
..
NK
Differentiation of NK Cells: In Vitro Requirements for
Growth and Maturation
- Stroma from normal animals
estrogen- or strontium-treated mice have functionally impaired NK cells
stroma from LTa-/- mice have functionally impaired NK cells
- Cytokines for growth and Differentiation
c-kit ligand; IL7; Flt3 ligand [stem cell factor]; IL15
- Cytokines and direct contact with stroma are required for
differentiation of phenotypically and functionally mature NK cells
– LY49- NK cells develop in cultures with cytokines but no stroma
NK Progenitors: Adult
Bone marrow
stroma
HSC
CLP
NKP
p-NK
Linc-kitlo
Thy-1IL7R+
Sca-1lo
CD122+
CD161cCD49b-
CD122+
CD161c+
CD49b-
.
.
..
..
NK
NK
..
CD122+
CD161c+
CD49b+
Ly49+
CD122+
CD161c+
CD49b+
Ly49+
Thymus
.
.
..
p-NK
NK
p-T
T
p-T/NK
Modified from Lian and Kumar, 2002
T
HSC
MY
X
pB
CLP
Bone Marrow
T/NKP
NKP
pT
.
.
..
NK
.
.
..
Periphery
NK
NK
. ..
.
CTX
.
.
..
NK
Enhanced
CTX
NK
Stimulus
NK
Cytokines:
IFNg
GM-CSF
TNFa
Modified from Yokoyama, et al, 2004
NK
NK
NK Progenitors: Adult
Periphery
Cytokines:
IFNg
GM-CSF
TNFa
.
.
..
NK
Stimulus: Hrs
IL2
IL12
IL15
IL23
IL27
IFNa, -b
NK
NK
Enhanced
CTX
NK
NK
NK Progenitors: Adult
Periphery
.
.
..
NK
Stimulus: Days
IL2
IL12
IL15
IL23
IL27
IFNa, -b
NK
NK
Cytokines:
IFNg
GM-CSF
TNFa
NK
NK
Enhanced
CTX, with
broader specificity
Proliferation
Knockouts/Transgenics: Transcription Factors
Gene Deleted
Ikaros
Effect
NK cells absent
PU.1
NK cell number decreased,
normal lytic function
NK cell number decreased,
Barton, 1998
decreased lytic function
NK cells decreased or absent,
Yokota, 1999
reduced lytic function
Ikawa, 2001
Altered acquisition of Ly49sHeld, 1999
Kunz,2001
NK cell number decreased,
Duncan, 1996
lytic function impaired
Ogasawara, 1998
NK cell number decreased,
Lohoff, 2000
lytic function impaired
Ets-1
Id2
TCF-1
IRF-1
IRF-2
Reference
Georgopoulos, 1994
Wang, 1996
Colucci, 2001
Knockouts/Transgenics: Receptors
Gene Deleted
LTbr
LTa1b2
Effect
NK cells severely decreased
NK cells severely decreased,
reduced lytic function
IL15Ra
IL2/15Rb
NK cells severely decreased
NK cells absent
c-kit
NK cells decreased, impaired
lytic function
Reference
Wu, 2001
Iizuka, 1999
Smyth, 1999
Ito, 1999
Lodolce, 1998
Gilmour, 2001
Suzuki, 1997
Colucci, 2000
Knockouts/Transgenics: Cytokines
Gene Deleted
IL15
Flt3-ligand
Effect
NK cells absent; no lytic
function
NK cells severely decreased,
impaired lytic function
Reference
Puzanov, 1996
Kennedey, 2000
McKenna, 2000
NK Cell Differentiation Pathway: Informative Gene
Knockout and Transgenic Mice
HSC >> CLP >> T/NKP >> NKP >> NK
Trnscrptn. Fctr./DBP
Ikaros
PU.1 (P)*
Cytokine/Rcptr.
Flt3L
Sgnl. Trnsdcr.
Jak3
Ets1
Id2
CD3e tg
FceR1g tg
IRF-1
IRF-2
STAT5a/b (P)
MEF (P)
IL15
IL15Ra
IL2/IL15Rb
LTa/LTbR (P)
III. NK Cell Function as Anti-tumor and
Anti-Viral Effector Cells
• NK cells were initially described as being
cells important for surveillance against
tumor development, or more importantly,
against tumor metastases
• NK cells were also found to be important as
anti-viral effector cells, particularly against
Herpes virus infection.
NK cells and Anti-tumor Activity
•
•
What is the evidence of NK cell
anti-tumor function?
In vitro – many tumor cells are
susceptible to lysis by NK cells
depending upon how you assess killing (Kashii,
Y., et al. J. Immunol. 163:5358-66 [1999]).
In vivo…..
Putative Evidence for Immunosurveillance by NK
Cells Using Transplantable Tumor Models
•
•
•
•
Elimination of NK cells resulted in increased tumor growth
Elimination of NK cells resulted in increased numbers of
metastastic lesions in lungs
Adoptive transfer of NK cells, into immunodeficient
animals challenged with tumors, results in tumor clearance
in metastases models
Best results almost always were derived in models of
metastatic disease (Barlozzari, T., et al., J. Immunol.
134:2783-2789, 1985)
This evidence did not initially garner robust
support for NK cell participation in immune
surveillance – Why?
•
•
•
There has been a growing belief that transplantable tumor
models have little value in assessing tumor immunity, and
particularly for “immune surveillance” of tumors
The only report providing evidence for disease in
individuals with reduced NK cells is for recurrent Herpes
virus infections (Biron, C.A., et al., NEJM 322:1731-1735,
1989)
Identification of receptors on NK cells with coordinate
tumor cell ligand was lacking
Studies Supporting Increased Incidence of
Cancer in Immunosuppressed Individuals
• An 11 year follow-up study of immune function and cancer
incidence in a general population of 3625 individuals was
carried out (Imai, K., et al., The Lancet 356:1795-1799,
2000)
• Immune function, i.e. NK cell lytic activity, was assessed
at baseline and cancer incidence
• Medium and high cytolytic function was associated with
reduced cancer risk; low cytolytic function was associated
with increased cancer risk
Support for NK Cells Providing a Mechanism for
Immune Surveillance of Cancer
•
•
•
•
•
Families of NK cell receptors (e.g. NKG2s) with activating and
inhibitory function have been defined
Tumor associated ligands similar to MHC Class I have been defined,
e.g. Rae-1 [mice], MICA/B [humans]
Binding of MHC Class I and Class I-related proteins (e.g. Rae-1(a-e);
ULBP-1, -2, -3; H60) by NKR has been demonstrated
In mice, binding of NKG2D to Rae1a (Cerwenka, A., et al., PNAS
USA 98:11521-11526, 2001) or Rae1b (Diefenbach, A., et al., Nature
413:165-171, 2001) has been demonstrated to activate anti-tumor lytic
function
Human orthologs of Rae-1 genes, e.g. ULBP-1 also are bound by
NKG2D; and this activates NK lytic function
NK Cells as Anti-viral Effector Cells:
Evidence for a role as anti-virus effector cells
• Natural defects in NK cells
Recurrent Herpes virus infections [Biron, 1989]
• Expansion of NK cells during viral infections
LCMV infections
• Viral antigens as ligands for NK cell receptors
ULBP1-4
NK-mediated Response to Virus Infection
NK Cells as Anti-viral Effector Cells:
Mechanisms of Evasion of NK Cell Function by Viruses
• Expression of virally encoded MHC class I protein
homologs
• Selective modulation of MHC Class I expression by viral
proteins
• Virus-mediated inhibition of activating receptor function
• Production of virally encoded cytokine-binding proteins or
cytokine-receptor agonists
• Direct viral effects on NK cells – infection/envelope
ligation of inhibitory receptors
Virus-infected Cell
NK Cell
MHC ClassI
Homolog
Selective
Expression
Activating
Receptor
Antogonist
Virus
Down Regulating
Activating Ligand
MHC Class I
Inhibitory
Receptor
1
MHC Class I
Inhibitory
Receptor
2
Activating
Receptor
3
Cytokine Binding
Protein
Cytokine
Receptor
Cytokine
Antogonist
4
Cytokine
Receptor
NK Cell
Infection
5
IV. NK Cell Recognition Receptors
•
•
•
•
•
“Missing Self” Hypothesis
Activation and Inhibition via Receptors
Recognition of “Self”
Recognition of Tumor Cells
Recognition of Virus-infected Cells
“Missing Self” Hypothesis
•
•
•
NK cells do not require expression of MHC Class I determinants for
recognition of target cells.
There is, in fact, an inverse relationship between expression of MHC
Class I and susceptibility to lysis by NK cells, i.e. less Class I equals
more lysis.
Led to the hypothesis* that NK cells surveyed the surface of target
cells for “self”. If it was present, the cell was presumed to be normal
and not lysed. If self was absent, as is often the case in tumor cells and
virus-infected cells, NK cells could be activated to lyse the “abnormal”
cell.
*Ljunggren, H.G. and K. Karre, 1990. Immunology Today 11:237-244.
Receptors in Innate and Adaptive Immunity
Characteristics
Innate
Adaptive
Specificity inherited in the
genome
Yes
No
Expressed by all cells of a
particular type
Yes
No
Trigger immediate response
Yes
No
Recognize broad classes of
pathogens
Yes
No
Encoded in multiple gene
segments
No
Yes
Require gene rearrangement
No
Yes
Clonal distribution
No
No
Yes
Yes
Able to recognize a wide
variety of molecular
structures
Recognition – NK cells
-
-
-
There is no evidence supporting clonally restricted recognition
molecules expressed by NK cells, nor for recombinatorial events being
important for development of an NK cell repertoire
NK cells recognize MHC determinants, but these structures, nor
peptides expressed by MHC, are target antigens for activation of NK
lytic function
Some NK cells express CD8 homodimers, but it is unclear whether
binding to MHC Class I affects activation
NK cell recognition of targets involves a balance between inhibitory
signals and activation signals
Receptor:ligand pairs providing inhibitory signals are fairly well
defined
Receptor:ligand pairs providing activation signals are rapidly being
defined
NK Cell Gene Complex (NKC)
• The NKC is a genomic region, first described on NK cells, encoding
structurally related receptors
• NKC maps to Chromosome 12p13, 6 and 4 in man, mouse and rat,
respectively
• Type II integral membrane proteins with external domain similar to Ctype (Ca++-dependent) lectins. However, they lack amino acid residues
that coordinate binding of Ca++, and do not bind carbohydrates in the
same manner as conventional C-type lectins. Can be expressed homoor heterodimers.
• Highly conserved evolutionarily – found in sea squirt and several
poxviruses
• Activating and inhibitory receptors for immune cells; can be either
primary or co-stimulatory receptors.
NK Cell Gene Complex (NKC)
- Contains genes encoding C type lectin related receptors
- Disease resistance elements mapped to this locus, e.g. Cmv1
- Conserved across species
Human – Chromosome 12
Mouse – Chromosome 6
Rat – Chromosome 4
Leukocyte Receptor Cluster (LRC)
LRC is a ~1 mb region located on chromosome 19q13.42
NK Cell Inhibitory Receptors: CLRR and KIR
Name
p58.1
p58.2
p70
p140
p49
LIR1
LIR2
CD94*
NKG2A
NKR-P1B, D
p40
IRC1
p75AIRM1
Alternative Name[s]
KIR2DL1
KIR2DL2
KIR3DL1
KIR3DL3
KIR2DL4
ILT2/LILRB1
ILT4/LILRB2
KLRD1
KLRC1/CD159A
CD161B, D
LAIR1
IRp60/CMRF35H
Siglec-7
*CD94 forms heterodimers with NKG2A, -C and –E
**CD94/CD159A heterodimer is specific for HLA-E
Cellular Ligand
HLA-Cw2,4,5,6
HLA-Cw1,3,7,8
HLA-Bw4
HLA-A3, -A11
HLA-G
HLA-G
HLA-F
HLA-E**
HLA-E
Clrb
?
?
Sialylated sugars
Viral Ligand
HCMV-UL18
Target Cell membrane
NH3
IgV
IRp60
NK Cell membrane
Cytoplasm
SHP-1
COOH
I/VxYxxL
Inhibition of lytic function
ITIM
• Immunoreceptor tyrosine-based inhibitory motif
• Based upon the amino acid motif: I/VxYxxL
• Commonly expressed in signaling receptors in
lymphocytes
• Recruits SHP-1/SHP-2 phosphatases
• Linked to inhibition of function in lymphocytes
NK Cell Activating Receptors
Name
NKp46
NKp30
NKp44
2B4
NTB-A
NKp80
CD16
CD2
DNAM-1
NKG2D
NKR-P1A
NKR-P1C
NKR-P1F
P40
IRC1
p75AIRM1
Alternative Name[s]
Ly94/NCR1
IC7/NCR3
Ly95/NCR2
CD244
KALI
KLRF1
FcgRIII
LFA-2
CD226
D12S2489E/CD159D
CD161A
CD161C
CD161F
LAIR1
IRp60/CMRF35H
Siglec-7
Cellular Ligand
Viral Ligand
?
SV-HA, IV-HA
?
?
SV-HA, IV-HA
CD48
?
?
IgG
CD58, LFA-3
PVR/CD155, Nectin-2/CD112
MICA, MICB, MULT1 ULBP1-4
[IC-21]*
?
Clrg
?
?
Sialylated sugars
*Rat NKR-P1A binds an undefined determinant on IC-21 tumor cells
NK cell activating receptors
•
•
•
•
•
•
Loss of the inhibitory signal does not, in and of itself, provide signals
to kill target cells
Some receptors able to activate NK cells to kill target cells have been
defined – NKG2D, Ly49D, Ly49H, NKp30, NKp44, NKp46, CD161A
Some activating receptors are members of the C-type lectin [e.g.
NKG2D] and IgSF [NKp30] superfamilies
IgSF members often referred to as KARs
Associate with an adaptor molecule [e.g. DAP12] containing an ITAM.
Associate via a charged residue in the TM domain
Some ligands for activating receptors have been defined, e.g. RAE-1
for NKG2D
NH3
NKp46:SV-HA or IV-HA
IgC2
IgC2
FceR1g
CD3z
R
NK Cell membrane
*D
COOH
I
T
A
M
I
T
A
M
I
T
A
M
Cytoplasm
ZAP70
SYK
I
T
A
M
Activation
NKp44:SV-HA or IV-HA
NH3
IgV
DAP12
K
NK Cell membrane
*D
Cytoplasm
I
T
A
M
I
T
A
M
ZAP70
SYK
COOH
Activation
NKp30:? [iDCs and some tumors]
NH3
IgV
CD3z
R
*
COOH
NK Cell membrane
D
I
T
A
M
I
T
A
M
I
T
A
M
I
T
A
M
I
T
A
M
I
T
A
M
Cytoplasm
ZAP70
SYK
Activation
ITAM
• Immunoreceptor tyrosine-based activating motif
• Based upon the amino acid motif: …YxxL/Ix6-8YxxL/I…
• Serves as a signaling partner to transmembrane receptors with a
charged residue in the transmembrane region which allows docking of
signal transducers such as DAP12, CD3z-CD3z homodimers, CD3zFcer1g heterodimers
• Activation of cells either via PI3 kinase, or ZAP70 or Syk tyrosine
kinases
NKG2D
•
•
•
•
Single gene
Distantly related to other NKG2 family members
Alternatively spliced isoforms (short and long) in mice
NKG2D-s and NKG2D-l, short from binds both DAP10
and DAP12
• Expressed in NK cells, CD8+ cells and macrophages
NKG2D:MICA, MICB, ULBPs
COOH
COOH
CTLD
CTLD
DAP10
R
NK Cell membrane
*D
Y
x
x
M
Cytoplasm
PI3K
Grb2
NH3 NH3
ERK1/2
MAPK
Cytokine secretion
Cytotoxicity
Ligands for NK Cell Activating Receptors
•
•
•
•
•
•
MICA, MICB: Stress-inducible molecules encoded within the human MHC,
also can be induced by some infections. Normally expressed by
gastrointestinal epithelium, but also by some epithelial, lung, breast, kidney,
ovary, prostate and colon tumors, and by some melanomas. Transmembrane
with a1, a2, and a3 domains; but do not associate with b2m and do not bind
peptides.
ULBP1-4: 1-3 are GPI-linked, cell surface molecules which bind human
cytomegalovirus UL-16; ULBP-4 is a cell surface molecule with
transmembrane and cytoplasmic domains. ULBPs have a1 and a2 MHC
Class I-like domains.
Rae1b: Retinoic acid inducible protein, in mice, that shares sequence
homology with ULBPs. Expressed in early embryogenesis and in some
tumors, but generally absent in normal tissues.
H60: Minor histocompatibility antigen expressed by Balb/c mice, target for
alloreactivity responses by C57Bl/6 mice.
DCs: Known that NKp30 is required for recognition of immature DCs by
activated NK cells.
IC-21: Known that rat CD161A is required for recognition of IC-21 tumor
cells to mediate their lysis.
Signal Transduction Pathway for NK cells*
(NKp44)
(KIR2DL1)
(NKG2D)
(DAP12)
Modified from Vely and Vivier, 2005, www.stke.org/cgi/content/full/sigtrans;2005/292/cm6
V. Non-adaptive vs. Adaptive Function
- Mediators of non-adaptive immunity
- Interface between non-adaptive and adaptive
immunity –
“Passive” interaction – antibody dependent cellular
cytotoxicity
“Active” interaction – reciprocal co-activation of NK cells
and DCs to induce adaptive responses
- New hypothesis regarding NK cells as mediators of
adaptive immunity is the topic of the journal club
article
Interactions with Dendritic Cells to Promote
Adaptive Immune Responses
VI. Therapeutic Applications of NK Cells
Biological Response Modifiers
IL2, IL12, IL15, IL21, IFNa, IFNg, IFNb, PolyI:C, b-glucan
Adoptive Cellular Immunotherapy
Freshly isolated NK cells – autologous/allogeneic-alloreactive
BRM/Cytokine activated NK cells – autologous/allogeneic
Long term established NK cell lines (NK-92)
NK Cells as Vehicles for Delivery of Therapeutic Agents
Chemotherapeutic agents - doxorubicin
Cytokines – IL2
Trials for:
melanoma, renal cell carcinoma, lung carcinoma, ovarian cancer,
Glioblastoma – variable results
Utilization of Modified NK-92
Modification of
functional activity
IL2
Prolonged
in vivo
activity
NK-92
Control of in
vivo Expansion
NK-92-CD20
Targeting specific
tumor types
NK-92-Her2/neu
NK-92-CD19
NK-92-CD38
Epithelial tumors
breast
ovarian
Myeloma
In vivo control
of proliferation
through suicide
gene binding
B-cell precursor
leukemia
Improved cytolytic efficacy
Accessibility to resistant tumors
Modified from Suck, G. 2006